NF-κB-dependent synergistic regulation of CXCL10 gene expression by IL-1β and IFN-γ in human intestinal epithelial cell lines

Background and aims Little is known about the intestinal epithelial expression and secretion of CXCL10 (IP-10), a chemokine involved in recruiting T cells and monocytes. We aimed to study CXCL10 gene expression and regulation by the pro-inflammatory cytokines interleukin (IL)-1β, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in intestinal epithelial cell lines.
Introduction
Intestinal epithelial cells (IECs) form the first immunologic barrier of the organism while the villus surface is continuously in contact with various agents. IEC gene expression is tightly regulated to induce appropriate responses to pathogens and to avoid deleterious reactions to microbial flora. When stimulated with pro-inflammatory cytokines or infected with microbial pathogens, IECs may secrete chemokines, a set of low-molecular weight cytokines, directing migration and activation of leucocytes, which play a major role in the perpetuation of inflammatory processes [1–5]. The final composition of leucocytes present in the inflamed intestine is most likely due to both secreted chemokines and the relative expression of chemokine cell surface receptors on different cell types. Chemokine production by colonic epithelial cells is thought to contribute to the characteristic increased infiltration of selected populations of leucocyte cells in inflammatory bowel disease (IBD) [6, 7].
CXCL10 is a CXC chemokine, known to be secreted by endothelial cells [8], keratinocytes [9], eosinophils [10] and neutrophils [11]. It is also constitutively expressed by normal human colon epithelium [12] and chemoattracts activated T cells as well as monocytes by binding to the CXCR3 receptors present on the respective cells [13, 14]. While CXCL10 up-regulation may occur in acute inflammation in response to pathogens, it is also known to be permanently over-expressed in IBD patients [15, 16]. Blocking CXCL10 could prevent mice from acute colitis, suggesting this chemokine to play an important role in IBD pathogenesis [17]. Moreover, CXCL10 is believed to possess anti-tumoural properties by promoting anti-tumour T cell immunity [18, 19]. Knowledge of its regulation in colonic cell lines may therefore give also insight into the pathobiology of colorectal cancer.
Most previous studies on chemokine expression in IEC concentrated on CXCL8 (interleukin [IL]-8), a neutrophil chemoattractant [20, 21], but there were only few studies regarding the expression and secretion of CXCL10 in IECs [12]. Although it is well known that interferon (IFN)-γ is a major inducer of CXCL10 and that CXCL10 expression may be enhanced by pathogens and other inflammatory cytokines, the relative contribution of cytokines on CXCL10 induction, their synergistic action and the kinetics of cytokine-induced CXCL10 expression in intestinal epithelial cells has not been fully elucidated. Especially, the role of IL-1β in CXCL10 gene regulation in IECs and also in other cell types has not been fully evaluated so far. Intestinal mucosa is composed of cell populations in continuous change from a proliferative and undifferentiated stage in the basal parts of crypts to mature surface villus epithelial cells [22, 23]. Little is known about the alteration of immunological functions as IECs mature. It has been shown that cellular differentiation affects IEC responsiveness to IL-1β [24].
In the present study, we aimed to elucidate the role of the pro-inflammatory cytokines IL-1β, tumour necrosis factor (TNF)-α and IFN-γ, which have established role in IBD pathogenesis, in inducing CXCL10 gene expression in IEC lines. Especially, the role of IL-1β, one of the most abundantly expressed cytokines, both in normal and inflamed mucosa [25], in inducing CXCL10 expression by IECs and its possible signalling mechanisms was given importance. To consider possible influences of cellular differentiation on cytokine responsiveness, we performed studies on three different colonic epithelial cell lines. Our findings will get an insight into the role of intestinal epithelium in immune responses and in evaluating pathways, which might be targets of present and future pharmacotherapy of IBD and possibly colorectal cancer.
Materials and methods
Biological reagents
The recombinant cytokines IL-1β, TNF-α and IFN-γ were purchased from Roche applied science (Mannheim, Germany). An enzyme-linked immunosorbent assay (ELISA) kit for CXCL10 was purchased from RnD systems (Weisbaden, Germany). The nuclear factor (NF)-κB inhibitor BAY11-7082 was purchased from Calbiochem (San Diego, CA).
Cell culture and stimulation protocols
The human colon adenocarcinoma cell lines Caco-2, HT-29 and DLD1 were obtained from DSMZ (Braunschweig, Germany). Caco-2 cells were grown in Eagle’s minimal essential medium (BioWhittacker, Germany) containing 20% foetal calf serum (FCS) supplemented with 100 U/ml each of penicillin and streptomycin and 1% non-essential amino acids at 37°C and 5% CO2. HT29 and DLD1 were grown in Roswell Park Memorial Institute medium containing 10% FCS and 100 U/ml penicillin and streptomycin at 37°C and 5% CO2. Caco-2, HT29 and DLD-1 cells were plated into six-well plates at a density of 5 × 105 cells per well for real-time polymerase chain reactions (PCRs), ELISA, Northern blotting and electrophoresis mobility shift assay (EMSA) experiments unless until stated and grown till they reached 70–80% confluence. These cells were then stimulated with IL-1β (0.1, 0.5, 1, 2, 10 and 50 ng/ml), TNF-α (2, 10 and 50 ng/ml) and IFN-γ (2, 10 and 50 ng/ml) based on the type of experiments.
RNA isolation, real-time PCR
Ribonucleic acid (RNA) was isolated by using Qiagen RNeasy mini kit according to the manufacturer’s protocol. The RNA concentrations were determined photometrically using a Gene Quant RNA/deoxyribonucleic acid (DNA) calculator (Pharmacia, Freiburg, Germany). RNA was subsequently used for real-time PCR and Northern blot analysis. Reverse transcription of messenger RNA (mRNA) was performed using 1 μg of total cellular RNA. To determine the mRNA expression of CXCL10, real-time PCR was carried out using gene-specific primers for human CXCL10 (Invitrogen GmbH, Karlsruhe) forward 5′-CCA GAA TCG AAG GCC ATC AA-3′, reverse 5′-CAT TTC CTT GCT AAC TGC TTT CAG-3′ and β-actin (Invitrogen GmbH) forward 5′-CTG GCA CCC AGC ACA ATG-3′, reverse 5′-CCG ATC CAC ACG GAG TAC TTG-3′ in an ABI Prism 7000 sequence detection system. PCR reaction was set up with Sybr® Green PCR Master mix (Invitrogen) containing 0.3 μmol/l primers each and 1 μl of RT-product in 25 μl volume. A two-step amplification protocol was chosen consisting of initial denaturation at 95°C for 10 min followed by 45 cycles with 15 s denaturation at 95°C and 30 s annealing/extension at 60°C. Finally, a dissociation protocol was performed to control specificity of amplification products.
Relative expression of CXCL10 was then calculated using the comparative threshold-cycle (CT) method. The amount of target mRNA in each sample was normalized to the amount of β-actin mRNA designated as calibrator, to give ΔCT (CT β-actin − CT CXCL10). The relative expression of CXCL10 was calculated as the . MRNA expression is presented as fold increase calculated in relation to unstimulated cells after normalization to β-actin.
Transient transfections
Caco-2, HT29 and DLD1 cells were plated in 24-well plates, 24 h before transfection (Costar, Corning, NY), at a density of 50,000/well in 1 ml medium. After 24 h, the cells were transfected with 250 ng of respective plasmids along with 20 ng of renilla plasmid using the non-liposomal formulation FuGENE (Roche Molecular Biochemicals, Mannheim, Germany). On the day of transfection, fresh medium was added. FuGENE was added to the plasmid DNA at a ratio of 3 μl/μg DNA. FuGENE was prediluted in 100 μl serum-free medium and added drop-wise to the concentrated plasmid DNA. After 15–25 min at room temperature, the mixture was added to the cells. The plasmids TGL-IP10, TGL-IP10 κB2 Mut were kind gifts from Dr. Richard M. Ranshoff (Cleveland, OH) and were described elsewhere [26].
Luciferase assays
Twenty-four hours after transfection, cells were stimulated with IL-1β (0.1,1, 2, 10 and 50 ng/ml), TNF-α (2, 10 and 50 ng/ml), IFN-γ (2, 10 and 50 ng/ml), IL-1β (1 ng/ml) + TNFα (50 ng/ml), IL-1β (1 ng/ml) + IFN-γ (50 ng/ml) and TNFα (50 ng/ml) + IFN-γ (50 ng/ml) for 24 h, harvested and lysed, and luciferase was assayed by using a luciferase kit (Promega). Values were normalised to renilla luciferase.
Enzyme-linked immunosorbent assay
To study the synergistic effects of cytokines on CXCL10 secretion, supernatants were collected at 0, 3, 6, 12 and 24 h in Caco-2 and HT29 cells stimulated with IL-1β (1 ng/ml), TNF-α (50 ng/ml), IFN-γ (50 ng/ml), IL-1β (0.5 ng/ml) + TNFα (50 ng/ml), IL-1β (0.5 ng/ml) + IFN-γ (50 ng/ml) and TNFα (50 ng/ml) + IFN-γ (50 ng/ml) and stored at −70°C until measured. CXCL10 protein secretion was measured by ELISA according to the manufacturer’s protocol.
Preparation of nuclear extract
Caco-2 cells were grown for 24 h, pre-treated for 1 h with BAY 11-7082 (10 μM) and then stimulated with respective cytokines for 30 min. Cells were washed with 10 ml cold phosphate-buffered saline (PBS) twice, harvested, centrifuged at 1,000 × g for 5 min and resuspended in 1 ml of cold PBS. Centrifuged briefly, excess PBS was removed, and the pellet was resuspended in ice-cold extraction buffer (20 mM hydroxyethyl piperazineethanesulfonic acid [HEPES] pH 7.8, 10 mM KCL, 0.1 mM ethylenediamine tetraacetic acid [EDTA], 1 mM dithiothreitol and 0.5 mM phenylmethanesulphonylfluoride [PMSF]) by gentle pippeting. Cells were then kept on ice for 15 min, and 30 μl of 10% Nonidet NP-40 was added and vortexed briefly for 10 s. The lysed cell suspension was centrifuged for 30 s, and the supernatant was discarded. The pellet was resuspended in 50 μl extraction buffer containing 400 mM NaCl, agitated vigorously for 15 min at 4°C. Then, the cell suspension was centrifuged for 5 min at 4°C, and the supernatant containing the nuclear extract was collected and stored at −80°C until further use.
Electrophoretic mobility shift assay and super-shift assay
For binding reactions, 5 μg of nuclear extracts were incubated in 20 μl reaction mixture containing 40 mM HEPES pH 7.5, 50 mM NaCl, 1 mM EDTA, 1mM DithitreitolHSCl, 1 mM PMSF, 1.28 μg/ml pBluescript vector DNA as half of the reaction and the rest being nuclear extract plus other components. Oligonucleotide duplex probes (Promega) were end-labelled with T4 polynucleotide kinase and [γ32P] adenosine triphosphate; 1 × 104 cpm were then added to the reaction mixtures and incubated overnight at 4°C. Reaction products were analysed by non-denaturing electrophoresis in a 5% polycralamide gel with 0.5× Tris–borate–EDTA buffer at room temperature. Gels were then exposed directly to X-ray film at −70°C for autoradiography. For competition, unlabelled oligonucleotides were added in molar excess at room temperature for 15 min before adding radiolabelled probe. In super shift experiments, nuclear extracts were incubated with anti-p65 anti-body (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C.
Statistical analysis
Statistical analyses were carried out using the Prism software packet (Version 3.0, Graphpad Software, San Diego, CA). Comparisons between two or more different treatment groups were made with the non-paired t test or analysis of variance (ANOVA) where appropriate. For time kinetics experiments, a two-way ANOVA test followed by the Bonferroni post-test was performed. In case of RNA expression, a log transformation was performed before. Statistical differences were regarded significant at a p level below 0.05. Data were expressed as means ± standard error of the mean (SEM).
Results
Differential expression of CXCL10 mRNA in intestinal epithelial cell lines under basal conditions
In the present study, the basal mRNA expression of CXCL10 in three different IECs, Caco-2, HT29 and DLD1 cells, were studied first. Real-time PCR experiments using gene-specific primers for CXCL10 revealed that the basal mRNA expression of CXCL10 was highest in Caco-2 followed by HT29 and DLD1 (Fig. 1).
Fig. 1Basal level expression of CXCL10 in IECs. Caco-2, HT29 and DLD1 cells were plated at a density of 5 × 105 cells in six well plates, grown for 24 h and total RNA from the cells was isolated. First-strand complementary DNA was prepared from 1 μg of total RNA. Real-time PCR was performed for CXCL10 and β-actin was used as an internal control. The bars indicate the mean of six individual experiments. Asterisk corresponds to p < 0.01
IL-1β, TNF-α and IFN-γ dose-dependent influence on CXCL10 gene expression in IECs
Caco-2, HT29 and DLD1 cells were tested for their ability to respond to pro-inflammatory cytokines by the induction of CXCL10 mRNA expression. Cells were stimulated for 4 h with various concentrations of cytokines, and CXCL10 expression was analysed by real-time PCR. In response to IFN-γ and TNF-α, Caco-2, HT29 and DLD1 cells showed a dose-dependent induction of CXCL10 mRNA (Fig. 2). Increased expression of CXCL10 mRNA was observed when 2 ng/ml IL-1β was used, while a higher concentration did not further enhance expression. Further, we checked the response of IECs to lower doses of IL-1β showing a dose response in the range of 0.1 to 1 ng/ml in the induction of CXCL10 mRNA. IL-1β at a concentration of 0.5 ng/ml was able to induce maximum expression of CXCL10 mRNA at 4 h (data not shown).
Fig. 2Dose-dependent expression of CXCL10 mRNA in IECs stimulated with cytokines. 5 × 105 cells were plated into six-well plates and grown for 24 h and then stimulated with IL-1β, TNF-α and IFN-γ at an increasing doses of 2, 10 and 50 ng/ml. The cells were harvested after 4 h, total RNA from the cells was isolated, and first-strand cDNA was prepared from 1 μg of total RNA. Real-time PCR was performed for CXCL10, and β-actin was used as an internal control. Graph represents the mean ± SEM of four individual experiments. All cytokine-stimulated expressions were significantly different from unstimulated controls (p < 0.05). Asterisk, no significant difference between different IL-1β concentrations (p < 0.05)
IL-1β, TNF-α and IFN-γ time-dependent influence on CXCL10 gene expression in IECs
Once we confirmed the responsiveness of IECs to pro-inflammatory cytokines, we next examined the mRNA kinetics of CXCL10 induced by the cytokines alone or in combination with each other. Each cell line had a different time course of response when stimulated with respective cytokines and their combinations (Fig. 3a–c). In Caco-2, stimulated with IL-1β or TNF-α, CXCL10 mRNA was induced within the first 40 min, and the induction reached to a peak after 4 h, while IFN-γ had a later initial effect inducing CXCL10 mRNA after 2 h and reached to a maximum at 8 h. Combination of IL-1β + IFN-γ had a stronger synergistic effect in the induction of CXCL10 mRNA within 20 min and reaching to maximum after 4 h, whereas TNF-α + IFN-γ synergistically led to a later induction of CXCL10 mRNA starting at 40 min but had a strong synergistic effect after 8 h when compared to IL-1β + IFN-γ (Fig. 3a). In HT29 (Fig. 3b) and DLD1 (Fig. 3c), CXCL10 mRNA time kinetics deviated from that in Caco-2 (Fig. 3a). CXCL10 mRNA was induced after 1 h of stimulation with the respective single cytokines in DLD1. In case of HT29, either TNF-α or IFN-γ alone led to delayed induction of CXCL10 mRNA after 1 h, whereas IL-1β alone led to an induction starting at 2 h. HT29 and DLD1 cells treated with combinations of IL-1β + IFN-γ showed a strong synergistic induction of CXCL10 mRNA within 40 min, whereas the TNF-α + IFN-γ combination led to an even stronger induction of CXCL10 mRNA within 20 min in HT29 and within 40 min in DLD1. IL-1β + TNFα in combination had no synergistic effect on CXCL10 mRNA induction in all the cell lines used for this study. The real-time PCR results could be confirmed by Northern blot analysis in Caco-2 and HT29 cells. However, the detection threshold was not low enough to display basal and single cytokine-induced CXCL10 mRNA (data not shown).
Fig. 3Time kinetics of CXCL10 mRNA expression in IECs stimulated with cytokines. a Caco-2, b HT29 and c DLD1 cells were plated at a density of 5 × 105 cells in six-well plates and grown for 24 h and then stimulated with respective cytokines and their combinations. Cells were harvested at different time points as indicated, and total RNA from the cells was isolated and first-strand cDNA was prepared from 1 μg of total RNA. Real-time PCR was performed for CXCL10, and β-actin was used as an internal control. Graphs represent the mean ± SEM of three to five experiments. IFN-γ led to a significant increase in CXCL10 expression from 2 h onwards. A significant increase (p < 0.05) from control was observed in all expect in those marked as N.S. (non-significant). Number sign indicates time points with significant differences (p < 0.05) of cytokine combinations or IL-1β compared to IFN-γ alone
IL-1β, TNF-α and IFN-γ time-dependent influence on CXCL10 protein secretion in IECs
To test whether cytokine stimulation of IECs would lead not only to the induction of CXCL10 mRNA but also to the synthesis and secretion of the protein, Caco-2, HT29 and DLD1 cells were incubated with respective cytokines (from 3 to 24 h), and secretion of CXCL10 was detected by ELISA. Under basal conditions, Caco-2 cells secreted 0.012 ± 0.003 ng/ml CXCL10. Treatment with IL-1β, TNF-α and IFN-γ led to a time-dependent increase in CXCL10 protein, with IL-1β being the most potent stimulus during the first 6 h (Fig. 4a). Of importance, after 12 h, CXCL10 secretion was stimulated strongly by IFN-γ. Co-incubation of Caco-2 with respective cytokines revealed a synergistic effect of IL-1β + IFN-γ and TNF-α + IFN-γ on CXCL10 secretion, reaching levels of 3.354 ± 0.690 and 7.808.1 ± 0.173 ng/ml after 24 h. The combination of IL-1β + TNF-α had no synergistic effect on CXCL10 secretion when compared to the respective cytokines alone. These time kinetic studies revealed that in Caco-2, IL-1β alone or in combination with IFN-γ induced the secretion of CXCL10 very early compared to the combination of TNF-α + IFN-γ, which induced a delayed but stronger synergistic secretion of CXCL10 protein, suggesting that in case of IL-1β + IFN-γ, a synergism could take place very early, e.g., at the level of intracellular signal cascades. In HT29 cultures, CXCL10 secretion was not detectable under basal condition. TNF-α or IFN-γ alone led to an induction of CXCL10 protein secretion within 3 h (0.0172 ± 0.005 and 0.011 ± 0.001 ng/ml), whereas IL-1β could not induce the secretion of CXCL10 protein. IL-1β, TNF-α and IFN-γ led to a time-dependent increase in CXCL10 concentrations in HT29 cell supernatants reaching maximum levels of 0.039 ± 0.0057, 0.632 ± 0.0222 and 19.929 ± 0.838 ng/ml showing that IFN-γ is the strongest inducer of CXCL10 protein secretion when compared to other two cytokines in this cell line (Fig. 4b). The combinations of TNFα + IFN-γ and IL-1β + IFN-γ induced CXCL10 protein secretion to 1.822 ± 0.109 and 0.410 ± 0.027 ng/ml within 3 h and led to a maximum of 492.728 ± 83.776 and 87.158 ± 7.913 ng/ml after 24 h (Fig. 4b).
Fig. 4Time kinetics of CXCL10 protein secretion in Caco-2 and HT29 cells stimulated with cytokines. 5 × 105 cells were plated into six-well plates and grown for 24 h and then stimulated with IL-1β, TNF-α and IFN-γ as indicated. Supernatants were collected at respective time points from a Caco-2 and b HT29 cells and ELISA for CXCL10 protein was performed. Data represent means of three to five experiments ± SEM done in duplicates for each sample. Asterisk indicates time points with significant differences (p < 0.05) of cytokine combinations compared to IFN-γ alone
CXCL10 promoter activation by cytokines
TGL-IP10, a promoter-reporter containing 435 bp of human CXCL10 sequence upstream of the transcriptional start site, was used to evaluate the role of IL-1β, TNF-α and IFN-γ alone or in combination with each other in the transcription of CXCL10. When TGL-IP10-transfected Caco-2 cells were stimulated with single cytokines, IL-1β (1 ng/ml) led to a stronger induction (6.53 ± 1.65-fold increase) than TNF-α (50 ng/ml) and IFN-γ (50 ng/ml) alone (2.76 ± 0.53 and 3.14 ± 1.2-fold increase). The combination of IL-1β + IFN-γ showed a stronger synergistic induction (26.18 ± 11.68) than the combination of TNF-α + IFN-γ (13.97 ± 3.25-fold increase) (Fig. 5a). In HT29 cells, when stimulated with single cytokines, IFN-γ (50 ng/ml) led to a maximum induction (6.52 ± 0.58-fold increase), followed by TNF-α (4.44 ± 0.16-fold increase) and IL-1β (1.43 ± 0.05-fold increase). The combination of IL-1β + IFN-γ synergistically led to 12.76 ± 3.21-fold induction, and TNF-α + IFN-γ led to 26.70 ± 7.5-fold induction of promoter activity (Fig. 5b). In DLD1 cells (Fig. 5c), IFN-γ (50 ng/ml) led to a maximum induction (8.06 ± 0.51-fold increase) of CXCL10 promoter activity followed by TNF-α (50 ng/ml) induction (3.59 ± 0.08-fold increase) and IL-1β (1 ng/ml) induction (2.32 ± 0.12-fold increase). The combination of IL-1β + IFN-γ led to a 40.72 ± 12.19-fold induction, and TNF-α + IFN-γ led to a 20.56 ± 6.27 fold induction. In all three cell lines, the combination of IL-1β + TNF-α had no synergistic effect on CXCL10 promoter activity.
Fig. 5Transient transfection analysis of TGL-IP10 in IECs. The CXCL10 promoter containing reporter construct, pTGL-IP10 and pRL-TK (renilla) plasmids, was transiently transfected into a Caco-2, b HT29 and c DLD1 cells. 24 h after transfection, cells were stimulated with cytokines as indicated for 24 h, harvested, and promoter activity was monitored by luciferase assay. Data were normalised with renilla luciferase values and included as fold increase vs control. Data represent the means ± SEM of three to five independent experiments done in duplicates for each sample. Asterisk indicates an induction significantly different (p < 0.05) from control (C). Significant differences between combinations of cytokines and the respective single cytokines are indicated with bars with a number sign (p < 0.05). N.S. is not significant
Evaluating the role of NF-κB in CXCL10 gene expression
To evaluate the possible role of NF-κB in cytokine-induced CXCL10 gene expression, experiments with a specific NF-κB inhibitor were performed. The effect of BAY 11-7082 on NF-κB inhibition was evaluated by reporter assays. BAY11-7082 was able to dose-dependently inhibit IL-1β-induced pNF-κB-SEAP gene reporter activity in Caco-2 cells (data not shown). Based on this experiment, we could determine that 10 μM of BAY11-7082 was sufficient to inhibit the NF-κB activation.
The induction of CXCL10 gene expression by cytokines was inhibited by addition of the NF-κB-specific inhibitor BAY11-7082 in Caco-2, HT29 and DLD1 cells, with a varying degree of inhibition in the individual cell lines. Inhibition of NF-κB led to almost complete inhibition (approximately 90–95% inhibition) of CXCL10 mRNA and protein secretion induced by cytokines in Caco-2 and DLD1 cells (Fig. 6a and c). In DLD1 cells, IL-1β (1 ng/ml) did not have any inducing effect on CXCL10 secretion. In HT29, inhibition of NF-κB had approximately 60–75% inhibitory effect on cytokine-induced CXCL10 mRNA expression and secretion (Fig. 6b).
Fig. 6Role of NF-κB in cytokine-induced CXCL10 mRNA expression by IECs. 5 × 105 cells were plated and grown for 24 h. Then, the cells were pre-treated with 10 μM of BAY 11-7082 for 1 h and then stimulated with respective cytokines and their combinations as stated in the figure for 2 and 24 h. After 2 h, cells were harvested for RNA isolation to perform real-time PCRs, and at 24 h, cell supernatants were collected and stored at −70°C until ELISA was performed for CXCL10. Data presented are the means ± SEM of three independent experiments done in duplicates for each sample in a Caco-2, b HT29 and c DLD1 cells. Significant CXCL10 inhibition by BAY 11-7082 is indicated by an asterisk (p <  0.05). N.S. is not significant
To further evaluate the role of NF-κB, transient transfections were done with a CXCL10 promoter construct bearing a mutated NFkB-binding site (TGL-IP10 κB2 Mut). In Caco-2 cells, TGL-IP10 κB2 Mut was not responsive to any of the cytokines and their combinations, suggesting that CXCL10 gene induction is strongly dependent on NF-κB in this cell line (Fig. 7a). In HT29 and DLD1 cells, the combination of TNF-α + IFN-γ led to 6.49 ± 2.13 and 4.87 ± 1.43-fold increase, and the combination of IL-1β + IFN-γ led to 2.26 ± 0.63 and 4.75 ± 0.53-fold increase of the TGL-IP10 κB2 Mut promoter activity (Fig. 7b and c), revealing that a smaller portion of CXCL10 gene expression might be regulated independently from NF-κB.
Fig. 7Transient transfection analysis of TGL-IP10 and TGL-IP10 κB2 mutant in IECs. TGL-IP-10 and TGL-IP10-κB2 Mut plasmid was transiently transfected into a Caco-2, b HT29 and c DLD1. 24 h after transfection, cells were stimulated with cytokines for 24 h, and promoter activity was monitored by luciferase assay. Data were normalised with renilla luciferase values. Data presented are the means ± SEM of three to five independent experiments done in duplicates for each sample. Significant differences vs controls (C) are indicated by an asterisk (p < 0.05). Significant differences of wild type vs mutant are indicated by a number sign (p < 0.05). N.S. is not significant
To confirm further our results from promoter and inhibitor studies, we did gel-shift and super shift assays to show that NF-κB is activated by cytokines and that the activated NF-κB complex contains the NF-κB functional sub-unit p65. In the nuclear extracts of Caco-2 cells, we could clearly see a shift in the samples treated with IL-1β, TNF-α alone or the combinations of IL-1β + IFN-γ and TNF-α + IFN-γ. The protein-DNA complex was efficiently competed by unlabelled κB oligonucleotides. There was no shift in the samples treated with the NF-κB inhibitor BAY11-7082, stating that the shift was indeed because of NF-κB binding. Furthermore, antibody super shift assay using anti-p65 antibody had clearly shown that the complex contains NF-κB functional sub-unit, p65 (Fig. 8).
Fig. 8Gel shift and super shift analysis for NF-κB. Caco-2 cells were seeded and grown for 24 h before pre-treating with BAY 11-7082 (10 μM) for 1 h and then stimulated with respective cytokines for 30 min; cells were then harvested, and nuclear extracts were prepared. 5 μg of nuclear extracts were used for gel shifts with a wild-type κB oligonucleotide. Super shifts were performed with anti-p65 antibody. Similar results were obtained in two separate experiments
Discussion
In addition to its many homeostatic functions such as barrier protection, mucus production and resorption, the intestinal epithelium plays an important role in regulating local inflammation and immune responses. The intestinal epithelium participates in inflammatory responses in part through the generation of numerous cytokines and chemokines that mediate recruitment and activation of inflammatory cells. The epithelium is involved in local cytokine networks allowing first response to noxious agents as well as cross-talk with immune cells to help provide an effective inflammatory response. These bi-directional inflammatory communications are necessary for normal host defence, but they also play an important role in the pathogenesis of IBD and might be also of importance in modulating anti-tumoural immunity in colonic malignancies. In this report, we demonstrate that pro-inflammatory cytokines, which may be produced by sub-mucosal immune cells, can differentially and co-ordinately regulate CXCL10 gene expression in IECs.
CXCL10 is one of several chemokines produced by IEC. Previous studies could show that IFN-γ in combination with TNF-α caused induction of RANTES (regulated upon activation, normal t-cell expressed, and secreted protein) and monocyte chemotactic protein-1 in colorectal tumour cell lines, while IFN-γ had no effect on CXCL8 (IL-8) secretion [27], the latter observation being confirmed in all three cell lines in the present study (data not shown).
Dwinell et al. [12] had previously shown that stimulation of IECs with TNF-α or IL-1α strongly potentiated IFN-γ-induced CXCL10. It is well known that among IL-1α and IL-1β, the latter is secreted and has an active role in inflammation. Importantly, IL-1β was shown to be over-expressed in IBD patients [25, 28]. This makes IL-1β an interesting cytokine to study its effect on CXCL10 gene regulation. Although the synergistic effects of TNF-α + IFN-γ on CXCL10 gene expression and the signalling mechanisms involved were studied in astrocytes [26] and their synergistic effect on inducible nitric oxide synthase expression in IECs and macrophages [29] had been studied in detail, the synergistic action of these cytokines on CXCL10 gene expression and the signalling mechanisms involved have not been evaluated in IECs. Especially, the role of IL-1β alone and its synergism with both TNF-α and IFN-γ on CXCL10 gene expression in IECs has been given no attention.
Previously, it has been shown that IECs may express elevated levels of CXCL10 [12], and its expression is increased in the mucosa of patients with IBD [16]. A critical role for CXCL10 in IBD has also been shown in animal models [30, 31]. Apart from this, CXCL10 has been shown to be expressed constitutively in colorectal carcinomas and primary metastatic sites exerting anti-tumour activity by recruiting mononuclear cells via the CXCR3 receptor. However, at the same time, it has also been shown that CXCL10 promoted metastatic adhesion and tumour cell migration [32] so that care should be taken considering CXCL10 as an anti-tumoural therapy option.
In light of these findings, we assessed whether stimulation of IECs in culture by inflammatory mediators could induce CXCL10 gene expression. All three IEC lines we used expressed CXCL10 mRNA in a concentration-dependent manner when stimulated with IFN-γ and TNF-α alone. These data support a vast body of evidence that demonstrates that IFN-γ can induce CXCL10 expression in a variety of cells types, including granulocytes, T lymphocytes, bronchial epithelial cells and keratinocytes [33, 34], while TNF-α is a potent inducer for CXCL10 in hepatocytes [17]. Importantly, in addition to these data, we could also show that Caco-2 cells stimulated with IL-1β up-regulate CXCL10 mRNA to a significant level, whereas in HT29 and DLD1 cells, induction of CXCL10 mRNA was less pronounced.
A number of authors have reported functional synergy between TNF-α and IFN-γ in promoting gene expression for proteins such as RANTES, IFN regulatory factor 1 (IRF-1), MIG and intercellular adhesion molecule-1[35–38] in a variety of cell types and have demonstrated that this synergy depends on the co-existence of TNF-α-responsive NF-κB-binding sites and IFN-γ-responsive signal transducers and activators of transcription (STAT) protein-binding elements within the promoters of the genes of interest [35, 36]. In our study, when IFN-γ and TNF-α were combined, the accumulation of CXCL10 mRNA and protein was elevated compared to unstimulated cells or cells stimulated with either cytokine alone, implying a synergistic effect on transcription of the CXCL10 gene. A synergistic effect by these two cytokines on CXCL10 expression was also demonstrated in astrocytes, and this synergy has been shown to be at least in part dependent on NF-κB activation and binding to a NF-κB-binding site within the CXCL10 promoter [26].
The synergistic action of IL-1β with IFN-γ in IECs has not been characterised so far. Recently, Takami et al. [39] showed the synergistic induction of hepatocyte growth factor in human skin fibroblasts by IL-1β + IFN-γ+ IFN-γ [39]. We here report for the first time that in intestinal epithelial cell lines, IL-1β alone or in combination with IFN-γ induced CXCL10 gene expression. In Caco-2 cells, the combination led to an early synergistic induction of both CXCL10 mRNA and protein compared to the combination of IFN-γ+TNF-α. This early induction of CXCL10 mRNA and protein by IL-1β+IFN-γ in Caco-2 might be due to the early convergence of signalling pathways that activates transcription factors like NF-κB, IRF-1, STAT1 or other factors that mediate transcription factor interactions.
Transient transfections with human CXCL10 promoter [26], in Caco-2, HT29 and DLD1 cells, showed that the synergistic effect of IL-1β + IFN-γ and IFN-γ + TNF-α on CXCL10 gene expression in these cell lines was indeed because of the transcriptional synergy employed at the level of the CXCL10 promoter. A number of studies had revealed that cytokines like IL-1β, TNF-α or enteroinvasive bacteria and the bacterial cell wall product lipopolysaccharide induce the expression of several inflammatory genes through the transcription factor NF-κB in IECs [40–43]. To find out the signalling mechanism involved in CXCL10 gene expression in IECs, we explored the role of transcription factor NF-κB. Inhibition of NF-κB with BAY11-7082 clearly inhibited both CXCL10 mRNA and protein induced by single cytokine alone or in combinations in Caco-2, HT29 and DLD1 cells. Gel shift in Caco-2 could confirm that NF-κB is activated and binds to consensus κB oligonucleotides in cells treated with either IL-1β or TNF-α alone and in combination with IFN-γ. It was shown previously that the binding complex of NF-κB to the κB2-binding site of the CXCL10 promoter contains the homodimer of p65 [26]. In our study, super shift assays in Caco-2 cells treated with IL-1β alone or in combination with IFN-γ confirmed the presence of p65 in the complexes bound to the consensus κB oligo nucleotides. In addition to these experiments, transient transfection experiments with a κB2 mutant of the CXCL10 promoter [26] clearly showed that in Caco-2 cells, none of the cytokines and their combinations were able to induce the CXCL10 promoter activity. In HT29 cells, the combination of IFN-γ + TNF-α led to a weaker induction of TGL-IP10κB-2 mutant activity, whereas in DLD1cells, the combination of IL-1β + IFN-γ and IFN-γ + TNF-α led to a weak induction of TGL-IP10 κB-2 mutant activity. Based on these data, we can conclude that NF-κB plays a critical role in CXCL10 gene expression induced by cytokines in IECs, and this is mainly dependent on the κB-2 site present in its promoter. Moreover, IFN-γ-induced CXCL10 mRNA and protein secretion was inhibited upon NF-kB inhibition.
Although the present results may reflect only a small aspect in the overall concert of chemokines, it seems apparent that the basic regulatory principles elucidated here might be shared also by a number of other chemokine genes like that of CCL19, which has recently been shown to be regulated by multiple NF-κB and IRF family transcription factors in human monocyte-derived dendritic cells [44]. Recently, Hiroi M et.al. [45] showed that co-operation between IFN-γ induced STAT1, and the constitutively active or inducible NF-κB is necessary for the transcriptional activity of IFN-γ-inducible genes like MIG and CXCL10. Therefore, we suggest a similar mechanism for IFN-γ-induced CXCL10 in IECs.
The responsiveness to cytokines might depend on the state of differentiation. Schlottmann et. al. [46] recently showed that in contrast to HT29 where IFNγ up-regulated CXCL8 secretion, the same was unaffected in Caco-2 cells or even down-regulated in primary colonic epithelial cells. Future studies on chemokine regulation should therefore include primary cells, keeping well in mind that the presence of contaminating immune cells, culture conditions and cell stress during isolation may influence results.
Taken together, our data suggest that while IFN-γ, TNF-α and IL-1β may work individually through disparate signalling pathways in CXCL10 gene regulation, a combination of these cytokines enhances transcription of the CXCL10 gene in an NF-κB-dependent manner. The fact that IL-1β via activation of NF-κB is able to mimic the effect of TNF-α in augmenting CXCL10 expression might be of importance in patients with IBD, who do not respond to anti TNF-α antibody therapy. In those cases, an additional inhibition of IL-1β might be desirable as a future therapy option in treating IBD.

intestinal epithelial cells
inflammatory bowel disease
cytokines and chemokines
nuclear factor kappa b
colon cancer


The prescribing of prisms in clinical practice

The use of prisms in cases of decompensated heterophoria is an established treatment modality. The clinical literature lacks consensus upon the appropriate use of prisms, and fails to provide the necessary evidence base. While the experimental literature can guide the practitioner, the lack of double-blind, placebo-controlled clinical studies needs to be addressed.
The use of prisms in cases of binocular dysfunction is an important treatment modality for dealing with such patients [1]. The decision to prescribe a prism, and what value of prism to give, is subject to varying clinical opinions and practices [2]. The conservative view in clinical practice is that prisms should not be prescribed in the absence of symptoms of binocular dysfunction [1]. Furthermore, the prescribing of prisms is contraindicated in certain cases, due to the risk of exacerbating an existing condition through the process of vergence adaptation [3–6].
The control of the vergence response has been described previously using linear systems modelling [7], and can aid our understanding of the aetiology of binocular visual dysfunction, and the effect of clinical intervention. The normal vergence response operates in a closed-loop feedback system, where the total response is the sum of the outputs of two control elements with differing temporal properties; a fast (phasic) control element and a slow (tonic) adaptive control element [7].
Previous reports have suggested that the strength of the adaptive vergence control element is often related to the presence of symptoms in patients with binocular dysfunction, and in severe cases the output of the adaptive vergence controller may be reduced to almost zero [8, 9]. In less severe cases, vergence adaptation may be present at a sub-normal level, but sufficient to produce an adaptive response to the introduction of a prism [8]. In such patients, strengthening the adaptive vergence control element is the treatment of first choice [8, 9]. Other studies have shown that in elderly patients vergence adaptation is very limited, and that while this may often be the cause of binocular dysfunction in these patients, remedial action with prisms is clinically viable because adaptation is very unlikely [10]. Indeed, we observe in our own binocular vision clinic that treatments designed to improve the strength of the adaptive vergence controller are generally unsuccessful in elderly patients.
Typical clinical measures of binocular function, three of which are investigated by Otto et al. [11] in the current issue, are:
Heterophoria (or dissociated phoria).This represents the fusion-free position of the eyes, and therefore the magnitude of the deviation which has to be overcome by the vergence system.Fixation disparity.First investigated by Ogle [12], this represents a small misalignment of the visual axes during binocular viewing, normally measured in seconds of arc. Many clinicians take the view that fixation disparity is indicative of stress within the binocular system [1]. Other authors regard fixation disparity to be a purposeful error, necessary for the vergence control system [5].Associated heterophoriaMallett [13, 14] is the author associated with describing the clinical characteristics of this measurement, which measures the amount of prism required to reduce any fixation disparity (in 2 above) present to zero. In Mallett’s opinion, the presence of an associated phoria in a patient with symptoms of binocular vision dysfunction is indicative of stress within the binocular system, and requires treatment [15].Fusional vergence reserves.This is a clinical measure of the overall ability of the vergence system to control heterophoria. It is generally used in the calculation of Sheard’s Criterion [16], which requires the vergence reserves to be at least twice the size of any heterophoria present. There is evidence that the vergence reserves are related to the strength of the adaptive component of the vergence system [8, 9].
Each of these measures assess one specific aspect of binocular function, and while some practitioners prefer to rely on a particular measure to assess binocular function, others may assess a combination of the measures above to decide the most appropriate clinical intervention for the patient [2].
There is a lack of consensus in the literature about which of these measures correlates most closely with symptoms of binocular dysfunction, and which provides the most accurate basis for prescribing a prism when this is appropriate. Previous work has shown that no single measure correlates well with patient symptoms in all types of binocular anomaly, suggesting that neither fixation disparity, nor associated phoria, provide a universal indicator of binocular dysfunction [17]. This work also found that measures of vergence reserves often showed a better correlation with symptoms [17].
The imperative question for the clinician remains; is there a reliable method to determine the magnitude of prism required to compensate the heterophoria in patients suitable for this method of treatment? Mallett [15] suggested that the associated heterophoria identified the uncompensated portion of the binocular anomaly, and therefore this value represented the prism required to give the patient binocular comfort and stability. Other studies have questioned this [5, 17], and indeed the presence of an associated phoria in patients without binocular visual problems casts doubt on the general applicability of this measure [18]. Sheard’s criterion provides a rationale for prescribing prisms [1, 18]; however, there is still a lack of randomised controlled clinical trials to prove the efficacy of any treatment of binocular vision [2], although a recent study has begun to address this [19]. The use of a measure such as the “comfortable prism”, while clinically appealing, is highly subjective in nature, and would also require a clinical evidence base to prove efficacy. There is considerable work still to be done in the field of clinical management of binocular vision dysfunction in order to provide the required evidence base.
In all cases of binocular dysfunction, the choice to prescribe prisms lies with the clinician, who must judge what is most appropriate for each individual patient. A relatively straightforward procedure has been described to assess the probability of vergence adaptation occurring and rendering any prescribed prism ineffective [20]. Where prism treatment is being considered, obtain a prior measurement of the associated phoria, insert the prism to be prescribed into the trial frame and measure the associated phoria with the prism in situ. Allow the patient to wear the prism for 10 minutes; if the associated phoria returns to the value measured prior to insertion of the prism, then the patient’s vergence adaptation mechanism has been strong enough to overcome the prism, and it will have little clinical benefit. Conversely, if no vergence adaptation is observed, the patient is likely to derive significant clinical benefit from this treatment.


Actin: its cumbersome pilgrimage through cellular compartments

In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days’ knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin.
Introduction
A common incident, ubiquitous and frequently observed by the experienced teacher: a student wants to purify protein X from an eukaryotic organism Y. Sooner or later the undergraduate stares at SDS polyacrylamide gels and is surprised, disappointed, depressed, furious about those horrible actin contaminations that seem to be everywhere. The student does not care about the advisor’s soothing explanations: “actin is the most abundant protein”, “actin exists as globular monomer (G-actin) in the soluble supernatant”, “but it is also sedimentable as filamentous actin (F-actin)”. It will be difficult to calm the young scientist down. Looking at these copious amounts of the protein, the student cannot believethat a long time ago it was a scientific breakthrough to show that actin exists at all,that it took years and years to convince truculent groups of actin pioneers that a non-muscle cell contains actin as well,that actin is not a singular and once for all invention in evolution, but is the founder of a large family of isoforms and related proteins, andthat quite a number of these proteins can be found in different subcellular compartments. For example, it has been established as a regular component of both, the cytoplasm and the nucleus; but this perception is the result of a fight that went on for decades.
The dawn: actin as a muscle component
The first traceable report that described contractile substances in muscle goes back to 1859 and Wilhelm Friedrich Kühne (Kuehne 1859). He belonged to the best known physiologists of his time and coined modern designations like “enzyme” or “myosin”. The latter one he described as a substance that could be isolated from frog muscle and forms a contractile clot under certain conditions. Today we would call this clot a contractile pellet, consisting mainly of actomyosin. Halliburton followed shortly afterwards and was intrigued by this clot formation (Halliburton 1887). He compared it to blood clotting and the transformation of fibrinogen to fibrin. In tedious experiments he extracted mammalian muscle tissue at different time scales, temperatures, salt concentrations and found that myosin apparently needed an additional component to perform fast aggregation and contraction. Without really knowing it, Halliburton worked with actin and produced with this so-called “myosin ferment” an actomyosin precipitate. In our days one reads Halliburton’s publication with some amazement. The report covers 70 pages and looks with its descriptions and tables like a lab-book, e.g.: “Extract #1—10 a.m.—Diluted with an equal amount of water—10 a.m. next day—No change”. Surely, at that time editors were not as busy as they are today.
Muscle actin comes of age
Then, it took almost 60 years until Brúnó F. Straub in Albert Szent-György’s laboratory at the Hungarian University of Szeged was able to purify actin in good qualities and quantities. At that time ATP was known and available, i.e. Straub could study the influence of ATP on the viscosity of myosin preparations in the presence and absence of other extractable muscle components. The decrease in viscosity upon addition of ATP was considered “activation” of myosin, the protein responsible for this activation was named “actin”, and the complex of these two proteins was “actomyosin”. Straub and Szent-Györgyi discovered these interrelationships 1941/1942 in the middle of World War II and, therefore, published the data only in local periodicals (“Studies from the Institute of Medical Chemistry”, University of Szeged). The research on actin and myosin, the political situation and the excitement in Szent-Györgi’s group in the early 1940s are nicely summarized by W.·F. Mommaerts who was a witness of the original experiments (Mommaerts 1992), and by A.G. Szent-Györgyi himself (Szent-Gyorgyi 2004).
It is far beyond the scope of this review to cover the avalanche of actin and myosin research that started after those first molecular characterizations. Of course, muscle was and still is the major source to purify actin from, and, consequently, the research during the following decades used exclusively muscle actin.
Actin conquers the cytoplasm
But as it is in science, one opens one door and discovers ten closed ones. Keeping that in mind a scientist should always be suspicious regarding dogmas of all sorts. For some time it was almost a law that actin exists only in skeletal muscle and that the well-known conventional myosin is the only motor protein which turns the world go round. Today it is a common place that both assumptions were wrong, although it was quite a task to convince the scientific community.
Many minor publications pointed at the presence of actin in non-muscle cells. For example, one found actomyosin-like components and Mg2+ATP-dependent viscosity changes in extracts from sea urchin eggs (Ohnishi 1962), and an actin-like protein from calf thymus nuclei (Ohnishi et al. 1963), and it was assumed that the filaments seen in fibroblasts are analogous to filaments in muscle, responsible for cell motility (Buckley and Porter 1967). Most of these publications never had a really strong impact, especially since they argued against a rock-solid preconception. But to be fair: these tiny reports were the pebbles that paved the road for better equipped groups and more detailed molecular studies. Notably, it was a slime mould, Physarum polycephalum that was used as a model organism to prove the existence of actin and myosin in non-muscle cells. The key experiments have been performed in Japan, in Sadashi Hatano’s group (Hatano and Oosawa 1966a, b; Hatano and Tazawa 1968) and the data were confirmed shortly afterwards by other laboratories. Especially the decoration of actin filaments in non-muscle cells with myosin was a breakthrough technique that unequivocally proved the existence of the actomyosin system in many non-muscle cells (for reviews see: Huxley 1973; Pollard and Weihing 1974; Pollard 1981; Tilney 1975).
Actin as the prototype of a large family
Today conventional actin is thoroughly studied, with respect to its polymerization and depolymerization equilibrium, its function in the cytoskeleton as a morphological stabilizer, its role during motile activities of a cell, its three-dimensional structure, its binding partners, etc. But the traitorous word in this sentence is “conventional”. What hides behind “conventional” actin? And what is an actin-related protein?
A “conventional” actin consists of 375 highly conserved amino acids, give or take a few. The polypeptide sequence is folded into a roughly U-shaped molecule, exposing well-characterized binding sites for many different ligands. There are several isoforms as products of different genes, coined alpha, beta and gamma actin, all of which are polymorphic proteins capable to form polymers. “Conventional actins” display a particular fold which enables them to interact with a variety of partners, to form specific suprastructures involved in intracellular motility, adhesion and locomotion (see below). Figure 1 shows the dynamics of the actin cortex in a Dictyostelium discoideum amoeba during random migration, pinocytosis, and phagocytosis of a yeast cell. An actin-related protein harbours an actin domain, but it differs from “conventional” actin in size and overall amino acid sequence, usually cannot form polymers and may differ from the conventional form in subcellular distribution and its physiological activity. The increasing number of completely sequenced genomes is an enormous help towards our understanding of actin and actin-related proteins. One can take any sequenced genome to analyze actin as a founder of a large protein family. We chose the recently unravelled D. discoideum genome since it represents the genome of a model organism that can locomote as a single cell and in cell assemblies, and can differentiate into simple tissues in a developmental cycle. Hence, it is a social amoeba at the evolutionary border between unicellular and multicellular organisms (Eichinger et al. 2005). Notably, the number of actin genes does not tell us very much about the complexity of an organism. Whereas the genome of budding yeast contains only one single and, not surprisingly, essential gene that codes for a conventional actin, mouse harbours 35, and the plant Arabidopsis thaliana 10 actin genes. The D. discoideum genome contains 33 genes that code for a bona fide conventional actin. Most puzzling, 17 of these actins share identical amino acid sequences, but are encoded by 17 distinct genes, and many of these are developmentally regulated. Why would evolution allow this seemingly luxurious feature? Elimination of redundant genes can only be avoided if they represent a selective advantage. If the identical actins are translated in a developmental pattern, then posttranslational modifications might play a much larger role than we are aware of today. Indeed, actin’s posttranslational modifications are numerous. Actins are acetylated, acylated, arginylated, Ser/Thr/Tyr-phosphorylated, ubiquitinylated etc. This suggests that a developmentally regulated expression of actin genes requires a similarly regulated expression of enzymes that catalyze posttranslational modifications. Such a hypothesis opens a completely new search for our understanding of actin and its isoforms.Fig. 1Dynamics of the actin cortex during amoeboid movement, pino- and phagocytosis. D. discoideum was transformed with a truncated LimE construct, tagged to green fluorescent protein (GFP). LimE binds specifically to filamentous actin (Bretschneider et al. 2004). The transformed cells are depicted in phase contrast (left panels) and fluorescence (right), which reveals the concentration of actin filaments by GFP-LimE. Upper panels: optical section through the actin cortex during random amoeboid movement. Bottom panels: during uptake of a rhodamine-labelled yeast cell the actin cortex forms a phagocytic cup around the particle. The engulfment of liquids by macropinocytosis requires the actin system as well (arrow). Size bar 5 μm
The picture became even more complicated after the discovery of the actin-related proteins (Arps). Calculation of the putative structures shows very nicely that actin and Arps share a common three-dimensional fold (Fig. 2, taken from Muller et al. 2005). Most characteristic is the nucleotide-binding cleft which divides the protein in roughly two halves. The depicted structures all show the same orientation. For actin itself, this means that, when incorporated into a “conventional” actin filament, the bottom part of the folded monomer points towards the fast growing (“barbed”) end, the upper part towards the slow growing (“pointed”) end (see also Fig. 3). Figure 2 shows strikingly (1) that all Arps can be moulded into a structure quite similar to the overall structure of conventional actin and (2) that they display a clearly distinct pattern of patches with conserved amino acids. As seen in the comparative scale (Fig. 2, right) Arps 1, 2 and 3 are most closely related to actin, whereas all the other Arps show a much lower percentage of identity. Arps 2 and 3 have been shown to form a tight complex that, after activation through several actin binding proteins and ATP, binds to actin filaments and induces their branching (Kiselar et al. 2007). However, it should be noted that the role of the Arp2/3 complex for branching of actin filaments in vivo is still heavily challenged (Koestler et al. 2008). It is quite amusing to read Vic Small’s statement in the Research Roundup of J Cell Biol: (Feb 25, 2008): “We haven’t disproven branching yet. We’ll need 3D imaging to put the nail in that coffin. But what we’ve seen makes the branching model unlikely”. Undoubtedly, we are still on thin ice with our knowledge on Arp2/3 function in vivo.Fig. 2Structural conservation in the ARP subfamily (adapted from Muller et al. 2005, with permission). The conservation scale weights identity from 0 (blue) to 100% (red). All Arps can adopt a global actin conformation (upper left) but only Arps 1, 2 and 3 show a pronounced sequence homologyFig. 3Generation of different actin polymers as a result of different subunit interactions (adapted from Jockusch et al. 2006, with permission). The subdomains of the actin monomer, as deduced from the crystal structure, are numbered 1–4. In the process that leads to the “conventional” actin filament (F-actin), three such monomers (a–c) form intermolecular contact sites involving the subdomains as indicated left of the trimeric seed. Under physiological conditions, these trimers convert subsequently into the polar F-actin filaments that are depicted in the electron micrograph (top right). In a more rapid process, the actin monomers may form an antiparallel dimer which, at least in vitro, can form bipolar filaments (LD filaments) that show a strikingly different appearance in the electron microscope (bottom right)
Let us risk some sort of a heretical view. Trivial as it may be, one has to repeat it again and again: not the three-dimensional fold, but the surface of a protein determines its function. There are curious examples for this. Hisactophilin, a histidine-rich actin-binding protein from D. discoideum, has a structure nearly identical to that of interleukin-1β and fibroblast growth factor, despite its unrelated amino acid sequence. Luckily for the authors, these data were published in a high ranking journal (Habazettl et al. 1992), although there was no functional similarity between these three proteins whatsoever. Today, their structural similarity is not considered important at all and, like many others, they are just members of the large trefoil protein superfamily (Liu et al. 2002). Sippl and coworkers systematically analyzed novel proteins based on known structures (Koppensteiner et al. 2000). They came to the conclusion that almost 30% of the studied proteins have similar structures but different functions.
Coming back to actins and Arps. Is it possible that the name “actin” for all of these proteins is utterly misleading? Are we wasting time in our search for cytoskeleton-related activities just because the fold triggered a wrong name? Is the so-called actin fold another example for a successful superfamily which contains only a few proteins that play a role in the cytoskeleton, while many other members are e.g. subunits of larger protein complexes in chromatin or hexokinases or proteins with yet unknown functions?
Actin-like proteins in the nucleus: the early period
The first reports on actin as a nuclear protein appeared already 5 years after its acceptance as a ubiquitous cytoplasmic component. Nuclear filament bundles resembling microfilaments were observed in oocytes when transcription was inhibited by actinomycin D (Lane 1969), and biochemical analyses identified a prominent protein present in isolated nuclei of the multinucleated plasmodia of Physarum polycephalum (Jockusch et al. 1971; Jockusch et al. 1974; Lestourgeon et al. 1975). The concentration of actin in the nucleus fluctuated with the cell cycle phase of the plasmodia, leading to suggestions that nuclear actin might either have a role in the constriction of the nuclear membrane in Physarum plasmodia that exhibit an intranuclear mitosis (Jockusch et al. 1971), or with changes in transcriptional activity during the cell cycle (Lestourgeon et al. 1975), or during transition from active growth to the formation of sklerotia (spherules) from plasmodia after starvation (McAlister et al. 1977). All this remained speculation at that time, and the majority of the cell biological community dismissed these findings as an artifact, caused by a contamination of nuclei with cytoplasmic actin. However, between 1979 and 1984 three reports appeared which supported the concept of actin being involved in transcription: (1) actin was identified in a complex of RNA polymerase II from Physarum (Smith et al. 1979), (2) it stimulated markedly the transcriptional activity of RNA polymerase II purified from HeLa cell extracts at the preinitiation phase (Egly et al. 1984), and (3) antibodies specific for actin, when injected into the large amphibian oocyte nuclei, caused a dramatic collapse of lampbrush chromosome loops, concommitant with a complete stop of mRNA transcription (Scheer et al. 1984). However, these observations still did not overcome the scepticism at that time—it took another two decades to recognize actin as a dual compartment protein that can execute different functions by selectively associating with ligands specific either for the cytoplasm or the nucleus.
Actin and Arps in the nucleus
In the last 20 years, studies on nuclear actin became fashionable, and now a new area began which firmly established actin, its relatives and binding partners as nuclear components.
Several important steps served as signposts in this process:Cell biologists began to understand that proteins are not necessarily confined to a single intracellular compartment. Numerous examples proved that proteins may shuttle between intracellular regions and even adopt different, compartment-specific activities. Shuttling of actin and actin-related proteins between the nucleus and the cytoplasm cannot be considered any longer as an exceptional, exotic behaviour.Genetic studies, for example performed with yeast or Drosophila, showed that activities specific for the nucleus, like chromatin remodelling or transcription required the presence of the beta actin isoform, in a configuration that could bind to nuclear protein complexes, but were inhibited with actin mutants that failed in this respect.The development of cell biological and immunological methods over the past 20 years, like cellular expression of flurorochrome-tagged proteins in conjunction with video microscopy, and the generation of epitope-characterized monoclonal antibodies allowed to follow actin trafficking throughout the cell and its location in the nucleus.And finally, some old observations made in structural biology came back into people’s mind: actin is a highly polymorphic protein, which can give rise to several different and distinct polymers (Aebi et al. 1981; Millonig et al. 1988). Thus, while there are functionally quite unrelated proteins that may share the same structure (see above), it is also conceivable that a particular protein may adopt slightly different “unconventional forms”, possibly induced by specific binding partners. Such “distortions” may then trigger the formation of specific oligomers or polymers and complex formation with still other partners. Thus, intranuclear actins need not necessarily adopt the same structures found so abundantly in the cytoplasm, like G- or F-actin. This topic is discussed in detail in (Pederson and Aebi 2002), and this concept is supported by monoclonal anti-actins that specifically decorate nuclear actin (Gonsior et al. 1999; Schoenenberger et al. 2005). In Fig. 3 (taken from Jockusch et al. 2006), we show actin in two different forms that both can form polymers. The conventional actin filament requires the interaction of three actin monomers in a specific orientation. Such a trimeric seed will then grow into the polarized F-actin filaments (Fig. 3, top). However, there is also evidence for another pathway: two actin monomers can bind to each other in another orientation, and this “lower dimer” may then lead to nonpolar, quite different filaments (Fig. 3, bottom). It is of course tempting to speculate that such “unconventional” actin polymers may play a role in vivo, for example in the nucleus, but so far, there is no solid evidence for this assumption.
Not surprisingly, the wealth of data on nuclear actin and its relatives stimulated numerous hypotheses on their function. The relevant findings and the conclusions on putative functions are the topic of a number of interesting reviews, and in this article, we can only refer the reader to them and to the list of original references covered there (Bettinger et al. 2004; Blessing et al. 2004; Franke 2004; Jockusch et al. 2006; Pederson and Aebi 2002; Rando et al. 2000). The brave reader who works himself through all this information will arrive at two conclusions: the actin-like proteins have definitely conquered a firm position among the group of respectable, important nuclear proteins, and they seem to be involved in a plethora of different activities. While the tasks for actin in the cytoplasm all are connected with the conventional G- and F-actin structures and the dynamic equilibrium between these two states, nuclear functions of these proteins seem associated with several different forms or configurations which are ill or even not at all defined.
In the following paragraphs, we will briefly mention the most important tasks proposed for nuclear actins.
Actin filaments and a caryoskeleton
It is generally accepted that nuclear activities require a precise topographical arrangement of chromatin, to spatially separate hetero- from euchromatin and allow for the complex mechanism of chromatin remodelling during gene activation and transcription. Hence, when electron microscopy provided elegant images of networks of intranuclear filaments, preferably in large objects like the amphibian oocyte nuclei (germinal vesicles), it seemed quite plausible to conclude that a “nuclear matrix” or scaffold is composed of F-actin filaments (Clark and Merriam 1977; Clark and Rosenbaum 1979; Gard 1999) that could provide mechanical stability to the nucleus and serve as a platform to anchor, at least temporarily, chromatin constituents. Indeed, there is evidence from both earlier and more recent investigations that actin associates with filamentous structures: actin antibodies and myosin subfragment 1 decorate such filaments in amphibian oocyte nuclei (Clark and Rosenbaum 1979; Gard 1999; Scheer et al. 1984), and fluorescently labelled actin is found in a polymeric form in Xenopus and HeLa cell nuclei (Kiseleva et al. 2004; McDonald et al. 2006). Yet, in general, these filaments do not stain with fluorescent phalloidin, a small drug binding with high affinity to actin filaments of the conventional “F-actin” type. It is only after subjecting cells to stress or fixation protocols that nuclear filaments bind phalloidin, thus, the suspicion arises that such treatments rearrange actin into conventional filaments (Gall 2006; Jockusch et al. 2006). This, of course, does not preclude the concept that actin participates in a nuclear matrix, especially in the very large, actin-rich nuclei of amphibian oocytes. There is good evidence that it engages in providing mechanical support to this cellular compartment (Bohnsack et al. 2006; Stuven et al. 2003).
The nuclear Arps from yeast, Arp 7 and Arp 9, have been shown to build heterodimers and have the potential to form filaments from tetramers (Szerlong et al. 2003). However, it is not clear whether they do in vivo, and whether they might coassemble with nuclear actin in polymers (Blessing et al. 2004).
Actin/Arps and their connection with the nuclear envelope
Several structural proteins that are essential for the assembly and maintenance of an intact, functional nuclear envelope and its underlying lamina, display well defined binding motifs for actin. These include spectrin, protein 4.1, the nesprins, lamin A and emerin, (Krauss et al. 2003; Shumaker et al. 2003, more references in Blessing et al. 2004; Rando et al. 2000). Hence, it has been speculated, but not proven, that at least part of the nuclear actin is involved in the structural organization of the nuclear envelope.
Actin/Arps and the nucleo-cytoplasmic traffic
Actin-containing, intranuclear filaments were seen to connect to nuclear pore complexes in the amphibian oocyte nucleus (Hofmann et al. 2001; Kiseleva et al. 2004). In the Xenopus oocyte, but also in dipteres and mammalian cells, actin is engaged in the transport and nucleocytoplasmic export of mRNA (Hofmann et al. 2001) and RNA-protein complexes, in particular in those containing a subset of hnRNPs (Percipalle et al. 2001, 2002, reviewed in Bettinger et al. 2004; Pederson and Aebi 2005).
Actin/Arps and chromatin remodelling
Actin and nuclear Arps were both reported as being critically involved in chromatin remodelling (reviewed in (Bettinger et al. 2004; Blessing et al. 2004; Olave et al. 2002; Percipalle and Visa 2006; Rando et al. 2000). Chromatin remodelling complexes operate as large, multiunit machines in mammals, insects, yeast and plants to reorganize the genetic material by unravelling nucleosomes and converting the genetic material into a form suitable for transcription. Many of these steps require energy, and thus many of these protein complexes contain ATPases, comprised of several subunits, that can be grouped into subfamilies. Actin was identified in complex with specific subunits of most ATPases, together with four nuclear Arps (4, 5, 6 and 8) in all organisms, with the exception of yeast. Here, the two yeast-specific nuclear Arps (7 and 9) are found in the corresponding ATPases. One of the histone acetyl transferases that also contribute to chromatin remodelling and subsequent transcription (NuA4, Doyon et al. 2004) is highly conserved from yeast to man and is also found in a complex with actin and Arp 4 (Harata et al. 2002). Structural organization and function of the actin and Arp molecules in these ATPase and acetyl transferase complexes are largely unknown, and speculations for their respective roles range from stabilizing the enzymatic and a chaperoning activity to connecting these gigantic protein complexes to nuclear filamentous scaffolds.
Actin/Arps and transcription
Actin has been identified as a regular component of all the three nuclear RNA polymerases (Pol I, II and III), apparently interacting with two subunits that are shared among all three enzyme complexes. In vitro transcription by all three enzymes is actin-dependent (reviewed in Grummt 2006; Percipalle and Visa 2006). In the nucleus, pre-mRNA is complexed with ribonucleoproteins to form hnRNPs, and, presumably, there is co-translational recruitment of beta actin to these particles during their formation, as deduced from findings that several hnRNP components bind directly actin. Actin’s association with the RNA polymerases may precede, be simultaneous with or follow the initiation of actin-dependent chromatin remodelling by ATP-dependent complexes and/or histone acetylation, as described above (Grummt 2006; Percipalle and Visa 2006). Most of the data available today are derived from studies with Pol II, in insects and vertebrates, supporting and confirming the conclusions drawn more than two decades earlier from work on Xenopus oocyte and HeLa cell nuclei (Egly et al. 1984; Scheer et al. 1984). There is solid evidence that actin’s role in Pol II-dependent transcription is executed by the beta actin isoform (Hofmann et al. 2004; McDonald et al. 2006), and no other isoform seems involved. While actin’s contribution to the formation of pre-initiation complexes and subsequent transport of pre-mRNPs through the nucleus towards their export through nuclear pores is well accepted today (Bettinger et al. 2004; Franke 2004), it is completely unknown whether actin might mediate such intranuclear movements in filamentous or even contractile structures (Pederson and Aebi 2005).
Actin ligands in the nucleus
The nuclear constituents harbouring actin, like those of the nuclear matrix, the nuclear envelope, the nuclear pores, chromatin remodelling complexes, RNA polymerases and pre-mRNPs are all highly insoluble, gigantic structures. However, there are also actin ligands that are found in the cytoplasmic as well in the nuclear compartment, possibly in rather dynamic and temporary complexes with actin. Since the discovery of actin as a prominent component in the cytoplasm of practically all eukaryotic cells, a plethora of such actin-binding proteins has been described and characterized. They arrange actin into the various suprastructures required for cytoplasmic functions, such as intracellular motility and locomotion, by either regulating the balance between monomeric and filamentous actin, or generating networks and bundles of filaments (Winder and Ayscough 2005). Remarkably, many of these actin ligands are evolutionary well conserved, like actin itself (Korn 1982; Schleicher et al. 1988). Members of the various subfamilies, with affinity to either monomeric or filamentous actin, are dual compartment proteins that can shuttle between the nucleus and the cytoplasm, their main location frequently being dependent on the differentiation state of cells. Comprehensive lists of these proteins that have previously been described as cytoplasmic actin ligands but were also identified in the nucleus are given in (Rando et al. 2000; Bettinger et al. 2004; Pederson and Aebi 2005). Among them are several which form complexes with actin and interfere with the formation of conventional actin filaments, like profilin, binding to monomeric actin, and cofilin, a ligand for both, monomeric and polymeric actin. Nuclear profilin is apparently involved in the regulation of the level of nuclear actin, as profilin–actin complexes are recognized and exported from mammalian nuclei by a specific exportin (Stuven et al. 2003), while actin free of profilin can apparently be exported by a different exportin, due to its nuclear export sequences (Wada et al. 1998). Profilin and cofilin both can sligthly change the fold of actin. Profilin induces a form which faciliates the exchange of bound ATP in G-actin (reviewed in Jockusch et al. 2007). Cofilin, a phosphoprotein, contains a nuclear location sequence, and in its dephosphorylated state can transport actin “piggy-back” into the nucleus (Pendleton et al. 2003). When bound to actin polymers, it distorts their conformation such that these filaments do not bind phalloidin anymore. Thus, these proteins might be critical in creating forms specific for nuclear actin, as detected by specific antibodies (Gonsior et al. 1999; Jockusch et al. 2006; Schoenenberger et al. 2005). Among the actin ligands solely found in the nuclear compartment are a nuclear actin binding protein identified in Acanthamoeba (Rimm and Pollard 1989), an actin binding protein composed of two different subunits in mammalian cells (Ankenbauer et al. 1989), and a nucleus-specific form of myosin I (Pestic-Dragovich et al. 2000). The latter is a small, monomeric myosin that does not form filaments as would be needed for contraction of putative actin filaments in the nucleus. It is apparently involved in transcription of ribosomal genes by Pol I, where it binds to a transcription initiation factor but also to components of a chromatin remodelling complex. It has been speculated that in this location it may recruit actin associated with Pol I to the site of rRNA transcription, but so far, there is no evidence that these two proteins interact directly in vivo (reviewed in (Grummt 2006; Percipalle and Farrants 2006).
Conclusions
So, have we now lost the student being confronted with actin for the first time in his scientific education, or have we been able to attract his curiosity for this abundant, highly conserved, mysterious protein?
Actin has come a long way from the times of its discovery in muscle and we have learned to know it as a member of a large family of structurally related proteins, some of which are dual compartment proteins. Concomitant with this, there is an overwhelming expansion of their putative functions, and both, the fine structural conformation as well as the different functions are probably determined by the numerous actin binding proteins. As a cartoon, Fig. 4 summarizes the most important players in the cytoplasmic and the nuclear compartment. It is obvious that the putative activities in the nucleus outnumber those in the cytoplasm. Taken together, these findings allow for the view that actin and its relatives were originally engaged in nuclear activities, and that their appearance in the cytoplasm is related to a specialization in functions connected solely with intracellular motility and locomotion (F-actin filament formation, actin dynamics). Further functional narrowing would then result in the special case of skeletal muscle, where a specific isoform, alpha actin, is overexpressed and knows nothing else than to allow myosin-driven contraction.Fig. 4The manifold functions of nuclear actin. The G-/F-actin equilibrium is only a highly specialized function of actin in the cytoplasm (top); in the nucleus (bottom) the actin system plays a quite different regulatory and structural role. Actin can execute its tasks as monomer or as nucleus-specific polymer. It interacts with heterogenous ribonucleoproteins (hnRNPs) and is thought to function as a track for RNA transport through NPCs. It remains to be shown how nuclear actin cooperates with nuclear myosin-1 to enhance movement of RNA polymerases or to re-organize chromatin (Nunez et al. 2008). Positioning of the nucleus in the cell is achieved by nuclear membrane (NM) proteins that, like the large nesprins, hook the nucleus to the actin cytoskeleton. Anchorage of nesprins is guaranteed by SUN proteins in the inner nuclear membrane (Worman and Gundersen 2006). The stabilization of the nucleoskeleton involves the interaction of inner nuclear membrane proteins like emerin or nesprins with lamins as well as polymeric actin (Libotte et al. 2005). It should, however, be borne in mind that the level of nuclear actin is apparently tightly regulated in mammalian cells by exportin 6 which selectively transports profilin–actin complexes from the nucleus into the cytoplasm (Bohnsack et al. 2006). The nuclear functions of many dual compartment actin binding proteins are not well understood, but one has to assume that their interaction can discriminate between nuclear and cytoplasmic actin (Pederson and Aebi 2005). For example, the recently described cyclase associated protein CAP2 is nuclear in myoblasts but sarcomeric in myotubes (Peche et al. 2007). Actin-related proteins are only summarized in one symbol and are either nucleus specific subunits in chromatin remodelling complexes (Arp4, Arp8 etc.), cytoplasmic or belong also to the group of dual compartment proteins
If one was to speculate further, one might imagine that prokaryotic precursors of present days’ actin, related to the proteins that spatially and temporally control macromolecular trafficking, chromosome segregation and cell polarity in recent bacteria (Carballido-Lopez 2006), appeared first as “nuclear actin” in an ancestor eukaryotic cell as part of the endosymbiontic engulfment. But, it will require several decades and lots of industrious students to decide whether this is science fiction or facts about actin.

transcription
chromatin remodelling
actin history
cytoplasmic motility
nucleocytoplasmic traffick


Respondent-Driven Sampling in a Study of Drug Users in New York City: Notes from the Field

Beth Israel Medical Center (BIMC), in collaboration with the Centers for Disease Control (CDC) and the New York State Department of Health (NYSDOH), used respondent-driven sampling (RDS) in a study of HIV seroprevalence among drug users in New York City in 2004. We report here on operational issues with RDS including recruitment, coupon distribution, storefront operations, police and community relations, and the overall lessons we learned. Project staff recruited eight seeds from a syringe exchange in Lower Manhattan to serve as the initial study participants. Upon completion of the interview that lasted approximately 1 h and a blood draw, each seed was given three coupons to recruit three drug users into the study. Each of the subsequent eligible participants was also given three coupons to recruit three of their drug-using acquaintances. Eligible participants had to have: injected, smoked or snorted an illicit drug in the last 6 months (other than marijuana), aged 18 or older, adequate English language knowledge to permit informed consent and complete questionnaire. From April to July 2004, 618 drug users were interviewed, including 263 (43%) current injectors, 119 (19%) former injectors, and 236 (38%) never injectors. Four hundred sixty nine (76%) participants were men, 147 (24%) were women, and two (<1%) were transgender. By race/ethnicity, 285 (46%) were black, 218 (35%) Hispanic, 88 (14%) white, 23 (4%) mixed/not specified, and four (<1%) native American. Interviews were initially done on a drop-in basis but this system changed to appointments 1 month into the study due to the large volume of subjects coming in for interviews. Data collection was originally proposed to last for 1 year with a target recruitment of 500 drug users. Utilizing RDS, we were able to recruit and interview 118 more drug users than originally proposed in one quarter of the time. RDS was efficient with respect to time and economics (we did not have to hire an outreach worker) and effective in recruiting a diverse sample of drug users.
Background
A variety of sampling methods have been used to recruit hard-to-reach populations, such as drug users, into research studies. These methods include snowball sampling, targeted sampling, and time-space sampling.1–3 Despite widespread use of these methods, they are not without important limitations.
Snowball sampling begins with a set of initial study participants, called seeds, who refer other eligible respondents. Once these persons are interviewed, they are also asked to bring in or provide references for other potential respondents. This process continues until the sample size is achieved. Snowball sampling can provide easy access to hidden populations, but it is most often biased because it is usually done out of convenience rather than randomly.1–3
Targeted sampling involves thoroughly collecting preliminary data to determine various characteristics such as locations where a sample congregates and demographic characteristics such as race, gender, and age.1,2,4 Similar to snowball sampling, using targeted sampling to study drug users may result in a selection bias because less-visible, isolated drug users may not be found at the sites where other drug users are sampled and, thus, may be overlooked.
Time-space sampling involves preliminary research by ethnographers to determine when and where hidden populations congregate.3,5 Once a list of times and sites is determined, researchers then randomly choose times to visit those sites to collect data. Time-space sampling has limited use with drug users because making an exhaustive list of where drug users congregate could be a very protracted and expensive process, as drug-use sites change frequently in response to environmental factors such as police presence.
Respondent-driven sampling (RDS),1 a relatively new method being used to draw probability samples of hidden populations, incorporates some methods of snowball sampling, such as chain referral sampling, but includes additional provisions to minimize bias. It produces population estimates that are asymptotically unbiased, which means that bias is only on the order of 1/[sample size], so bias is minimal in samples of substantial size.6 This allows for probability-based inferences based on the social network of the sample.
This paper reports on the use of RDS to recruit participants for a study of risk behavior and HIV seroprevalence among drug users in New York City. The study was conducted by Beth Israel Medical Center in collaboration with the Centers for Disease Control and Prevention and the New York State Department of Health. We discuss the operational issues faced with RDS such as recruitment, coupon distribution, storefront operations, police and community relations, and the overall lessons learned.
Methods
In April 2004, Beth Israel Medical Center staff recruited eight seeds from a syringe exchange in Lower Manhattan. Each seed was screened for eligibility before being given a coupon to come back for an interview. The seeds were asked about their drug use, including mode of use and drug preparation techniques. Those who claimed to be an injection drug user (IDU) were also asked to show track marks. Although not a requirement of RDS, the seeds were recruited to resemble the race and gender profile of drug users in Lower Manhattan.7 They were asked to come to a research storefront in Lower Manhattan the following day to complete a computer-assisted interviewer-administered personal interview (CAPI) and to have their blood drawn for an HIV test. Each seed, and subsequent study subject, received $20 compensation for their time.
When subjects arrived at the storefront, they were questioned by the study screener to ensure eligibility. Once they were deemed eligible by the screener, each subject was assigned a unique code to serve as their study identification. This code included the following information: the first two letters of the last name, first letter of mother’s first name, the last two digits of the birth year, one letter for the person’s race, and one letter for gender. This code was used to identify blood work and questionnaires and could be regenerated if the subject forgot it. No names or other identifying information were asked.
After being assigned a study code, subjects met with an interviewer to be consented and interviewed. The interview had three parts, an hour-long questionnaire, administered by the interviewer, HIV pre-test counseling and a blood draw for an HIV test. The interview consisted of a structured questionnaire which took approximately 1 h and asked about drug-use frequency, drug and sexual risk behavior, syringe acquisition, and knowledge of HIV and hepatitis B and C. After the interview, HIV counseling, and blood collection, each subject was given three coupons to recruit three other drug users into the study. The subjects were briefly trained on how to recruit others, with specific emphasis on the recruitment of friends and acquaintances who use drugs. Eligible respondents had to have done the following: injected, smoked, or snorted an illicit drug in the past 6 months (those who smoked only marijuana were not eligible for the study); turned age 18 years or older by the time of the interview; been able to speak English adequately to consent to the study and complete the questionnaire; and lived, bought, and/or used drugs on the Lower East Side of Manhattan. Subjects could have, however, lived in the larger New York metropolitan area, as long as they bought or used their drugs on the Lower East Side of Manhattan.
Coupons contained the time that the storefront opened (9:00 a.m.) and a unique number (to make each one distinct). To make them difficult to duplicate, coupons were printed on thick cardstock paper with color images. To track the coupons and payment for each respondent, we used custom-developed software for RDS called IRIS Plus. Information such as respondent’s unique code, physical traits, coupon number, and the numbers of the coupons each respondent distributed were all recorded in IRIS Plus. This information enabled us to link coupons together, determine when respondents should be paid and who gave coupons to whom. This software also helped to prevent the redemption of duplicated coupons because the database would not accept duplicate coupon numbers. When respondents came in to make an appointment, the coupon was checked in the IRIS Plus database to verify that it had not been used previously. Additionally, each person’s code was checked in IRIS Plus after being screened to determine whether a person with that code had previously been enrolled in the study. If another study subject had that code, the screener looked at the physical traits listed for that person for verification. Additionally, if the screener felt that a person looked familiar, she would search for the person's physical traits in IRIS Plus to see if another person with similar characteristics was previously enrolled.1
On July 2, 2004, we ceased coupon distribution to give potential subjects 2 weeks to redeem remaining coupons. On July 16, 2004, we concluded data collection, and the study officially concluded on July 30, 2004. The last 2 weeks of July were set aside to allow the remainder of subjects to come in for their HIV test results.
Results
From April to July 2004, 618 respondents were interviewed, including 263 (43%) current injectors (had injected drugs within the past 6 months), 119 (19%) former injectors (had injected drugs more than 6 months prior to the interview—who used drugs in the past 6 months by other modes of administration), and 236 (38%) never injectors (those that had never injected any drugs but used non-injection drugs in the past 6 months). The mean age of respondents was 44 years, with a mean age of first drug use at 19 years and mean age of first injection drug use at 22 years (among current and former drug injectors). By gender, 469 (76%) were men, 147 (24%) were women, and two (<1%) were transgender. By race/ethnicity, 285 (46%) were black; 218 (35%), Hispanic; 88 (14%), white; 23 (4%), mixed race or unspecified race; and four (<1%), native American.
Seed Recruitment
To control the flow of recruitment, we recruited only eight seeds: two white men, one white woman, two Hispanic men, one Hispanic woman, one black man, and one black woman, all of whom identified themselves as active IDUs. A relatively small number of seeds was recruited so that we could assess how productive they were and then recruit more if necessary, without being too overwhelmed with respondents.
Respondent Recruitment
Our initial goal was to interview 500 drug users within 1 year. After 4 months, 618 drug users had been interviewed. Respondent recruitment (those people recruited after the seeds) happened much faster than we had expected. In the first couple of weeks, recruitment was slow but quickly picked up as more coupons got out into the community. Interviews were initially done on a drop-in basis, but this system changed to appointments after 1 month into the study due to the large volume of respondents coming in for interviews. As the flow of potential subjects continued to be rapid and at times overwhelming, we were also forced to post-date all coupons by 2 days. Therefore, when a respondent was given a coupon, that person could not be interviewed or make an appointment until the date written on the coupon. This system was helpful in pacing the number of people coming into the storefront for interview appointments but perhaps choosing a smaller number of seeds may have alleviated the need to implement these changes altogether.
Police and Community Relations
As previously noted, as the study progressed and more coupons were distributed in the community, the number of people coming into the storefront for interviews increased dramatically. For the first 2 weeks, the interviewers would arrive in the morning to find 10 to 20 people waiting for them. People reported arriving as early as 7:00 a.m. so that they could be interviewed that day. Once crowds began to form outside the storefront, neighboring businesses complained to our staff or called the police to file complaints. The police came to speak with staff on three separate occasions regarding complaints of noise and loitering. At that point we decided to move to an all-appointment system. Instead of telling people that their recruits could show up and be interviewed, recruits were told to first come into the storefront to be screened, and that if they passed the screener, they could make an appointment.
Lessons Learned
Were we to do another RDS study in New York City, we would do the following:
We would have a phone number so that screening and appointments for potential participants could be done by telephone. This would lessen the amount of traffic in the storefront and reduce some confusion. Once participants arrived for their interviews, we would then screen them about their drug use.
If we were looking to recruit a large number of drug users in a short amount of time, we would again recruit eight seeds. The eight seeds recruited for this study seemed to be an appropriate number because we were trying to maximize our time in the field for data collection. Although the first 2 weeks of the study were relatively slow-paced, it gave the field staff time to adjust to their new environment and new roles and make any necessary changes before they became too overwhelmed. Drug users in New York City are generally a very overstudied population, and thus we expected a large portion of the seeds to show up for their interviews. In cities where the population is not as familiar with research, researchers may want to recruit more seeds, considering that many may not return for the initial interview. Another alternative, however, if time permits, is to recruit a smaller number of seeds, give them a few weeks to return for their interview and then recruit more seeds, if necessary. It was our intention to recruit more seeds if our initial eight were not productive, but this was not necessary.
We would start with an appointment-only system. We began with a drop-in system for interviews because our time to collect data was very limited and this was the most efficient use of the interviewers’ time. For instance, if a person dropped in and wanted to be interviewed, we could do it on the spot if the person with a scheduled interview had not shown up. Even though this system worked for us initially, it soon became unwieldy, and we were forced to change a month into the study. People adjusted very quickly, and many people reported being relieved because they didn’t have to wait in front of the storefront for 2 h in the morning before staff arrived.
We would be very rigid about our appointments. In this study we were very strict about appointment times and conveyed this to each participant. If persons with appointments were more than 5 min late and someone else arrived to schedule an appointment but was free to complete it then, we took the person who dropped in rather than waiting for the person with the scheduled appointment to show up. We rarely had interviewers sitting around with nothing to do. There was almost always someone who dropped in when someone else was late for an appointment.
We would meet with local business owners to inform them of who we are and what we were doing prior to the start of the study. We would invite them to tell us if they had any concerns. We would not disclose the type of research being done or with whom, but it would be helpful to let the surrounding business owners know that they may see an increase in the number of people in or around the storefront.
We would remain at the storefront 6 weeks after ending coupon distribution. Coupon distribution ended a full 4 weeks before we vacated the storefront, and data collection ended 2 weeks before. The timing of both of these allowed for a fair amount of those with coupons to redeem them, for those with outstanding incentives to collect their money, and for those with outstanding test results to come in for them. Two additional weeks would have been helpful to allow for remaining coupons to be redeemed. Because the storefront we used stayed open for other research studies, we were able to leave contact information for our interviewers in case any respondents showed up for their HIV test results. Within the month after we left the storefront, we received two additional calls for HIV test results. Our interviewers met the respondents at a park and a coffee shop to give them their results. The other storefront staff reported a handful of people coming in with coupons after the study closed, but they did not report any incidents with those people that were unable to redeem them. Finally, we did not pay out any uncollected incentives after July 30, 2004.
Some of the lessons that we learned about conducting a study using RDS may be specific to New York City. Because there was an abundance of willing and able participants, we had to take several measures to make data collection more manageable. These measures may not be necessary or practical for researchers in cities where the drug-using population is smaller, less willing to participate in research, or both.
Conclusions
Using RDS, we were able to recruit and interview 118 more drug users than originally proposed in one third of the time. In our experience, RDS was efficient with respect to time and economics (we did not have to hire an outreach worker) and effective in garnering a diverse sample of drug users. We were able to interview many more drug users than expected in a short period of time with very minimal recruitment effort. In summary, RDS can be an efficient and effective form of recruitment, particularly for research involving drug users or other hidden populations.

drug users
respondent-driven sampling (rds)
time-space sampling
capi
iris plus