Hypertonicity-induced cation channels rescue cells from staurosporine-elicited apoptosis

Cell shrinkage is one of the earliest events during apoptosis. Cell shrinkage also occurs upon hypertonic stress, and previous work has shown that hypertonicity-induced cation channels (HICCs) underlie a highly efficient mechanism of recovery from cell shrinkage, called the regulatory volume increase (RVI), in many cell types. Here, the effects of HICC activation on staurosporine-induced apoptotic volume decrease (AVD) and apoptosis were studied in HeLa cells by means of electronic cell sizing and whole-cell patch-clamp recording. It was found that hypertonic stress reduces staurosporine-induced AVD and cell death (associated with caspase-3/7 activation and DNA fragmentation), and that this effect was actually due to activation of the HICC. On the other hand, staurosporine was found to significantly reduce osmotic HICC activation. It is concluded that AVD and RVI reflect two fundamentally distinct functional modes in terms of the activity and role of the HICC, in a shrunken cell. Our results also demonstrate, for the first time, the ability of the HICC to rescue cells from the process of programmed cell death.
Introduction
It has become increasingly evident in recent years that the mechanisms of cell volume regulation are employed in a variety of physiological processes, in addition to just the maintenance of cell homeostasis. Among these processes are the coordination of transport across the apical and basolateral membranes in epithelia, the locomotion of cells, the regulation of metabolic processes in the liver and, most notably, the control of cell proliferation and apoptosis [1–3].
Since the rates of ion transport through channels are some 4–5 orders of magnitude higher than those achieved by transporters (carriers and pumps), any modulation of ion channel activity may serve as a rapid and efficient mechanism of osmolyte transport and cell volume regulation. Accordingly, hypertonicity-induced cation channels (HICCs) were found to be the main mediators of the “regulatory volume increase (RVI)” of a shrunken cell, whenever a system was analysed in a quantitative fashion [3–5]. Despite the low selectivity of HICCs for Na+ over K+, activation of these channels leads to an actual net uptake of cations, which is mainly due to the membrane voltage, which is negative intracellularly. Anions and osmotically obliged water follow, allowing the cell to complete the process of RVI [3–5].
Persistent isotonic shrinkage of a given cell, as opposed to hypertonicity-induced shrinkage, is one of the hallmarks of apoptosis. In many instances, this “apoptotic volume decrease (AVD)” employs the activation of K+ channels, which serve as the main routes of K+ exit [6–9]. Cl− channels also should open, with this parallel transport strongly depending, however, on whether or not the electro-chemical driving forces actually favour conductive Cl− export [1, 2, 10–12].
The question is thus raised of how hypertonic stress might interfere with AVD. Would osmotic shrinkage facilitate the process? Or would the activation of HICCs or RVI oppose the induction of apoptosis?
Materials and methods
Cell culture
Human cervix HeLa cells were grown as monolayers in minimum essential medium supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin and 40 IU/ml penicillin, under 95% air/5% CO2 at 37°C. For the experiments, cells were mechanically detached from the plastic substrate of the culture flasks with a jet of culture medium and then kept in suspension for 15–120 min.
Determination of cell volume
Changes of HeLa cell volume were quantified by means of electronic cell sizing on a Coulter-type analyzer (CDA-500; Sysmex, Kobe, Japan), as previously described [13]. The experimental solution contained (in mM): NaCl, 95; KCl, 4.5; MgCl2, 1; CaCl2, 1; NaHCO3, 10; HEPES, 5. Osmolality was adjusted to values from 300 to 600 mosmol/kg-H2O by adding mannitol under osmometric control (OM802; Vogel, Giessen, Germany). Measurements were performed at room temperature.
Patch-clamp experiments
Membrane currents were recorded in the fast whole-cell mode of the patch-clamp technique using 2 MΩ borosilicate pipettes. Currents were recorded with an Axopatch 200B amplifier (Molecular Devices, Union City, CA), filtered at 5 kHz with a four-pole Bessel filter and digitized at 20 kHz. pClamp 9.02 software (Molecular Devices) was used for control of the pulse protocol, as well as for data acquisition and analysis. Series resistance was <5 MΩ and was compensated (by 70–80%) to minimize voltage errors. In the experiments, voltage ramps from −80 to +20 mV and of 1 s duration were applied every 10 s; holding voltage was −30 mV.
The bath solution (pH 7.5) contained (in mM): NaCl, 94; Na-gluconate, 6; MgCl2, 1; CaCl2, 2; TEA-Cl, 2; HEPES, 10; d-glucose, 10. Osmolality was adjusted to 310 (isotonic) and 400 mosmol/kg-H2O (hypertonic) by the addition of mannitol.
The pipette solution (pH 7.3, 300 mosmol/kg-H2O) contained (in mmol/l): NaCl, 26; Na-gluconate, 69; MgCl2, 1; TEA-Cl, 2; Na2-ATP, 2; Na2-GTP, 0.5; HEPES, 10; EGTA, 1. With these ion gradients, the equilibrium potentials for Na+ and Cl− (as the only permeant ions) are set at 0 and −30 mV, respectively [13]. Experiments were performed at room temperature.
Monitoring apoptotic cell death
Cell viability and caspase-3/7 activity were determined by use of a calorimetric MTT assay (Cell Counting Kit-8; Dojindo, Kumamoto, Japan) and a fluorometric probe (Apo-ONE Homogeneous Caspase-3/7 Assay; Promega, Madison, WI), respectively.
Fragmentation of DNA was determined by means of a photometric enzyme immunoassay (Cell Death Detection ELISAPLUS, Roche Applied Science, Diagnostics, Mannheim, Germany) with HeLa cells plated on 96-well plates. Following exposure to the various experimental conditions, cells were lysed and centrifuged at 200g. The cytoplasmic (DNA containing) fraction was then transferred to streptavidin-coated microtiter plates that had been incubated with a biotinylated monoclonal anti-histone antibody. Finally, the amount of the fragmented DNA on bound nucleosomes was determined with a peroxidase-conjugated monoclonal anti-DNA antibody and the substrate ABTS [2,2′-azino-di-(3-ethylbenzthiazoline sulfonate)], which was quantified photometrically at 405 nm. In all measurements, cells were incubated for 6 h at 37°C.
Testing for necrosis and late apoptosis
Necrosis and late apoptosis of cells were quantified fluorometrically on the basis of a propidium iodide staining of nuclei (excitation/emission at 530 nm/620 nm) performed with reference to an overall staining of cells by Hoechst 33342 (excitation/emission at 350 nm/460 nm).
Chemicals
All reagents were purchased from Sigma–Aldrich (Tokyo, Japan).
Statistical analysis
Data are presented as mean values ± SEM with n denoting the number of observations. Student’s t tests for paired and unpaired data were used as appropriate and P < 0.05 was considered significant.
Results and discussion
As was determined by means of electronic cell sizing, increasing osmolality from 300 mosmol/kg-H2O to 330, 400, 500 and 600 mosmol/kg-H2O led to an initial shrinkage of HeLa cells to 96.6 ± 0.4, 82.6 ± 1.9, 72.5 ± 0.2 and 66.2 ± 0.6% of the control level, respectively (n = 10; Fig. 1a). When plotted as a function of 1/osmolality (which is equivalent to a ‘Boyle-van’t Hoff graph’ [3, 14]), these data could readily be fitted by a straight line; its intercept with the y-axis yields the osmotically active versus inactive space of the cells, with the latter amounting to 30.6% of total cell volume (Fig. 1a, Insert). This osmometric behaviour of HeLa cells was then followed by an active volume recovery to 101.7 ± 0.4, 92.6 ± 0.5, 89.7 ± 0.8 and 88.4 ± 0.9%, in 120 min of hypertonic stress (Fig. 1a, b). The process of “regulatory volume increase (RVI)” occurred at essentially constant rates that equalled 0.4 ± 0.2, 0.9 ± 0.2, 1.6 ± 0.2 and 1.8 ± 0.2%/10 min, respectively, in the range of 5–120 min after application of hypertonicity.
Fig. 1Hypertonicity-induced HeLa cell shrinkage and RVI determined by electronic cell sizing. (a) The volume response of cells to increases in osmolality (from 300 mosmol/kg-H2O) to 330, 400, 500 and 600 mosmol/kg-H2O at time zero (arrow) are depicted. The insert gives the initial (passive) amount of cell shrinkage as a function of 1/osmolality (equivalent to a ‘Boyle-van’t Hoff plot’ of these data; see text for details). 3.33 kg-H2O/osmol = 1/(0.300 osmol/kg-H2O) is the 100% value under isotonic conditions. (b) The volume of HeLa cells at 120 min of various levels of hypertonic stress, after partial volume recovery (RVI) had been achieved. n = 10 for each experimental condition
It has become increasingly evident in recent years that persistent cell shrinkage is one of the hallmarks of apoptosis [6, 15]. Also, it has recently been found that after 2-h stimulation with staurosporine (STS), TNFα or Fas ligand, HeLa cells lose their ability to undergo RVI (12). To test whether HeLa cell shrinkage and/or RVI interferes with apoptotic volume decrease (AVD), 4 μM STS was used to induce apoptosis in HeLa cells [6], and a series of volume measurements was made. This was done under isotonic conditions (300 mosmol/kg-H2O) and under hypertonic conditions in which osmolality was increased to 330, 400, 500 and 600 mosmol/kg-H2O (n = 10). Within an experimental time frame of 120 min, STS was found to reduce cell volume to 65.8 ± 1.6% that of the control, under isotonic conditions (Fig. 2a: diamonds). 5 min after osmolality was increased to 330, 400, 500 and 600 mosmol/kg-H2O, cell volume equalled 89.6 ± 0.7, 81.4 ± 0.2, 71.4 ± 0.9 and 64.5 ± 0.8% of control, respectively. These values are virtually identical to the values of purely passive or physical responses to hypertonic stimulation in the absence of STS (cf. to Fig. 1a). Moreover, an osmotically inactive space of 35.4% could be calculated from these measurements; this value is very similar to that obtained in the control experiments (see above). Of note, no RVI followed hypertonicity-induced cell shrinkage when STS was present. This indicates that the RVI response was completely inhibited by STS (or surpassed by the STS-induced AVD process), a phenomenon which clearly holds for the full 2 h period after STS application. Using the respective values for volume at 5 min as passive reference values, AVD was computed to be 30.8 ± 0.7, 22.2 ± 0.7, 12.1 ± 0.5 and 7.2 ± 0.7% for 330, 400, 500 and 600 mosmol/kg-H2O, respectively (calculated as the volume at 5 min minus the volume at 120 min). So as summarized in Fig. 2b, a decrease of AVD was observed with increasing hypertonic stress. On the one hand, then, hypertonic stress appears to suppress STS-induced AVD; on the other hand, STS seems to abolish hypertonicity-induced RVI. Also of note, in this respect, is a recent study on HeLa cells in which tyrosine kinases, PKC, and p38 MAPK were identified as part of the signalling machinery employed in the activation of HICC and RVI [16]. Given the non-specificity of STS with respect to its inhibition of various protein kinases, it is not surprising that RVI is inhibited in the presence of the compound, while AVD is induced.
Fig. 2Effects of hypertonic stress on apoptotic volume decrease (AVD). (a) At time zero (arrow), 4 μM staurosporine (STS) was added to induce apoptotic shrinkage of HeLa cells under isotonic conditions (◆) or together with an increase of osmolality to 330 (■), 400 (▲), 500 (●) and 600 (▼) mosmol/kg-H2O. Hypertonic cell shrinkage, at 5 min, was very similar in the presence and absence (see Fig. 1) of STS. (b) AVD under isotonic conditions (volume at 0 min minus volume at 120 min) and hypertonic conditions (volume at 5 min minus volume at 120 min). n = 10 for each experimental condition
To study the interference of STS-induced AVD with hypertonicity-induced RVI in more detail, experiments were performed in which osmolality was increased from 300 to 500 mosmol/kg-H2O, at time zero (see Fig. 3a: arrow). In addition, 4 μM STS was applied either 2 h before (t = −120 min), 1 h before (t = −60 min), at the same time as (t = 0) or 1 h after (t = 60 min) hypertonic stimulation (see Fig. 3a: arrowheads). AVD was then computed from the overall changes in cell volume (i.e. the volume at 0, −60 or −120 min, respectively, minus the volume at 120 min) minus the amount of passive cell shrinkage (i.e. the volume at 0 min minus the volume at 5 min). Hypertonic stress (500 mosmol/kg-H2O) significantly reduced AVD from the control value of 34.2 ± 0.5% (Fig. 2b: 300; also marked as STS-ISO, in Fig. 3b) to 12.1 ± 0.5 or 12.5 ± 0.5% (Fig. 3b: STS-HYPER or HYPER/1hSTS) when given simultaneously with, or 1 h before, application of STS (n = 10, P < 0.001 each), respectively. Also apparent from these experiments is that no RVI occurred in the presence of STS. It is noteworthy that increasing the osmolality to 500 mosmol/kg-H2O after 1-h pretreatment with STS still led to a markedly reduced AVD of 25.9 ± 0.7% (P < 0.001; Fig. 3b: STS1h/HYPER). A hypertonic stimulation after 2-h pretreatment with STS, however, was not effective at suppressing AVD (Fig. 3b: STS2h/HYPER). These results suggest that hypertonic stimulation of HeLa cells is somehow interfering with the process of AVD when applied in the time range of 1 h before to 1 h after the application of STS.
Fig. 3Interference of AVD with RVI. (a) Osmolality was increased from 300 to 500 mosmol/kg-H2O at time zero (arrow), with 4 μM STS applied (at arrowheads) either 2 h before (STS2h/HYPER: ■), 1 h before (STS1h/HYPER: ●), at the same time as (STS-HYPER:▼) or 1 h after (HYPER/1hSTS: ▲) the start of hypertonic stress. (b) Total AVD computed from the overall volume changes shown in (a) minus the initial amount of passive cell shrinkage in the first 5 min of hypertonic stress (i.e. volume at 0 min minus volume at 5 min). STS-ISO refers to the AVD observed under isotonic conditions. n = 10 for each experimental series
Next, the effects of STS on activation of the HICC were tested. This was done by increasing osmolality at various times relative to STS application. As shown in Fig. 4 (STS-HYPER), STS had no significant short-term effect on activation of the HICC when given together with hypertonic stress, i.e. the peak activation of HICC current was virtually unaffected for 5–7 min by STS applied simultaneously with hypertonic stress. This finding fits with the observation that HICC activation appears to occur on a considerably faster time scale than the initiation of AVD does. In this respect, it is also of note that even with STS applied 1 h ahead of hypertonicity (STS1h/HYPER), the HICC was still activated to a level approximately half of the control value, i.e. to 1.59 ± 0.39 pA/pF. With STS given 2 h before hypertonic stress (STS2h/HYPER), HICC currents were almost completely inhibited (0.62 ± 0.20 pA/pF, n = 5–33).
Fig. 4Effects of STS on HICC activation. (a) HICC currents in the absence (Control) or presence of STS, which was applied simultaneously with hypertonic stress (STS-HYPER), 1 h ahead of hypertonic stress (STS1h/HYPER) or 2 h ahead of hypertonic stress (STS2h/HYPER). Representative traces of 5 to 33 experiments. (b) Typical I/V-relationships under these conditions. (c) Summary of HICC current densities obtained under the four different conditions shown in (a)
Whenever studied in a quantitative way, HICCs have been proven to be the main mechanism of the RVI of a given system [3–5]. This also holds for HeLa cells, in which the relative contribution of HICC activity and Na+/H+ antiport to RVI is approximately 2:1 (with no detectable contribution of Na+-K+-2Cl− symport at all) [5, 13]. This raises the question of whether the apparent interference of hypertonic stress with AVD may occur via activation of the HICC. In the experiments shown in Fig. 5a, osmolality was increased from 300 to 500 mosmol/kg-H2O, at time zero (arrow). Following 60 min of hypertonic stress, 300 μM flufenamic acid (FFA, an established blocker of the HICC in HeLa cells [4, 5, 13]), STS (4 μM) or FFA plus STS were then applied (arrowhead). As expected, FFA significantly blunted the progress of RVI, presumably by inhibition of the HICC. The volume change from 60 to 120 min (volume at 120 min minus volume at 60 min) was −2.0 ± 0.6% in the presence of FFA; in the control experiments, on the other hand, the 60-to-120 min volume change due to RVI equalled 10.2 ± 0.9% (n = 10, P < 0.001; Fig. 5b: Control). Of note, STS led to a pronounced decrease in cell volume of −12.5 ± 1.6% (Fig. 5b: STS), and the effects of FFA plus STS were additive, resulting in a decrease in cell volume of −19.0 ± 1.0% (P < 0.01; Fig. 5b: STS+FFA). Experiments with 1 mM 2-aminoethoxydiphenyl borate (2-APB) as an alternative blocker of the HICC [17] yielded virtually identical results. With 2-APB, the 60-to-120 min volume change equalled 2.9 ± 1.1% (Fig. 5b: 2-APB), and again, the effects of HICC blockage and STS (volume change of −16.3 ± 0.6%; P < 0.05) were additive (Fig. 5b: STS+2-APB).
Fig. 5Effects of HICC channel blockers and STS on RVI and AVD. (a) Osmolality was increased from 300 to 500 mosmol/kg-H2O at time zero (arrow) for all groups. 300 μM flufenamic acid (FFA), STS or FFA plus STS were applied at t = 60 min of hypertonic stress (arrowhead) as indicated. (b) Summary of the volume changes from 60 to 120 min (volume at 120 min minus volume at 60 min) observed in the experiments shown in (a) and two additional experiments in which 1 mM 2-aminoethoxydiphenyl borate (2-APB) was used as a blocker of HICC channels, alone or in combination with STS. n = 10 for each experimental condition. *** Significantly different from Control with P < 0.001; #,## significantly different from STS with P < 0.05 and P < 0.01, respectively
It may be argued that, due to the limited specificity of FFA and 2-APB, the effects of both compounds described above may be due to an inhibition of volume-sensitive outwardly rectifying (VSOR) Cl− channels (see [2, 18] for review), rather than blockage of the HICC. In an additional series of measurements, however, it was found that even with application of 10 μM DCPIB (4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid), an effective blocker of the VSOR Cl− channel [19], an RVI value of 10.6 ± 0.6% (when added at t = 60 min; n = 10) was obtained; this is virtually identical to the value of 10.2 ± 0.9% observed under control conditions (see above). Furthermore, 10 μM DCPIB inhibited VSOR currents in HeLa cells by 67.6 ± 3.6% (n = 6) whereas it did not exhibit an effect on HICC currents at all (n = 8; Wehner and Numata, unpublished results).
These data strongly imply that, even in early-stage apoptosis, HICC activation is an effective mechanism of RVI in HeLa cells. Furthermore, they provide additional strong evidence for the functional interplay of AVD and RVI. In this regard, the apparent lack of RVI in HeLa cells undergoing AVD (Fig. 2a) could be accounted for as follows: In early-stage apoptosis (say, within 1 h after STS application) the AVD process surpasses the RVI process, which involves HICC activity, whereas in the later stage of apoptosis, the RVI process is suppressed because of inhibition of the HICC by STS.
Finally, we characterized the interplay between HICC activation and STS-induced cell death and apoptosis. As determined by an MTT assay (Fig. 6a), a caspase-3/7 activity assay (Fig. 6b), and an apoptosis assay measuring nucleosome-bound DNA (Fig. 6c), increasing osmolality from 300 mosmol/kg-H2O to 400, 500, and 600 mosmol/kg-H2O led to significant and parallel reductions of both cell death and apoptosis observed 6 h after STS application; the maximal effect was observed at 500 mosmol/kg-H2O and above. This is a surprising finding because, based on what has been reported so far concerning the interplay of cell volume and apoptosis, one might expect a synergistic effect of cell shrinkage on the induction of programmed cell death [15]. Furthermore, FFA and 2-APB almost completely blocked these effects, the fact providing strong further evidence that it is the activation of the HICC that is actually rescuing HeLa cells from apoptosis.
Fig. 6Rescue from STS-induced apoptosis by hypertonic stimulation and inhibition of rescue by HICC blockers. (a) Cell death observed 6 h after STS treatment at various osmolalities, and at 500 mosmol/kg-H2O with 300 μM FFA or 1 mM 2-APB as indicated (n = 12–34). (b) Apoptosis monitored as caspase-3/7 activity induced by 6-h exposure to STS at various osmolalities, and when FFA or 2-APB was also applied, at 500 mosmol/kg-H2O (n = 12–26). (c) DNA fragmentation analyzed with a nucleosomal ELISA assay after a 6-h exposure to STS at various osmolalities, and when FFA or 2-APB was also applied, at 500 mosmol/kg-H2O (n = 6)
To insure that the above measurements were not corrupted by the necrosis of cells, propidium iodide staining was performed. Propidium iodide is known to selectively bind to cell nuclei, and because it does not permeate intact cell membranes, it provides a reliable measure for the necrosis and late apoptosis of cells. Following a 6-h incubation, the percentages of necrotic cells were 0.1 ± 0.1% (for 300 mosmol/kg-H2O), 1.8 ± 0.3% (for 300 mosmol/kg-H2O—STS), 0.8 ± 0.1% (for 400 mosmol/kg-H2O—STS), 0.6 ± 0.1% (for 500 mosmol/kg-H2O—STS), and 0.5 ± 0.1% (for 600 mosmol/kg-H2O—STS; n = 8–10, for each condition). It is concluded from these experiments that STS, at 4 μM, is an effective and reliable tool for the induction of apoptosis in HeLa cells, as reported also in an earlier study from this laboratory [6].
It may be argued that hypertonic stress (and HICC activation) just delays, rather than prevents, STS-induced cell death in HeLa cells. As propidium iodide staining after 24 h of STS exposure revealed, however, the necrosis or late apoptosis of cells was 94.7 ± 0.7% for 300 mosmol/kg-H2O, and 76.1 ± 2.8% for 500 mosmol/kg-H2O. Clearly, even under these harsh experimental conditions (i.e. stimulation with STS for 1 day), hypertonicity rescued HeLa cells from death to a significant degree (P < 0.001; n = 8).
In the last series of measurements, the timing of apoptosis induction by STS versus hypertonic stress was analysed. As depicted in Fig. 7, increasing osmolality from 300 to 500 mosmol/kg-H2O rescued HeLa cells from apoptosis induced by 6-h application of STS if this maneuver was performed 2 h before (HYPER2h/STS), 1 h before (HYPER1h/STS) or at the same time as the application of STS (STS-HYPER). Moreover, even with STS given 1 h ahead of hypertonic stress, apoptotic cell death was reduced to some 50% of the isotonic control value (Fig. 7). Of note, the overall profiles of the effects of the timing of STS application versus hypertonic stress on cell death and apoptosis, as shown in the figure, are undistinguishable from those observed on AVD (see Fig. 3) as well as on HICC activation (Fig. 4).
Fig. 7Interference of HICC activation with STS-induced apoptosis. For the control, STS was applied for 6 h under isotonic conditions (STS-ISO). For the other groups, it was applied 2 h before (STS2h/HYPER), 1 h before (STS 1h/HYPER), at the same time as (STS-HYPER), 1 h after (HYPER1h/STS) or 2 h after (HYPER2h/STS) osmolality was increased from 300 to 500 mosmol/kg-H2O. Apoptosis was monitored as a decrease of cell viability (a, n = 12 to 42), caspase-3/7 activity expressed as relative fluorescence units (FU; b, n = 6 and 12), and DNA fragmentation measured in an enzyme-linked (ELISA) assay as absorbance values (OD, optical density; c, n = 6)
It has long been known that AVD occurs in parallel with a loss of cellular K+ and a gain of cellular Na+ [20–22]. To some extent, these effects might reflect reduced activity of the Na+/K+-ATPase [23]. Nevertheless, if the activation of the HICC were triggered as a consequence of conductive K+ export and AVD, this would lead to very similar results. Also of interest in this respect is a recent study on human leukemia U937 cells reporting two distinct phases during STS-induced apoptosis: an initial phase in which cellular K+ and Cl− decreased in parallel, followed by a second phase in which there was a further decrease in cellular K+ and an enormous increase in cellular Na+ (accompanied by a rise in cellular Cl−) [24]. Of note, U937 cells were shown to express HICC currents [25]. Thus, the ionic scenario described could also reflect the interplay of HICC activation and AVD, as proposed in the present study.
In rat hepatocytes, 405 mosmol/kg-H2O was insufficient to induce apoptosis but it sensitized cells to CD95-triggered cell death, whereas 505 mosmol/kg-H2O functioned as an effective apoptosis inducer per se [26–29]. Since rat hepatocytes express (amiloride-sensitive) HICC currents [4, 30–32], it would be interesting to examine the degree to which blockage of the HICC might interfere with the induction of cell death in this system.
Conclusions
Taken together, our results indicate that hypertonic stress caused a significant reduction in the staurosporine (STS)-induced apoptosis of HeLa cells. When analysing the functional interplay between apoptotic volume decrease (AVD) and regulatory volume increase (RVI) through the use of different activation protocols and effective blockers, the activation of hypertonicity-induced cation channels (HICCs) could be identified as the molecular mechanism by which HeLa cells are actually rescued from STS-induced apoptosis in hypertonic conditions. The precise mechanism by which HICC activation inhibits AVD and apoptosis remains to be elucidated.
Nevertheless, the results reported herein demonstrate a novel role of the HICC in the induction of programmed cell death. They are also in perfect agreement with a recent study in which inhibition of the HICC was found to sensitize HeLa cells to shrinkage-induced apoptosis [33]. We have only begun to understand the role of the HICC in these processes [4, 5], and the general relevance of the channel in cell proliferation and apoptosis remains a question of considerable interest.

cation channel
apoptosis
cell shrinkage
hela cells
volume regulation


Femoroacetabular impingement in 45 professional athletes: associated pathologies and return to sport following arthroscopic decompression

Femoroacetabular impingement (FAI) occurs when an osseous abnormality of the proximal femur (cam) or acetabulum (pincer) triggers damage to the acetabular labrum and articular cartilage in the hip. Although the precise etiology of FAI is not well understood, both types of FAI are common in athletes presenting with hip pain, loss of range-of-motion, and disability in athletics. An open surgical approach to decompressing FAI has shown good clinical outcomes; however, this highly invasive approach inherently may delay or preclude a high level athlete’s return to play. The purpose of this study was to define associated pathologies and determine if an arthroscopic approach to treating FAI can allow professional athletes to return to high-level sport. Hip arthroscopy for the treatment of FAI allows professional athletes to return to professional sport. Between October 2000 and September 2005, 45 professional athletes underwent hip arthroscopy for the decompression of FAI. Operative and return-to-play data were obtained from patient records. Average time to follow-up was 1.6 years (range: 6 months to 5.5 years). Forty two (93%) athletes returned to professional competition following arthroscopic decompression of FAI. Three athletes did not return to play; however, all had diffuse osteoarthritis at the time of arthroscopy. Thirty-five athletes (78%) remain active in professional sport at an average follow-up of 1.6 years. Arthroscopic treatment of FAI allows professional athletes to return to professional sport.
Introduction
In publications dating back several decades, irregularities in the morphology of the femoroacetabular articulation have been implicated as a possible source of hip pain in young, active athletes. A French article published in 1979 described hip pain associated with structural abnormalities of the proximal femoral neck in athletes participating in hockey, football, soccer, rugby, martial arts, and tennis [5]. More recently, developments pioneered by open hip surgeons have shown that morphologic abnormalities of both the femur and acetabulum underlie a large number of labral and chondral injuries in the hip [1, 29]. Wenger et al. [29] noted the presence of osseous abnormalities, including dysplasia and femoroacetabular impingement (FAI), in the majority of patients with labral tears. This suggests that isolated treatment of soft tissue pathologies may not be adequate without concomitantly addressing underlying structural abnormalities.
The concept of FAI has been defined by Ganz and colleagues [1, 8, 27]. In this condition, a structural or spatial abnormality of the femur (cam) or acetabulum (pincer) damages the chondrolabral structures during normal joint movement. The most common situation is a mixed cam and pincer pathology, occurring along the anterior femoral neck and the anterior–superior acetabular rim. In high flexion and internal rotation movements, abutment and impingement of the labrum and cartilage occurs. The precise etiology of FAI is not well understood. Sub-clinical slipped capital femoral epiphyses [15, 25], mal-union of femoral neck fractures [6], and decreased femoral anteversion [26] are described causes of cam impingement. Relative posterior opening of the acetabulum (acetabular retroversion) or global (coxa profunda) overcoverage of the femur by the acetabulum are described causes of pincer impingement [23, 24]. Cam and pincer lesions lead to distinct patterns of labral and chondral damage [1, 10] and long-standing impingement is likely a significant cause of previously described idiopathic hip joint degeneration [1].
Ganz et al. developed an open surgical dislocation approach to decompress FAI [7, 13]. This technique has demonstrated good results of FAI decompression in a general population [2]. However, we believe that an arthroscopic approach involves less post-operative morbidity and allows patients, including professional athletes, to return to high-functioning lifestyles [28]. The purpose of this study was to define associated pathologies and determine if professional athletes could return to high-level athletics following arthroscopic decompression of FAI.
Methods
Patient selection
A retrospective chart review was performed of all professional athletes presenting for arthroscopic treatment of FAI by the senior author between October 2000 and September 2005. All athletes presented with debilitating hip pain and an inability to participate in their sport. All were diagnosed with FAI based on physical and radiographic examination. Physical examination criteria included a positive impingement test [14] or flexion–abduction–external rotation (FABER) test. A positive impingement test was defined as groin pain with 90 degrees of hip flexion and maximal internal rotation. A positive FABER test was defined as asymmetry in the distance between the lateral knee and the exam table between the injured hip and the non-injured hip. Radiographic criteria included decreased anterior and superior femoral head–neck offset, acetabular retroversion (as defined by a positive cross-over sign), or coxa profunda (as defined by the medial acetabular border overlapping the ilioischial line).
For these athletes with documented physical and radiographic evidence of FAI, conservative treatment was limited to only 6 weeks. Based on evidence described by Beck and colleagues [1], our thought was that conservative treatment could not address the underlying bony abnormalities and any further treatment delay in these highly active patients would likely precipitate further irreversible damage to the articular cartilage. Inclusion criteria for this study included professional athletes with at least one positive physical exam finding, at least one positive radiographic finding, and failure of at least 6 weeks of conservative therapy. Forty-five professional athletes from various sports met the inclusion criteria and were included in the study (Table 1). There were 42 male and 3 female patients.
Table 1Pre-operative sports activities of the 45 professional athletesSportNo. of patientsHockey24Golf6Football5Soccer3Dance2Baseball2Martial arts1Tennis1Jockey1Total45
Arthroscopic procedure
The modified supine approach to hip arthroscopy was used [3]. Two portals (anterior, anterolateral) were established and a third portal (distal lateral accessory) was established as needed [12]. At the time of arthroscopy, the central compartment of the hip was first inspected.
Pincer lesions were typically identified in the superior acetabular quadrant. For the treatment of small pincer lesions (<2 to 3 mm), osteoplasty of the proximal femoral neck was occasionally sufficient to relieve the impingement [28]. Patients with moderate or large-sized lesions underwent acetabular rim trimming, using an arthroscopic osteotome or 5.5 mm round motorized burr [22].
Acetabular labral tears were arthroscopically identified, and based on previous research on the vascularity of the labrum [9, 11], detached and peripheral midsubstance tears were typically repaired [12, 22]. Care was taken to preserve as much healthy, viable labrum as possible, however, degenerative or frayed tissue was debrided to a stable remnant.
Labral repair was performed with suture anchors to repair detached labral tears or to re-fix the labrum following iatrogenic detachment for complete resection of pincer lesions. The previously described technique involved placement of the anchors high on the acetabular rim in the area of detachment [12]. Typically, one bioabsorbable anchor (BioRaptor, Smith+Nephew, Andover, MA, USA) was placed at the 12:00 acetabular position, and re-inforcement was placed either anteriorly or posteriorly of this area as needed. One limb of suture (Ultrabraid, Smith+Nephew, Andover, MA, USA) was passed between the labral tissue and the rim, and was retrieved through the substance of the labral tissue. In cases of inadequate availability of labral substance or in patients with highly degenerative, friable tissue, the suture was passed around the labral tissue. Standard arthroscopic knots fixed the repair to the rim. For midsubstance labral tears, a suture passer looped an 0-Vicryl around the torn tissue to approximate the edges of the midsubstance split.
The condition of the articular cartilage was evaluated according to the Outerbridge Classification System [18]. For this study, the Outerbridge Classification System was defined as follows: grade 0, normal cartilage; grade 1, cartilage with softening and swelling; grade 2, a partial thickness defect with fissures on the surface that do not reach the subchondral bone or exceed 1.5 cm in diameter; grade 3, fissuring to the level of the subchondral bone in an area with a diameter more than 1.5 cm; and, grade IV, exposed subchondral bone [18]. Chondral procedures performed in this group included chondroplasty and microfracture of lesions of the femoral head and acetabulum as needed.
Following inspection of the central compartment, traction was released and the peripheral compartment was evaluated for cam lesions. A dynamic examination of the hip in all motion planes allowed for direct visualization of impingement at the femoroacetabular interface. For the treatment of cam lesions, an osteoplasty was performed with a 5.5 mm round motorized burr, restoring the anatomic concavity of the femoral head–neck junction [28]. Burring was performed from superior to inferior along the anterior femoral head–neck junction, to a depth of approximately 5–7 mm and a width of approximately 8–12 mm. Care was taken to avoid resecting too distally along the femoral neck to avoid the lateral epiphyseal vessels. A final dynamic examination was performed to confirm adequate decompression, and a lack of entrapment of the labrum during joint motion.
In general, the above-described procedure took approximately 2–3 h, depending on the degree of labral and chondral pathology. Traction time was limited to less than 2 h to reduce the risk of neurovascular compromise.
Post-operative management
Post-operative medical prophylaxis included an aspirin or a low-molecular weight heparin, a non-steroidal anti-inflammatory, and an antibiotic. Each of the 45 athletes underwent post-operative rehabilitation according to the following generalized protocol that was devised based on personal experience. For basic procedures involving decompression of FAI and labral treatment, weightbearing was restricted to 20 pounds, flatfoot for 4 weeks. A brace was prescribed to be worn for 10 days to protect the hip and limit abduction and rotation. Continuous passive motion (CPM) was used to apply 0–90 degrees of hip flexion for up to 8 h per day for 4 weeks. Night boots were worn for ten post-operative days to limit rotation during sleep. If capsular modification procedures (thermal capsulorrhaphy or capsular plication) were performed, rotation precautions were extended for a total of 21 days to avoid capsular stress. If microfracture was performed, weightbearing restrictions and CPM use were extended for a total of 8 weeks.
Physical therapy exercises were implemented within four post-operative hours. It is our belief, based on clinical observation, that this, in addition to an early emphasis on passive hip motion (particularly internal rotation) reduces the incidence of adhesion formation. Active hip flexion was limited for 4 weeks, based on clinical observation, to minimize the risk of hip flexor tendonitis.
Athletes were typically allowed to return to full competitive activity between 12 and 16 post-operative weeks. Return-to-play statistics were determined by retrospective chart review and personal follow-up communication with the athletes.
Results
The average age of the athletes at the time of surgery was 31 years (range: 17–61). Eleven athletes (24%) previously underwent hip arthroscopy by multiple primary surgeons for the treatment of labral and chondral pathologies and experienced a recurrence of hip symptoms. The average time to follow-up was 1.6 years (range: 6 months to 5.5 years).
Twenty-two athletes (49%) were treated for an isolated cam lesion and three athletes (7%) were treated for an isolated pincer lesion. Twenty-one athletes (47%) had a mixed pathology of both cam and pincer lesions.
All of the athletes had acetabular labral tears. Twenty-five patients (56%) underwent either labral repair or re-fixation following rim trimming with suture anchors (average 1.3 anchors per patient, range: 1–3). Twelve patients (27%) had intra-substance labral repair. Five patients had labral debridement; one patient had a detached tear, one had frayed labral tissue, and three patients had complex tears consisting of detached, frayed, and flap components. Two patients were labral deficient from a previous arthroscopic debridement. They underwent labral grafting using an iliotibial band autograft.
Twenty-one patients (47%) had a grade IV acetabular chondral defect; 14 underwent arthroscopic microfracture, 5 underwent thermal chondroplasty, and 2 patients had no treatment due to the diffuseness of their disease. Three patients (7%) had a grade IV femoral head chondral defect; one was treated with microfracture, one with chondroplasty, and one patient had no treatment due to the diffuseness of his disease. Seventeen (38%) had a grade I–III acetabular chondral defect (13 treated with chondroplasty and 1 treated with microfracture) and 11 (24%) had a grade I–III femoral head chondral defect (all 11 were treated with chondroplasty). Four patients (one baseball player, one football player, one hockey player and 1 golfer) had extensive diffuse OA at the time of arthroscopy, but opted to delay arthroplasty.
Twenty-six patients (58%) had a partial tear of the ligamentum teres and 3 patients (7%) had a complete ligamentum teres avulsion. Thirteen (29%) patients underwent thermal capsulorrhaphy and 9 (20%) underwent plication for capsular redundancy. Seventeen patients (38%) had loose bodies in the joint and two patients required excision of myositis ossificans of the rectus femoris. Three patients (7%) underwent cheilectomy of a stenotic cotyloid fossa and 9 patients (20%) underwent arthroscopic release of a tight iliopsoas.
Figure 1 illustrates restoration of femoral head–neck concavity in an athlete treated for cam impingement (Fig. 1) and Fig. 2 illustrates successful removal of the anterior acetabular overhang in an athlete treated for pincer impingement (Fig. 2).
Fig. 1A 27-year-old professional hockey player was evaluated for right hip pain. a A pre-operative cross-table lateral radiograph demonstrated convexity at the anterior femoral head–neck junction. b The presence of a cam lesion was verified arthroscopically. The femoral head (FH) and acetabulum (Ac) are visualized in the peripheral hip compartment with the camera in the anterior portal. c A motorized burr was used to restore anterior concavity at the head–neck junction. This was visualized at the superior acetabular (Ac) position (12:00) with the camera in the anterior portal. d A post-operative film verified successful decompression of the cam impingement and restoration of the femoral head–neck junction concavityFig. 2A 22-year-old professional football player was evaluated for right hip pain. a A pre-operative AP radiograph demonstrated a cross-over sign of the right acetabulum, indicating acetabular retroversion. b The presence of a pincer lesion was verified arthroscopically with the camera in the anterolateral portal with excessive bone along the anterior–superior acetabulum (Ac). c A motorized burr in the anterior portal resected the lesion with the burr shield placed against the labrum (L) for protection. d The labrum was fixed back to the rim using two suture anchors. e A post-operative film verified the successful removal of pincer impingement and lack of retroversion in the superior region of the acetabulum
Forty-two athletes (93%) returned to professional sport following hip arthroscopy. Three players (1 football player, 1 hockey player, and 1 baseball player) did not return to play following arthroscopy. Each of these patients had diffuse osteoarthritis at the time of arthroscopy.
Five athletes (11%) required re-operation. Three underwent lysis of adhesions and two had symptomatic treatment of extensive osteoarthritis. All of the patients who underwent revision surgery for lysis of adhesions returned to professional play and the two with extensive osteoarthritis did not return to play.
Thirty-five of the 45 athletes (78%) remained active at the professional level at an average of 1.6 years after hip arthroscopy.
Discussion
Intra-articular hip injuries are a recognized cause of pain, mechanical symptoms, and disability in athletes. Traumatic intra-articular injury results from acute injury, including hyperabduction injuries, direct hip contact, and joint dislocation or subluxation. Atraumatic hip injury, however, is less well understood. Various motions exerted during sport, particularly flexion combined with internal rotation (hockey-goalie stance), have been suggested as potential causes of overuse hip injury [19, 21]. In this position, a cam lesion extending anteriorly or superiorly from the proximal femoral neck would impinge at the anterosuperior acetabular rim. Combining this understanding with recent reports by Wenger et al. [29] and Ganz et al. [1, 2, 8, 13], morphological abnormalities of the femoroacetabular joint have been closely examined as possible underlying sources of atraumatic hip injuries in athletes. Ganz and colleagues described FAI resulting from structural abnormalities of the proximal femoral neck (cam), acetabular rim (pincer), or most commonly, a combination of the two pathologies (mixed). Damage to the articular cartilage and acetabular labrum results from this pathologic bony contact and FAI is a likely trigger of early hip joint degeneration. In our experience, FAI is a common problem that has only recently been recognized in athletes with a primary hip complaint.
An open surgical dislocation approach was first described to treat hip impingement [7]. This technique showed good midterm results in a general population [2]. However, increasing recognition of FAI in a high-demand, competitive athletic population encouraged the development of a less invasive arthroscopic approach to facilitate prompt return to competitive play.
Various studies have demonstrated that hip arthroscopy is a safe and effective intervention in athletes with intra-articular injuries [4, 17, 19, 20]. Philippon reported on a series of ten elite athletes who underwent hip arthroscopy for labral debridement with thermal capsulorrhaphy [20]. All of the athletes returned to high-level athletic activities. McCarthy et al. [17] reported 80% excellent results following hip arthroscopy in elite athletes (average follow-up of 23.6 months). Byrd et al. [4] reported a series of 44 hip arthroscopies in 42 recreational, high school, collegiate, elite, and professional athletes. Post-operative improvement, as quantified by the modified Harris Hip Score, was present in all classes of athletes and in athletes undergoing any arthroscopic procedure (removal of loose bodies, debridement of ligamentum teres, excision of osteophyte, labral excision, microfracture, chondroplasty). Byrd et al. reported better results in athletes who recalled a traumatic onset to their hip symptoms, when compared to those with an acute or insidious onset. The authors suggested that an unaddressed pre-disposition to injury might have had a negative impact on self-reported outcomes in athletes [4].
FAI may be the unaddressed pre-disposition to which Byrd and colleagues were alluding. While these above articles demonstrated successful outcomes following debridement procedures in athletes, there are a few caveats that must be addressed. First, it is likely that simple excision of damaged or diseased tissue, like with meniscal tears or loose bodies, can provide short-term abatement of pain and mechanical symptoms, and allow an athlete to return to professional athletics. However, recent studies have shown that simple hip debridement procedures failed to address the primary pathology in a vast majority of cases [1, 29]. A recent unpublished study performed by the senior author showed that a major cause of revision hip arthroscopy in all patients was treatment of labral and chondral re-injury and decompression of previously unaddressed FAI. Future studies examining the clinical and radiographic long-term outcomes of athletes treated with debridement procedures need to be compared to those athletes treated for FAI at an early stage of this disease. The purpose of this study was to simply state that despite the fact that FAI surgery is a more extensive procedure than debridement, professional athletes can still return to play following this procedure.
An arthroscopic approach to treat FAI was recently reported [28]; however, early outcome studies are lacking. The results of this study show that professional athletes with FAI can return to high-level competitive sport following this procedure. Ninety-three percent of athletes returned to their previous level of sport. Three athletes did not return to sport (1 hockey player, 1 football player, and 1 baseball player), however all had diffuse hip osteoarthritis present at the time of arthroscopy. One patient (a senior PGA golfer) was able to return to sport following arthroscopy, despite his diffuse osteoarthritis. Based on this information, it seems that arthroscopic treatment of FAI in the presence of OA can allow patients to return to low-impact, however, not likely high-impact professional sports. A larger cohort of patients is needed to test this hypothesis.
Avascular necrosis (AVN) and femoral neck fracture are recognized risks of osteoplasty. A recent study showed that up to 30% of the femoral neck may be resected before it is structurally compromised [16]. In this series of patients, no post-operative cases of AVN or femoral neck fracture were diagnosed. Additional potential complications include adhesion formation at either the capsulolabral margin or between the capsule and the site of bony resection. In this series of 45 athletes, 3 patients (7%) required revision arthroscopy for the lysis of adhesions (LOA). All athletes returned to competitive sport following revision arthroscopy for LOA. Two additional athletes had extensive OA at the time of primary arthroscopy and subsequently underwent revision arthroscopy for symptomatic treatment of OA, including osteophyte debridement. Both did not return to professional athletics due to the extent of their disease. There were no reported cases of heterotopic ossification or neurovascular compromise, including lateral femoral cutaneous, sciatic, and pudendal neuropraxia, reported, although this statistic may have been underestimated due to the nature of this professional athlete cohort.
A limitation of this study was the inherent selection bias involved with the study of professional athletes. This patient sub-group was financially motivated to return to play and may have been less likely to report post-operative symptoms and complications than the rest of patients treated for FAI. Despite this, we believed that a return-to-play analysis was critical to assess the outcomes of FAI treatment in a high-demand population. This cohort of professional athletes was debilitated prior to hip arthroscopy, and following intervention, was able to perform in physically intense professional sport activities. Also, the athletes’ precise reason for retirement and whether it was related to their hip injuries was difficult to discern. For this reason, given the relatively short span of all professional athletes’ careers, we chose to include all athletes at a minimum of 6 months post-operative. This time period was selected as an ample time period for completion of post-operative rehabilitation and return to sport. Another limitation of this study was the lack of follow-up subjective and objective data. Current outcomes instruments (modified Harris Hip score and the Non-Arthritic hip score) have not been validated for use in high-level athletes. It is our belief that these scoring systems fail to address the activities that are most limiting to athletes, and hence, underestimate the degree of debilitation in professional athletes. Evaluating the applicability of current scoring systems in athletes, and developing new outcomes instruments for athletes will be the focus of future studies. Other limitations of this study included unequal distribution of gender (42 males and 3 females) and professional sports. Because different sports place different demands on the hip, we cannot draw conclusions among the various sports.
The arthroscopic treatment of FAI represents the evolution of hip arthroscopy. In the past, soft tissue pathologies were treated in isolation, without addressing underlying impinging osseous abnormalities. Recent developments, particularly by Ganz and colleagues, have enlightened us to the pathology of FAI and the associated treatment options. In order to treat high-demand patients with FAI, an arthroscopic technique was developed. This study has demonstrated that full return to professional competitive sport is possible following arthroscopic treatment of FAI. Additional studies are needed to determine the effect on long-term joint degeneration of early surgical intervention to treat FAI.

femoroacetabular impingement
cam
pincer
hip arthroscopy
osteoplasty


A novel family of diversified immunoregulatory receptors in teleosts is homologous to both mammalian Fc receptors and molecules encoded within the leukocyte receptor complex

Three novel and closely related leukocyte immune-type receptors (IpLITR) have been identified in channel catfish (Ictalurus punctatus). These receptors belong to a large polymorphic and polygenic subset of the Ig superfamily with members located on at least three independently segregating loci. Like mammalian and avian innate immune regulatory receptors, IpLITRs have both putative inhibitory and stimulatory forms, with multiple types coexpressed in various lymphoid tissues and clonal leukocyte cell lines. IpLITRs have an unusual and novel relationship to mammalian and avian innate immune receptors: the membrane distal Ig domains of an individual IpLITR are related to fragment crystallizable receptors (FcRs) and FcR-like proteins, whereas the membrane proximal Ig domains are related to several leukocyte receptor complex encoded receptors. This unique composition of Ig domains within individual receptors supports the hypothesis that functionally and genomically distinct immune receptor families found in tetrapods may have evolved from such ancestral genes by duplication and recombination events. Furthermore, the discovery of a large heterogeneous family of immunoregulatory receptors in teleosts, reminiscent of amphibian, avian, and mammalian Ig-like receptors, suggests that complex innate immune receptor networks have been conserved during vertebrate evolution.
Introduction
Immune receptors that bind similar ligands often form clusters that are encoded on distinct chromosomes in the human genome. For example, receptors that bind to the Fc portion of Ig [fragment crystallizable receptors (FcRs)] are found on chromosome 1q21-23 and consist of the high-affinity IgG and IgE receptors (FcγRI and FcεRI) and the related low- or moderate-affinity Ig receptors (FcγRII, FcγRIII, and FcγRIV) (Hullet et al. 1991; Ravetch and Kinet 1991; Hullet and Hogarth 1994; Daeron 1997; Falk et al. 2005; Falk and Ravetch 2006). Alternatively, immune receptors involved in the recognition of major histocompatability class 1 (MHC I) antigens, such as killer cell Ig-like receptors (KIRs) and leukocyte Ig-like receptors (LILRs), are clustered together on chromosome 19q13.4 in a region known as the leukocyte receptor complex (LRC) (Kremer et al. 1992; Wagtmann et al. 1997; Wende et al. 1999; Martin et al. 2002). Recently, comparative studies have suggested that FcRs and LRC-encoded receptors, although genomically and functionally distinct, appeared to have evolved from a common ancestor. This contention is supported in part by recent attempts to find LRC regions in other species.
Mouse chromosome 7 is syntenic to the human LRC and encodes the paired Ig-like receptors (PIRs) that were originally discovered in an attempt to find a murine IgA receptor ortholog (CD89; Kubagawa et al. 1997; Hayami et al. 1997). Subsequently, searches for PIR relatives resulted in the identification of the chicken Ig-like receptors (CHIRs) in birds (Dennis et al. 2000), representing a multigene family encoded within an avian LRC-like region (Nikolaidis et al. 2005a; Viertlboeck et al. 2005). More importantly, CHIR Ig domain sequences are not only related to the mammalian PIRs and KIRs, but also to various FcR Ig domains (Dennis et al. 2000). This previously unrecognized relationship was supported by phylogenetic and comparative structural analysis and was the first evidence suggesting that certain genomically and functionally diverse mammalian and avian immune receptor families probably evolved from a common ancestor (Dennis et al. 2000; Nikolaidis et al. 2005b).
Compared to mammals and birds, bony fish are among the most phylogenetically distant vertebrates with characteristic innate and adaptive immune systems that have mammalian-like B and T cells (Yoder 2004). Furthermore, due to the establishment of clonal lymphoid cell lines, catfish are known to also possess natural killer (NK)-like cells capable of killing xenogeneic targets by an antibody-dependent cell-mediated cytotoxicity (ADCC) process (Shen et al. 2002–2004). This functional evidence suggested that catfish NK cells possess a putative FcμR (Shen et al. 2003). Additionally, catfish NK cells can kill allogeneic targets in an ADCC-independent manner, indicating that target recognition may also involve KIR and/or LILR-like receptors (Shen et al. 2002, 2004). However, to date, FcR and/or KIR/LILR homologs have not been described in ectothermic vertebrates, making it difficult to elucidate the evolutionary history of these important receptor families. Large families of putative activatory and inhibitory novel immune-type receptors (NITRs) also belonging to the Ig superfamily (IgSF) have been described in teleosts. Pufferfish (Spheroides nephelus), zebrafish (Danio rerio), catfish, and rainbow trout (Oncorhynchus mykiss) all express NITRs (Strong et al. 1999; Yoder et al. 2001, 2002; Hawke et al. 2001). Because some NITRs are also expressed by catfish NK cells (Hawke et al. 2001), it has been suggested that these receptors may represent the “functional orthologs” of the mammalian KIRs and/or LILRs (Yoder et al. 2001; Hawke et al. 2001). However, NITRs do not exhibit any sequence identity or phylogenetic relationship to such receptors (Yoder et al. 2001) and have only been identified in bony fishes.
Herein is described a novel set of IgSF receptors present in the channel catfish, and possibly other teleosts, that are composed of Ig domains related to functionally diverse immunoreceptor families present in mammals. Detailed sequence analysis supports that individual catfish leukocyte immune-type receptors (IpLITRs) contain a unique composition of Ig C2-like domains sharing homology with both FcRs and receptors encoded within the LRC. Thus, IpLITRs provide further evidence supporting the common evolutionary origins of genomically and functionally distinct immunoregulatory receptor families present in mammals.
Materials and methods
Experimental animals and cell lines
Catfish (1–2 kg) were obtained from a commercial source (ConAgra, Isola, MS, USA) and maintained in individual tanks as described previously (van Ginkel et al. 1992). Catfish peripheral blood leukocytes (PBL) were isolated from heparinized blood by centrifugation on a cushion of Ficoll-Hypaque (Lymphoprep, Accurate Chemical, Westbury, NY, USA) as described previously (Miller et al. 1994a). Catfish cells lines were grown at 27°C in AL-3 medium consisting of equal parts AIM-V and L-15 (Life Technologies, Gaithersburg, MD, USA) adjusted to catfish tonicity with 10% (v/v) deionized water and supplemented with 1 μg/ml NaHCO3, 50 U/ml penicillin, 50 μg/ml streptomycin, 20 μg/ml gentamicin, 50 uM 2-ME, and 3% heat-inactivated, pooled, normal catfish serum (Miller et al. 1994a,b). 3B11 is a cloned autonomous B cell line generated from an outbred catfish by mitogen stimulation (Wilson et al. 1997). G14D is an automonous T cell line derived from a gynogenetic catfish (Hogan et al. 1999), whereas 42TA is a macrophage cell line derived from a different outbred fish (Miller et al. 1994b). TS32.15 and TS32.17 are clonal nonautonomous antigen-dependent cytotoxic T cell lines developed from an alloantigen immunized outbred fish (Stuge et al. 2000). 1F3 is a clonal nonautonmous NK-cell line developed from an alloantigen-stimulated culture of PBL from a naive outbred fish (Shen et al. 2004). The nonautonomous cytotoxic T cell and NK cell lines require weekly restimulation with irradiated allogeneic cells for continuous proliferation. The channel catfish ovary fibroblast cell line (CCO; Browser and Plumb 1980) was provided by Dr. V.G. Chinchar (University of Mississippi Medical Center). CCO cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FCS.
Channel catfish LITRs and sequence analysis
In a search for IgSF receptors expressed by alloantigen stimulated catfish PBL and various catfish clonal cell lines, several expressed sequence tag (EST) libraries were analyzed. Subsequently, IpLITR1 and IpLITR3 cDNAs were identified by single pass sequencing of a cDNA library generated from a catfish 7-day-old mixed lymphocyte culture (MLC) enriched in NK-like cells, MLC52-1 (Shen et al. 2004; Stuge et al. 1997). IpLITR2 was obtained from the catfish 42TA macrophage cell line (Miller et al. 1994b) cDNA library. The cDNAs were sequenced on both strands using universal forward and reverse primers and gene-specific primers. Nucleotide and amino acid sequences were analyzed using DNASTAR software (Madison, WI, USA) and aligned using CLUSTALW (Thompson et al. 1997). Neighbor-joining (NJ) trees with pairwise gap deletions were drawn using MEGA v3.0 (Kumar et al. 2001). Maximum parsimony and maximum likelihood trees were drawn with essentially the same results (data not shown). Similarity searches were performed with the extracellular Ig domains of IpLITR1, IpLITR2, and IpLITR3 using position-specific iterative (PSI) and Basic Local Alignment Search Tool (BLAST) analysis (Altschul et al. 1990) against the National Center for Biotechnology Information (NCBI) nonredundant database. SOSUI (signal) beta version, Simple Modular Architecture Research Tool (SMART) (Letunic et al. 2004), Pfam databases (Bateman et al. 2004) and the three-dimensional position-specific scoring matrix server (Kelly et al. 2000) were used for predictions of signal peptides, Ig domains, transmembrane (TM) segments, and secondary structure. Sequence decorations were performed using GeneDoc (http://psc.edu/biomed/genedoc).
Southern and Northern blot analyses
Genomic DNA was prepared from erythrocytes of 16 catfish siblings. These siblings were representatives of a 78-member family obtained from the Catfish Genetics Research Unit, United States Department of Agriculture, Agricultural Research Service (Stoneville, MS, USA; designated family 1). The sibling catfish 1–16 used were A10, A5, A32, A22, A38, A36, A16, A29, A18, A49, A159, A15, A23, A25, A186, and A37, respectively (Quiniou et al. 2005). The DNA (10 μg) was digested to completion with Pst1 or EcoRI, separated on 1% agarose gels and transferred by capillary action onto Hybond-N+ membranes (Amersham Pharmacia Biotech, Arlington Heights, IL, USA) using standard techniques. Hybridizations were performed in Rapid-hyb buffer (Amersham Pharmacia Biotech) at 65°C and membranes were washed at high stringency (65°C with 0.1× saline-sodium citrate, 0.1% sodium dodecyl sulfate). Specific probes (Table 1) were amplified by PCR using IDPol DNA polymerase (ID Labs Biotechnology, London, Ontario, Canada) according to the manufacturer’s recommended protocol. Typical parameters were: 1 min 94°C, followed by 29 cycles of 94°C 30 s, 62°C 30 s, 72°C 1 min, then extension at 72°C for 10 min. Probes were random primed labeled with [32P] 2′-deoxycytidine 5′-triphosphate using a Megaprime labeling kit (Amersham Pharmacia Biotech).
Table 1IpLITR PCR primers used for generating Ig domain-specific probes and gene-specific primers used in RT-PCR experimentsPrimer  IpLITR1 D1Forward5′ GTCGGACAAGGTCAAGTTCTG 3′Reverse5′ GGCTTTTGGTCTCTCTATCAC 3′IpLITR1 D4Forward5′ AATCCTGATGAACAGGTGTACC 3′Reverse5′ GTGTTTACAGTGGTAGAAACC 3′IpLITR2 D3Forward5′ AGTCGTGAAGGAGCTGTACTGA 3′Reverse5′ TTCACTGCCAGAATGATGGTCAC 3′IpLITR3 D6Forward5′ CTGAGGGACATCCTCTGACCTT 3′Reverse5′ GTGTTTACAGTGGTAGAAACC 3′IpLITR1 GSPForward5′ GTCGGACAAGGTCAAGTTCTG 3′Reverse5′ CTGCAGACATGATGAACTTCT 3′IpLITR2 GSPForward5′ GTCGGACAAGCTCAAGAGTTT 3′Reverse5′ TGTGTAGTAGAGTGGGTTTCC 3′IpLITR3 GSPForward5′ GTCGGACAAGGTCAAGTTCTG 3′Reverse5′ GCTTTTGGGTGACTCTCCTCT 3′IpEF1α GSPForward5′ GACTGCCACACTGCTCACATTG 3′Reverse5′ TTAGTTACTCAGCAGCTTTCTTCCGSP gene-specific primers
Total RNA from catfish PBL and various tissues was prepared using RNA-Bee (Tel-test, Friendswood, TX, USA); 10 μg of each sample were electrophoresed on 1.5% formaldehyde-agarose gels and transferred by capillary action onto Hybond-N+ (Amersham Pharmacia Biotech), hybridized, and washed as above.
Reverse transcription (RT)-PCR
Total RNA from PBL, MLC, various tissues, and catfish clonal cell lines were prepared as described above, and treated with DNase I (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s recommended protocol. One μg RNA was reverse transcribed using an oligo-T primer and 200 units of Superscript III reverse transcription (RT) (Invitrogen Life Technologies). RT-PCR was performed using specific primers for IpLITR and the housekeeping gene elongation factor 1 alpha (IpEF1α; Table 1). Typical parameters were: 3 min 94°C, followed by 30 cycles of 94°C 45 s, 58°C 45 s, 72°C 2 min, then extension at 72°C for 10 min. PCR products were visualized on 1.2% Tris-acetate-EDTA agarose gels. PCR products were also cloned into pCR4-TOPO® (Invitrogen Life Technologies) and verified by sequencing. The Genbank accession number for IpEF1α is CB938718.
Results
Catfish LITR sequences
In a search for IgSF receptors expressed by alloantigen-stimulated catfish PBL and various catfish clonal cell lines, several EST libraries were analyzed. Three IpLITR sequences were subsequently identified that encoded type I TM proteins with extracellular C2-like Ig domains, which were predicted using SMART (see Electronic Supplementary Material). Even though these sequences vary in length, they are highly similar (Fig. 1a,b). All three receptors have identical signal peptides and IpLITR1 and IpLITR3 have almost identically encoded Ig domains D1, D2, D3, and D4 (Fig. 1a). Comparatively, IpLITR2 D1 and D2 are 77.2 and 86.8% identical at the amino acid level to their IpLITR1 and IpLITR3 counterparts (Fig. 1c). IpLITR1 encodes a 346 amino acid extracellular region consisting of four Ig domains, a 23-amino acid TM segment and a 116-amino acid cytoplasmic tail (CYT). The IpLITR1 CYT contains two immunotyrosine-based inhibition motifs (ITIMs) centered at Y439 and Y461 and an ITIM-like motif (SEYTTE) centered at Y479 (Fig. 1b) (Daeron and Vivier 1999; Ravetch and Lanier 2000; Billadeau and Leibson 2002). An overlapping immunotyrosine-based switch motif (ITSM; TVYSQL) centered at Y465 is also present in the CYT of IpLITR1 (Shlapatska et al. 2001). In contrast, the smaller IpLITR2 and the longer IpLITR3 transcripts encode for molecules with identical 25-amino acid TMs containing a lysine residue and very similar charged CYTs (Fig. 1b). Overall, the individual IpLITRs are composed of a membrane distal to membrane proximal ordering of Ig domains, each containing similar D1s and D2s. However, the membrane proximal domains vary, i.e., IpLITR3 D5 and D6 are only 15.7–24.7% and 15.2–25% identical, respectively, to all other IpLITR Ig domains, and IpLITR2 D3 is only 17.9–39.3% identical to other IpLITR domains (Fig. 1c).
Fig. 1Predicted amino acid sequence, domain comparisons, and schematic representation of IpLITR1, IpLITR2, and IpLITR3. a Alignment of the extracellular and b TM/CYT regions of IpLITR1, IpLITR2, and IpLITR3. Signal peptide (SP), and immunoglobulin (Ig) domains are labeled; cysteine residues predicted to be involved in intrachain disulfide bonds are marked with asterisks; gray shaded residues represent differences from IpLITR1 in a and differences from IpLITR2 in b. TMs are underlined, ITIM-like motifs are boxed, an overlapping ITSM within the CYT of IpLITR1 is indicated by a bracket, and TM charged residues are shaded black and marked (+). c Phylogenetic analysis of Ig domains in IpLITRs. NJ trees with pairwise gap deletions were drawn using MEGA v3.0 (Kumar et al. 2001) with 10,000 bootstrap replications, and bootstrap values >50% are shown. Branch lengths were measured in terms of amino acid substitutions and a scale bar are shown below the trees. The predicted SP, Ig domains, TM, and CYT are indicated. ITIM-like motifs are shown as boxes, N-linked glycosylation sites are marked as ballpoint lines. Individual IpLITR domains are shaded according to their relatedness between IpLITRs and percent amino acid identity with IpLITR1 Ig domains indicated to the left of IpLITR2 and IpLITR3. IpLITR2 D3 and IpLITR3 D5 and D6 are 15.2–39.3% identical to all IpLITR1 Ig domains
IpLITRs belong to polymorphic and polygenic gene families
Sibling restriction fragment length polymorphism (RFLP) patterns using domain-specific probes for the various IpLITRs show a large number of hybridizing bands with each probe used, as well as differences in the banding patterns among siblings (Fig. 2). These findings indicate that the IpLITR gene complex is both polygenic and polymorphic. However, it is presently unknown whether these polymorphic differences are due to variability within the exons encoding these receptors and/or within the introns. Some of the high-intensity bands found in each of the Southern blots may reflect multiple IpLITR copies, further increasing the possible number of genes. Importantly, the Southern blot segregation patterns show that there is more than one IpLITR locus. For example, among the 16 sibling fish used there are at least nine, perhaps more, different RFLP patterns for IpLITR D1 (Fig. 2b). If these genes were closely linked then only four RFLP patterns (barring cross-over events) would be observed, as shown for the MHC class IIβ RFLP patterns (Fig. 2a). When all Southern blots are compared, at least three distinct linkage groups (marked by ●, ▲, and ■ ) are observed, indicating the presence of multiple independently segregating loci, and none of these are linked to the MHC II RFLP (Fig. 2b–d). Similar results were also obtained using the IpLITR2 D3 probe (data not shown). Mining of the zebrafish genome for IpLITR relatives [whole genome shotgun database of the NCBI (Sanger Genomic Institute, Cambridge, UK)] further supports that these receptors are present on different chromosomes because five contigs were identified with IpLITR-like homologs on zebrafish chromosomes 3, 7, and 8 (Table 2).
Fig. 2Southern blot analyses. Genomic DNA from 16 sibling catfish was digested, separated, transferred to nylon membranes, and hybridized with a MHCIIβ, b IpLITR1 D1, c IpLITR1 D4, and d IpLITR3 D6-specific probes. Letters a–i indicate different segregation patterns (b′, e′, and h′ represent variant segregation patterns that may be the result of recombination). Representative RFLP bands illustrating different linkage groups are indicated by ●, ▲, and ■. Kilobase size markers are indicated to the left of each blotTable 2Identification of IpLITR-like genes in the zebrafish genomeAccession numbersChromosomeBX0051568BX0005248BX5111763BX6496273CAAK01000490.17Search (04/05) of the zebrafish whole genome shotgun database of the NCBI (Sanger Genomic Institute)
IpLITR expression
Northern blot analyses using a cross hybridizing IpLITR1 D1 probe revealed that LITR message is highly expressed in the hematopoietic pronephros and mesonephros, with predominant transcript sizes of ∼6.6, ∼4.0, and ∼3.8 Kb (Fig. 3a). Low levels of IpLITR D1 expression were detected in the spleen, with little to no message detected in the heart, liver, gill, or muscle. The use of the more specific IpLITR2 D3 probe revealed message expression not only in the pronephros and mesonephros, but also in the spleen, gill, and perhaps heart with transcripts of ∼6.0, ∼3.7, ∼2.7, and ∼1.9 Kb (Fig. 3b). RNA load levels for each of the tissues tested were similar based upon hybridization with a catfish EF1α probe. RT-PCR, using primers specific for the putative inhibitory IpLITR1 and the putative activating IpLITR2 as representative IpLITRs, was also used to assess tissue expression. As shown in Fig. 3c, IpLITR1 and IpLITR2 are in most cases coordinately expressed in a variety of different tissues and confirm the Northern blot data. In addition, multiple PCR products were amplified using the IpLITR1 and IpLITR2 specific primers, which is reminiscent of the multiple transcripts observed following RNA blotting. Because the predicted sizes of PCR products for the prototype IpLITR1 and IpLITR2 are 1,461 and 780 bp, respectively, at least two additional IpLITR1 products (1,545 and 2,392 bp) and one additional IpLITR2 product (1,298 bp) were amplified (Fig. 3c).
Fig. 3Northern blot and RT-PCR analyses of IpLITR tissue expression. Catfish LITR tissue expression was performed by Northern blot analysis using a an IpLITR1 D1-specific probe, and b an IpLITR2 D3-specific probe. Total RNA from pronephros (head kidney), mesonephros (trunk kidney), spleen, heart, liver, gill, and muscle were examined. Kilobase markers are on the left margin and arrows indicate the major hybridizing bands observed. RNA integrity and load levels were determined by hybridization using a catfish EF1α probe as a housekeeping gene. c RT-PCR analyses of IpLITR1 and IpLITR2 in various catfish tissues. The sizes of the IpLITR bands verified by sequencing are indicated at the right margin and base pair sizes are at the left margin
To further examine the expression of IpLITR1 and IpLITR2, RT-PCR was performed using RNA from catfish clonal macrophage, B, T, and NK-like cell lines, as well as a MLC (Fig. 4a). RT-PCR products of 1,461, 1,545, and 2,392 bp were obtained using IpLITR1-specific primers, and these products were detected variably among the cell lines examined. It was also found that three RT-PCR products were also variably amplified from the different catfish leukocyte cell lines using IpLITR2-specific primers, the expected 780-bp product, a 1,298-bp product (previously observed in tissues; see Fig. 3c), and an additional 1,060-bp product (Fig. 4a). In contrast, no IpLITR message was detected in the fibroblast CCO cell line (data not shown), indicating that these genes are primarily expressed in cells of immune function.
Fig. 4a RT-PCR analyses of IpLITR1 and IpLITR2 expression in various catfish clonal cell lines and a polyclonal MLC. Total RNA was obtained from the catfish 42TA macrophages, 3B11 B cells, TS32.15 and TS32.17 nonautonomous cytotoxic T cells, autonomous G14D T cells, a MLC, and 1F3 NK-like cells. RT-PCR was performed using primers specific for IpLITR1, IpLITR2, and IpEF1α. The sizes of the IpLITR bands verified by sequencing are indicated at the right margin and base pair sizes are at the left margin. b Schematic representation of IpLITR-types identified by sequencing of RT-PCR products obtained using IpLITR1 and IpLITR2-specific primers. The originally identified prototype IpLITRs are boxed, and arrows indicate the relative positions of the primer pairs used in RT-PCR reactions. Sizes in base pairs corresponding to the bands observed in a are indicated above each schematic. The predicted SP, Ig domains, TM, and CYT are indicated. ITIM-like motifs are shown as hatched boxes and noncanonical immunotyrosine-based activation motif-like motifs shown as white boxes within the CYT. Individual Ig domains are shaded according to relatedness as described for Fig. 1, and percentages above each receptor represent percentage of amino acid identities of the predicted extracellular region of the IpLITR-like sequences when compared with the prototype IpLITRs
The sequencing of the various IpLITR1 and IpLITR2 RT-PCR products from all sources (both tissues and cell lines) resulted in the identification of multiple different IpLITR-related sequences (Stafford et al. manuscript in preparation). Schematic diagrams of representative IpLITR1 and IpLITR2 variant forms are shown in Fig. 4b. These highly related IpLITR1- and IpLITR2-like sequences differed in the number of Ig-like domains present, and in the case of some IpLITR1-variants, also differed in the length of the CYT.
Homology between IpLITRs, LRC, and FcR genes
When the extracellular region of IpLITR1 was used in BLASTP analysis, various adhesion molecules, FcRs, FcR-like proteins (FCRLs), LILRs, and KIR gene family members were identified with significant homology (i.e., E values <10−7), albeit with moderate amino acid identities ranging from ∼25–30% (Table 3). Examples include the adhesion molecules CD22 and carcinoembryonic antigen-related cell adhesion molecule 5, Xenopus leukocyte FCRLs, XFL, PIRA1, LILRA2, CHIRA2, KIR3DL1, FCRL5, and FcγRIIB. When the Ig domains of IpLITR2 were used for screening, most matches were members of the FcR gene family, e.g., FcγRI, FcγRII, FcγRIII, and FcεRIα, from humans, primates, and rodents, as well as various FCRL proteins (Table 3). Searches using the extracellular region of IpLITR3 were essentially the same as the IpLITR1 BLASTP results, and PSI-BLAST analysis further reinforced the evolutionary relationships between IpLITRs and mammalian FcRs, FCRLs, KIRs, and LILRs, as well as avian CHIRs (data not shown).
Table 3Representative IpLITR BLASTP resultsReceptor AccessionScoreE-valueChromosomeIpLITR1CD22HumanNP_001762884e-1619q13.1XFL1.4XenopusAAQ56585845e-15?CEACAM5HumanCAA34474845e-1519q13.1PIRA1RatAAD50905731e-11?LILRA2 (ILT1)ChimpanzeeNP_001009044722e-1119FCRL5 (FcRH5)MouseNP_899045716e-113XFL1.3XenopusAAQ63874716e-11?LILRA2 (ILT1)HumanAAD50364701e-1019q13.4KIR3DL1HumanAAC83928693e-1019q13.4KIRChimpanzeeAAF86243684e-1019CHIRA2ChickenCAG33731661e-9?KIR3DL1OrangutanAAM78465661e-919FCRL5 (FcRH5)HumanCAH71428662e-91q21KIR3DS1HumanAAV32446662e-919q13.4FcγRIIBRatAAL29888631e-813q24XFL1.6XenopusAAQ63873631e-8?XFL1.2XenopusAAQ56587622e-8?CHIRB3ChickenCAG25957623e-8?LILRA3 (ILT6)HumanAAB87661608e-819q13.4LIRBovineXP_586587601e-7?PIRA2MouseAAC53213592e-77IpLITR2IRTA2BovineXP_595289761e-12?FcγRIIGuinea pigA34636613e-8?FCRL5 (FcRH5)MouseNP_899045606e-83FCRL3 (FcRH3)MouseAAS91578606e-83FcγRIMacaqueAAL92095591e-7?FCRL5 (FcRH5)HumanNP_112571574e-71q21FcγRIαHumanCAI12557575e-71q21FcγRIIBMouseAAH19758569e-71XFL1.4XenopusAAQ56585569e-7?IRTA1BovineXP_614632552e-6?FcεRIαHumanAAH05912538e-61q23FREB2HumanAAX11390538e-61q23.3FcγRIHumanAAA58414538e-61q21.2Similarity search (BLASTP) of the NCBI’s nonredundant protein database using the four Ig domains of IpLITR1 and the three Ig domains of IpLITR2CEACAM5 carcinoembryonic antigen-related cell adhesion molecule, XFLXenopus leukocyte FCRL
Phylogenetic analyses and alignments
The IpLITR D1 and D2 Ig domains are related to those of mammalian FcRs and FCRLs (Fig. 5a) in that these domains clustered with the FcR/FCRLs and away from catfish NITRs and from representative LRC members in a phylogenetic tree. In contrast to the D1/D2 sequence analyses, the membrane proximal D3 and D4 sequences of IpLITR1 and IpLITR3 resulted in a significant shift within the phylogenetic tree; IpLITRs now clustered with the LRC-encoded genes (Fig. 5b). The LRC relationship is supported by high bootstrap values when comparing IpLITR1 and IpLITR3 D3 and D4 sequences with D2 and D3 sequences of representative LILRs and KIRs, as well as with D1 and D2 sequences of NKp46 and CHIRs. The D5 and D6 sequences of IpLITR3 are related to the D1 and D2 domains of the FcR family (Fig. 5c). Thus, IpLITR3 is the best example of the unique domain structure of these teleost receptors, as it encodes for combinations of Ig domains that are phylogenetically related to the FcRs and FCRLs (D1 and D2; D5 and D6), as well as LRC encoded receptors (D3 and D4).
Fig. 5Phylogenetic analyses reveal unique Ig domain composition within individual IpLITRs. The Ig domains compared are indicated by gray shading in the schematics to the left of each phylogenetic tree. a Comparisons of IpLITR, representative FcR and FCRL, and human LRC D1 and D2 sequences. b Comparisons of IpLITR1 D3 and D4 with representative FcR, FCRL, KIR, LILR, and PIRA1 D2 and D3 sequences, as well as NKp46 and CHIRA2 D1 and D2 sequences. c Comparison of IpLITR3 D5 and D6 with D1 and D2 of representative mammalian FcRs, FCRLs, and LILRs/KIRs. The accession numbers for the various Ig domain sequences used are: human (hu)FcγRI(CAI12557), huFcγRII (CAA35642), huFcγRIII (CAA36870), huFcεRI (AAH05912), huFCRL3 (AAH28933), huFCRL4 (AAK93970), huFCRL5 (NP_112571), huFcαR/CD89 (AAH27953), huLILRB1 (AAH15731), huLILRB2 (AAB87662), huKIR3DL1 (AAC83928), huKIR3DS1 (AAV32446), huSIRP (CAA71404), human NKp46 (AJ001383), mouse (mo)FcγRI (AAD34931), moFcγRII (AAA37608), moFcγRIII (NP_034318), moFcεRI (NP_034314), moFCRL3 (AAS91578), moNKp46 (AJ223765), rat (ra) FcγRIII (AAA42050), raPIRA1 (XP_341773), raNKp46 (AF082533), guinea pig (gp) FcγRII (D13692), macaque (mac)FcγRI (AAL92095), bovine (bo)FCRL5 (XP_595289), boKIR3DS1 (AAP33626), boSIRP (CAA71943) chimpanzee (cp)LILRA2 (NP_001009044), orangutan (or)KIR (AAM78473), monkey (mk)KIR3DL20 (AAU50562), CHIRA2 (CAG33731), catfish (Ip)NITR1 (AF397454), and IpNITR2 (AF397455). For all analyses, SIRPs and/or IpNITRs were included as outgroups to root the trees. NJ trees with pairwise gap deletions were drawn using MEGA v3.0 (Kumar et al. 2001) with 10,000 bootstrap replications, and bootstrap values >50% are shown. Branch lengths were measured in terms of amino acid substitutions, and scale bars are shown below the trees
Subsequently, the D1 and D2 Ig domain sequences of IpLITR2 (as a representative) were aligned with various mouse and human FcRs and three representative human FCRLs. A majority of the predicted β-strands in IpLITR2 is present in similar positions as the β-strands found in FcR/FCRL family members (Fig. 6a), indicating a predicted degree of secondary structural conservation between IpLITRs and these mammalian immune receptors. In most cases, the predicted IpLITR2 β-strands were present in regions of high amino acid identity/similarity; however, none of the FcR residues known to contact the Fc portion of Ig were present (Fig. 6a). The D3 of IpLITR2, a domain specific to this IpLITR type, appears to be related to the membrane distal domain (D1) of several CHIRs (Fig. 6b), and alignment of IpLITR2 D3 with representative CHIR D1s indicated a degree of amino acid identity/conservation between these Ig domains (Fig. 6c). To reinforce the unique nature of domains present within this receptor, IpLITR2 D1 did not cluster with the CHIRs but was related to mammalian FcR D1 domains (Fig. 6b).
Fig. 6IpLITR2 contains Ig domains related to both FcRs/FCRLs and CHIRs. a Amino acid alignment of IpLITR2 D1 and D2 sequences with representative mammalian FcRs and FCRLs. Accession numbers are as in Fig. 5. Gray shading: residues similar/identical to IpLITR2. Boxed residues: contacts for Ig Fc in huFcεRI. Black and gray arrows represent the predicted β-strands for IpLITR2 and huFcεRI, respectively. Hatched boxes indicate conserved cyteines and dashes represent gaps. b Phylogenetic analysis of IpLITR2 D1 and D3 sequences (gray shaded) compared with representative mammalian FcR sequences (as in Fig. 5) and D1 sequences of representative CHIRs. The accession numbers for the various Ig domain sequences used are: CHIRB2 (XP_422905), CHIRB1 (CAH55757), CHIRAB2 (CAG33733), CHIRA2 (CAG33731), CHIRB5 (AJ879908), CHIR A1 (AF306851), CHIRB4 (XP_428342), CHIRB6 (CAI53861), and CHIRAB3 (AJ879909). NJ trees with pairwise gap deletions were drawn using MEGA v3.0 (Kumar et al. 2001) with 10,000 bootstrap replications, and bootstrap values >50% are shown. Branch lengths were measured in terms of amino acid substitutions, and scale bars are shown below the trees. c Alignment of IpLITR2 D3 and CHIR D1. Gray shading indicates residues that are similar/identical to IpLITR2, black and gray arrows represent predicted β-strands for IpLITR2 and CHIRA2, respectively; “dotted” lines indicate predicted helices and hatched boxes indicate conserved cyteines. Gaps in alignment are indicated by dashes
Attempts to model IpLITR Ig domains were unsuccessful as no sufficient modeling templates were identified using SWISS-Model. However, similarity searches against the NCBI’s protein data bank (PDB) indicated a significant match between D1 and D2 of IpLITRs and the Ig domains of human FcεRIα (E value <10−8). Conversely, the membrane proximal D3 and D4 Ig domains of IpLITR1 and IpLITR3 had significant alignment scores (E value <10−7) with the first two Ig domains of the human natural cytotoxicity receptor NKp46. Taken together, these PDB search results further support that IpLITRs D1 and D2 Ig domain sequences are related to mammalian FcRs and FCRLs, and that the D3 and D4 Ig domains are related to LRC encoded receptors.
Mining of the zebrafish genome identified five contigs (Table 2) containing sequences encoding IpLITR-like proteins predicted to contain several Ig domains and, in some cases, signal peptides, TM segments, and CYTs with signaling motifs (Fig. 7). These findings indicate that IpLITR-like molecules are not unique to the catfish, but are likely common in other teleost species. Because IpLITR3 was identified as the closest match from the NCBI’s database to the zebrafish (Zf) LITR-like proteins, it was used for further comparisons. Phylogenetic analysis of the various Ig domain sequences indicated that the ZfLITR-like proteins were closely related to those of IpLITRs (Fig. 7a). In most cases, the membrane proximal domains of ZfLITRs are highly related to their IpLITR3 counterparts with >60% amino acid identity (Fig. 7b). However, one of the ZfLITRs (XP_692267) contains two Ig domains similar to IpLITR3 D6 in the membrane distal region. This zebrafish receptor also contains three Ig domains that are phylogentically related (Fig. 7a) with high amino acid identity (∼50%) to IpLITR3 D4 (Fig. 7b). The other ZfLITR-like proteins are also composed of combinations of Ig domains that are related to IpLITR3 Ig domains with varying degrees of amino acid identities (e.g., 25–66%). However, none of the predicted ZfLITR-like molecules contained membrane distal Ig domains that were related to IpLITR D1 and D2, which are shared among all the IpLITRs identified to date. This suggests either that LITR membrane distal domains may have coevolved with distinct ligands within individual fish species or that ZfLITRs with D1 and D2 domains similar to IpLITRs have yet to be identified. It should be noted, however, that these zebrafish domains were obtained from genomic sequence and that they do not accurately represent the precise organization of the Ig domains likely to be present in encoded ZfLITRs, which will only be determined from cDNA sequences. Nonetheless, the genomic segments are useful for domain-to-domain comparisons and reinforced the presence of IpLITR-like sequences in another teleost species.
Fig. 7Annotation of zebrafish LITR-like proteins. a Phylogenetic analysis of ZfLITR and IpLITR3 Ig domains. Ig domains were predicted using SMART (Letunic et al. 2004) and the amino acid sequences aligned using CLUSTALW (Thompson et al. 1997). NJ trees with pairwise gap deletions were drawn using MEGA v3.0 (Kumar et al. 2001). Only branch values >50 are shown, and the major Ig domain groups are designated by brackets and shaded according to their relatedness with IpLTR3 Ig domain. b Schematic representation of ZfLITR-like proteins compared with IpLITR3. Domains are shaded according to their phylogenetic relationship with IpLITR3 domains. Hatched bars on IpLITR3 and Zf XP_692267 indicate signal peptides, TM segments are solid black lines, and ITIMs are shown as gray boxes for XP_694305. Amino acid identity vs corresponding IpLITR3 Ig domains are indicated as percentage value above each domain, and only IpLITR3 D3, D4, D5, and D6 were related to the Ig domains of the representative ZfLITR analyzed, as indicated by the dashed box. Predicted size in amino acids for each molecule is indicated on the left
Discussion
The novel IpLITRs described herein represent a polymorphic and polygenic gene complex that are primarily expressed in hematopoietic tissues. The polymorphic nature of this gene complex was indicated by RFLP analysis, which revealed differences in where various restriction enzymes cut the genomic DNA among the sibling catfish. However, it should be noted that only detailed sequence analyses of these IpLITR genes in a number of individuals would be required to determine if any polymorphic differences are also found within the coding regions. In addition, segregation analyses suggested that IpLITR genes are encoded within multiple independently segregating but homologous loci, similar to the situation seen with the ZfLITR homologs identified by genome mining. These paralagous relationships strongly suggest that IpLITRs are products of multiple gene duplication and translocation events from a common ancestoral gene. Whether or not the receptors encoded by the different IpLITR loci are functionally redundant remains to be determined. However, IpLITRs are related to functionally and genomically distinct mammalian FcRs and LRC encoded receptors, suggesting that such paralogous loci could have evolved to give rise to an array of immunoregulatory receptors with different functions.
The coordinate expression of IpLITRs by myeloid and lymphoid cell lines in combination with their potential activating and inhibitory signaling capabilities suggests that they play an important role in immune cell function(s). This possible importance of IpLITRs in immune responses is highlighted by the RT-PCR expression studies where a large number of highly related, but unique, IpLITR sequences were identified. These variant IpLITR sequences differed in the number of Ig domains present, and in some cases in the length of the CYT. Therefore, in addition to the prototype IpLITR1, IpLITR2, and IpLITR3 described here, other IpLITR family members are coexpressed by catfish myeloid and lymphoid cells. Whether or not some of the variously sized transcripts represent messages from different IpLITR genes or are the result of alternative splicing is unknown. However, the large number of unique IpLITRs found expressed suggests that IpLITRs represent a complex immune receptor family that can potentially generate a large number of IpLITR types.
The prototypical IpLITR1, IpLITR2, and IpLITR3 encode receptors with four, three, and six Ig domains, respectively. Pairwise comparisons and phylogenetic analysis of these domains suggest a high degree of conservation and a conserved membrane distal to membrane proximal ordering. Each of these three receptors has very similar D1 and D2 sequences, and IpLITR1 and IpLITR3 have almost identical D3 and D4s. However, variation does occur between predicted membrane proximal Ig domains. The IpLITR2 D3 sequence is unique to this receptor and IpLITR3 D5 and D6 are not similar to any other of the prototypical IpLITR Ig domain sequences. Identification of LITR-like sequences on different zebrafish chromosomes and comparisons with IpLITRs supports the notion that these polymorphic and polygenic immune receptors are not unique to catfish. Like IpLITRs, the predicted ZfLITRs are also composed of unique combinations of Ig domains. Such differences in the Ig domain compositions of IpLITRs and ZfLITRs may influence their ligand binding capabilities, formation of homo- or heterodimers and overall immunomodulatory functions.
IpLITR1 has a long CYT with two consensus ITIMs and a third ITIM-like motif, whereas both IpLITR2 and IpLITR3 have shorter CYTs devoid of recognizable signaling motifs. However, these latter two receptors each contain a lysine residue within their TM segments, indicating that associations with an adaptor molecule(s) would be necessary for them to initiate intracellular signaling (Daeron and Vivier 1999; Ravetch and Lanier 2000; Billadeau and Leibson 2002). Within the CYT of IpLITRI was also an overlapping ITSM suggesting that multiple motifs (ITIMs and ITSM) within the CYT of this receptor may participate in downstream signaling cascades through recruitment of various adaptor molecules (Sidorenko and Clark 2003). Similarly, predicted ZfLITR family members were shown to either contain long CYTs with ITIM motifs or short CYTs with charged TM segments. Genes for immunotyrosine-based-activation-motif-bearing adaptor molecules (e.g., FcRγ chain, CD3ζ, and DAP) have been identified in several ectothermic vertebrates, including frogs and fish (Guselnikov et al. 2003a,b). Hence, it seems likely that IpLITR2 and IpLITR3 would each associate with such molecules and function in a similar manner as mammalian receptors with charged TM segments. Thus, the paired expression pattern of IpLITRs with activatory and inhibitory signaling potential is very similar to that shown for several mammalian and avian families of immune receptors, including PIRs, LILRs, KIRs, FcRs, and CHIRs.
Similarity searches for IpLITR relatives revealed a striking dichotomy. The majority of matches were members of two distinct mammalian receptor families. While IpLITR2 appeared to be more closely related to FcRs and FCRLs, IpLITR1 and IpLITR3, exhibiting very similar D1–D4 sequences, were related to both FcR/FCRLs and LRC-encoded receptors. Thus, it is postulated that the D1 and D2 domains common to all IpLITRs are evolutionarily more closely related to domains encoded by the mammalian FcR and FCRLs, a contention supported by the retrieval of the human FcεRI alpha chain as the most similar structure from a PDB search. Phylogenetic analyses also supported this hypothesis because the IpLITR D1 and D2 clustered with members of the FcR and FCRL receptor families and away from representative LRC gene family members. Although the D1D2 of IpLITRs are phylogenetically related to FcR and FcRLs, there is no evidence to support that IpLITRs bind Ig. It appears more likely that the relatedness of these membrane distal Ig domains, as also demonstrated for FCRLs, PIRs, and CHIRs, are only evident at the phylogenetic level and that they are not functional homologs. Furthermore, during the preparation of this manuscript, a “bona fide” Ig-binding catfish FcR homolog, which is not a member of the IpLITR receptor family, was identified and characterized (Stafford et al. 2006). In contrast to the D1 and D2 domains, IpLITR1 and IpLITR3 D3 and D4 Ig domains are related to several members of the LRC (human chromosome 19q13.4). As above, these two domains clustered with one family of receptors (the LRC) and not the other (i.e., FcR/FcRLs). The identification of NKp46 as a structural relative from the PDB also supports this notion. Interestingly, D5 and D6 of IpLITR3 are related to FcR and FCRLs, but their membrane proximal location suggests that they are unlikely to be involved in ligand recognition (i.e., Ig). Finally, the third domain of IpLITR2 clustered with the Ig domains of several representative CHIRs. Taken together, IpLITR extracellular domain sequences appear reminiscent of both FcR/FCRLs and members of the mammalian and avian LRC, implying that catfish LITRs are distant relatives to each of these genomically and functionally diverse receptor gene families. At present, the functional significance of these novel teleost immune receptors is unknown.
Recently, key insights into the evolution of the FcR and LRC gene families and their potential common origin has been presented: (1) human chromosomes 1 and 19 are believed to be in paralogous regions of the genome due to an entire genome duplication event (Kasahara 1999; Shiina et al. 2001); (2) attempts at cloning rodent FcαR (CD89) orthologs identified the PIR gene family located on a mouse chromosome, which is syntenic to the human LRC (Kubagawa et al. 1997; Hayami et al. 1997); (3) the subsequent search for distant PIR relatives resulted in the discovery of the CHIRs, providing the first molecular evidence for a common ancestor of LRC and FcR gene families (Dennis et al. 2000); (4) sequencing of genomic segments encoding the CHIR gene family identified the chicken equivalent of an LRC that appears to have also been expanded by gene duplication events (Nikolaidis et al. 2005a); and (5) the identification of Ig-like domains in chickens, Xenopus, and bony fish that are related to Ig domains of mammalian FcRs and members of the LRC (Nikolaidis et al. 2005b). Taken together, these findings suggest that immune receptors encoded by human chromosome 1q21 and 19q13 most likely evolved from a common ancestor that has since undergone multiple rounds of duplication and homologous recombination events.
Although there is functional evidence for the existence of FcR- or KIR/LILR-like homologs in ectothermic vertebrates (Coosemans and Hadji-Azimi 1986; Shen et al. 2003, 2004), their genetic identification has not been previously reported and the inability to clone these receptors has made it difficult to elucidate their evolutionary history. The recent discovery of mammalian FCRL genes located on chromosome 1q21-22, however, has expanded the view of immune receptor evolution (Davis et al. 2001; Hatzivassiliou et al. 2001; Miller et al. 2002; Guselnikov et al. 2002). For example, FCRL3, FCRL4, and FCRL5 not only encode D1, D2, and D3 Ig domains homologous to the corresponding domains of FcγRI, but also contain variable numbers of unique membrane proximal Ig domains (Davis et al. 2001; Hatzivassiliou et al. 2001). Homology searches demonstrated that the FCRLs, also termed IFGP (for IgSF, FcR, and gp42) exhibited a “chimeric” Ig domain compositions and consequently these receptors were proposed to be a phylogenetic link between the FcRs, rat NK cell-specific gp42 antigen, and adhesion molecules (Guselnikov et al. 2002). This feature of similar Ig domains encountered in homologous but functionally diverse receptors is also a striking feature of the IpLITRs and supports the hypothesis that certain mammalian immune receptor families (i.e., FcRs and KIRs) have evolved from a common ancestor. Notably, although the IpLITRs D1 and D2 domains are phylogenetically and structurally similar to the mammalian FcRs and FCRLs, the IpLITR ligand(s) is unknown. This is similar to the situation with mammalian FCRL molecules, whose membrane distal Ig domains are clearly related to the classic FcRs, but are unlikely to be functional homologs because they do not bind Ig (Hatzivassiliou et al. 2001; Davis et al. 2005). Therefore, although certain Ig domains encoded by several receptor gene families identified in humans, rodents, birds, and fish (i.e., FCRLs, PIRs, CHIRs, and IpLITRs, respectively) are related to FcRs, they appear to have evolved to recognize different ligands. Recently, it was shown that rodent PIRs recognized MHC class 1 and β2M (Takai 2005), raising the possibility that related immunoregulatory receptors in other species (i.e., CHIRs and IpLITRs) could also be involved in MHC recognition.
In summary, the discovery of individual catfish receptors that encode Ig domains related to both FcR and LRC gene families provides further support for the common origin of these important mammalian immune receptors. Identification of LITR-like sequences in the zebrafish genome also demonstrates that these receptors are likely found in all teleosts. Annotation of these genomic segments will be necessary to determine if zebrafish LITR homologs exhibit conserved genomic features and syntenic genomic location with mammalian 1q21-23 and/or 19q13. Thus, the discovery of IpLITRs is an important step towards further understanding the common origin and evolutionary history of the different families of mammalian immunoregulatory receptors. The investigation of the functional significance of these novel teleost receptors is underway and will be aided by the availability of channel catfish cell lines that coordinately express IpLITRs and the eventual production of IpLITR specific antibodies.
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teleosts
innate immunity
evolution
molecular immunology
inhibitory receptors