Lipoxin transport system and uses therefor

A lipoxin transport system which mediates uptake of lipoxin by cells is disclosed. Lipoxin transport into cells by the lipoxin transport system is inhibitable by 3,5-diiodosalicylic acid (DISA), pentachlorophenol (PCP), .alpha.-cyano-.beta.-(1-phenylindol-3-yl)acrylic acid (UK-5099), mersalyl and p-hydroxymercuribenzoate (pHMB). The invention provides methods for identifying inducers, enhancers and inhibitors of the lipoxin transport system. The invention further provides methods for inducing, enhancing or inhibiting lipoxin transport by the lipoxin transport system. Methods for identifying molecules which are transported by the lipoxin transport system, including lipoxin analogs, are also disclosed. Modulation of physiological responses by modulation of lipoxin transport into cells by the lipoxin transport system of the invention are also contemplated.

BACKGROUND OF THE INVENTION 
Lipoxins are a group of biologically active mediators derived from 
arachidonic acid through the action of lipoxygenase enzyme systems. 
(Serhan, C. N. and Samuelsson, B. (1984) Proc. Natl. Acad. Sci. USA 
81:5335). Formation of lipoxins in human cell types is initiated by 
5-lipoxygenase or 15-lipoxygenase. (Serhan, C. N. (1991) J. Bioenerg. 
Biomembr. 23:105). Single-cell types generate lipoxins at nanogram levels 
during human neutrophil-platelet and eosinophil transcellular biosynthesis 
of eicosanoids. (Serhan, C. N. and Sheppard, K. -A. (1990) J. Clin. 
Invest. 85:772). Lipoxins are conjugated tetraene-containing eicosanoids 
that modulate cellular events in several organ systems. 
The two major lipoxins are lipoxin A.sub.4 (LXA.sub.4) and lipoxin B.sub.4 
(LXB.sub.4). The lipoxins have been demonstrated to elicit a variety of 
physiological effects that may play a role in regulating cellular 
responses involving host defense, vascular tone and inflammation. Lipoxins 
have a stimulatory effect on certain responses whereas they have an 
inhibitory effect on other responses. Some effects of the lipoxins 
include: LXA.sub.4 and LXB.sub.4, at a concentration of about 10 nM, 
enhance protein kinase C (PKC) activity in nuclei of erythroleukemia cells 
(Beckman, B. S. et al. (1992) Proc. Soc. Exp. Biol. Med. 201:169); 
LXA.sub.4 and LXB.sub.4 at nM levels elicit prompt vasodilation (Busija, 
D. W. et al. (1989) Am. J. Physiol. 256:H468; Katoh, T. et al. (1992) Am. 
J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32):F436); LXA.sub.4 in 
the 10.sup.-10 M range stimulates cell proliferation in combination with 
suboptimal concentrations of granulocyte-macrophage colony stimulating 
factor (GM-CSF) to induce myeloid bone marrow colony formation (Stenke, L. 
et al. (1991) Biochem. Biophys. Res. Commun. 180:255); LXA.sub.4 
stimulates human mononuclear cell-colony formation (Popov, G. K. et al. 
(1989) Bull. Exp. Biol. Med. 107:93); and LXA.sub.4 inhibits chemotaxis of 
polymorphonuclear leukocytes (Lee, T. H. et al. (1991) Biochem. Biophys. 
Res. Commun. 180:1416. 
Lipoxins can act as antagonists to leukotrienes (LT), which are mediators 
of inflammation. Leukotriene-induced inflanunation occurs, for example, in 
arthritis, asthma, various types of shock, hypertension, renal diseases, 
allergic reactions, and circulatory diseases including myocardial 
infarction. LXA.sub.4 modulates LTC.sub.4 -induced obstruction of airways 
in asthmatic patients. For example, administration of LXA.sub.4 in 
micromolar amounts via inhalation blocks bronchoconstriction in asthmatic 
patients. (Christie, P. E. et al. (1992) Am. Rev. Respir. Dis. 145:1281). 
LXA.sub.4 inhibits LTD.sub.4 - and LTB.sub.4 -mediated inflammation in 
animal in vivo models. (Badr, K. F. et al (1989) Proc. Natl. Acad. Sci. 
USA 86:3438; Hedqvist, P. et al. (1989) Acta Physiol. Scand. 137:571). 
Prior exposure to LXA.sub.4 (nM) blocks renal vasoconstrictor actions of 
LTD.sub.4 (Katoh, T. et al. (1992) Am. J. Physiol. 263 (Renal Fluid 
Electrolyte Physiol. 32) F436). Thus, the vasodilatory effects of lipoxins 
can counteract vasoconstriction caused by other agents. 
The functional effects of lipoxins are mediated, at least in part, through 
uptake of lipoxins by cells. Specific binding of LXA.sub.4 to cell surface 
LXA.sub.4 receptors has been demonstrated. (Fiore, S. et al. (1992) J. 
Biol. Chem. 267:16168-16176; Fiore, S. et al. (1993) Blood 81:3395-3403). 
Additionally, carrier-mediated transport systems for prostaglandins, 
compounds which are also derived from arachidonic acid, have been 
characterized in lung, kidney, choroid plexus and retinal epithelium. 
(Bito, L. Z. et al., (1977) Am. J. Physiol. 232:E382-E387; 
Boumendil-Podevin, E. F. et al., (1985) Biochim. Biophys. Acta 812:91-97; 
Bito, L. Z. et al., (1976) J. Physiol. 256:257-271). Identification of a 
transport system for lipoxins in cells would provide a pathway for lipoxin 
uptake which could be targeted therapeutically to either inhibit or 
enhance lipoxin uptake by cells to down- or upregulate physiological 
responses mediated by lipoxins. 
SUMMARY OF THE INVENTION 
This invention pertains to methods for inhibiting or enhancing lipoxin 
uptake by a cell by inhibiting or enhancing the transport of lipoxin into 
a cell that is mediated by a lipoxin transport system. The invention is 
based, at least in part, on the discovery of a transport system for 
lipoxins which is different than, and independent from, binding of 
lipoxins to specific receptors on a cell. The transport system of the 
invention has properties of a carrier-mediated transport system. The 
transport system is inhibitable by anionic inhibitors including 
3,5-diiodo-salicylic acid (hereinafter DISA), pentachlorophenol 
(hereinafter PCP) and .alpha.-cyano-.beta.-(1-phenylindol-3-yl)acrylic 
acid (hereinafter UK-5099) and organo-mercurial agents including mersalyl 
and p-hydroxymercuribenzoate (hereinafter pHMB). Influx of lipoxin by the 
transport system is Na.sup.+ - and membrane voltage-independent but 
dependent on extracellular pH. For example, transport is inhibited by an 
alkaline extracellular pH (e.g., pH=8.4) and enhanced by an acidic 
extracellular pH (e.g., pH=6.4). Inhibitors of the transport system of the 
invention do not affect binding of lipoxin to specific lipoxin receptors 
on cells. The characteristics of the transporter system are consistent 
with an H.sup.+ +lipoxin cotransport system. 
The invention provides a method for identifying an inhibitor of this 
lipoxin transport system. In the method, a cell, or membrane vesicle 
thereof, which has the lipoxin transport system is provided. Uptake of a 
lipoxin by the cell by the lipoxin transport system is characterized by 
being inhibitable by DISA, PCP, UK-5099, mersalyl or pHMB. The cell is 
contacted with a labeled lipoxin in the presence of a molecule to be 
tested, uptake of labeled lipoxin by the cell is measured and the ability 
of the molecule to inhibit uptake of labeled lipoxin by the cell is 
determined. An inhibitor of a lipoxin transport system is identified by 
the ability of the molecule to inhibit uptake of labeled lipoxin by the 
cell by the lipoxin transport system. Preferably, the labeled lipoxin is 
LXA.sub.4. In preferred embodiments, the cell is a neutrophil or 
differentiated HL-60 cell. 
The invention further provides a method for inhibiting transport of a 
lipoxin into a cell which has a lipoxin transport system. The method 
comprises contacting the cell with a molecule which is an inhibitor of the 
lipoxin transport system. In preferred embodiments, the inhibitor is an 
anion or organomercurial agent. An inhibitor of the lipoxin transport 
system can also be used to regulate lipoxin-mediated responses in a cell 
which has a lipoxin transport system by contacting the cell with the 
inhibitor. For example, an inhibitor of the lipoxin transport system can 
be used to down-regulate a response which is stimulated by a lipoxin by 
preventing transport of the lipoxin into a cell. Alternatively, an 
inhibitor of the lipoxin transport system can be used to up-regulate a 
response which is inhibited by a lipoxin by preventing uptake of the 
lipoxin by the cell. 
The invention further provides a method for identifying a molecule which 
induces or enhances lipoxin transport by a lipoxin transport system. The 
method includes providing a cell, or membrane vesicle thereof, which has a 
lipoxin transport system or in which a lipoxin transport system can be 
induced. The cell is contacted with a labeled lipoxin in the presence of a 
molecule to be tested, uptake of labeled lipoxin by the cell is measured 
and the ability of the molecule to induce or enhance uptake of labeled 
lipoxin by the cell is determined. A molecule which induces or enhances 
lipoxin transport by a lipoxin transport system is identified based upon 
this ability. Preferably, the labeled lipoxin is LXA.sub.4 and the cell is 
a neutrophil or differentiated HL-60 cell. 
The invention also provides a method for identifying a lipoxin analog which 
is transported at a faster rate than a natural lipoxin by a lipoxin 
transport system. The method includes providing a cell, or membrane 
vesicle thereof, which has a lipoxin transport system, contacting the cell 
with a labeled lipoxin analog to be tested, measuring the rate of uptake 
of the lipoxin analog by the cell, and comparing the rate of uptake of the 
lipoxin analog by the cell to the rate of uptake of a natural lipoxin by 
the cell to identify a lipoxin analog which is transported at a faster 
rate than a natural lipoxin by a lipoxin transport system. 
The invention still further provides a method for enhancing transport of a 
lipoxin into a cell which has a lipoxin transport system. The method 
comprises contacting the cell with a molecule which enhances transport of 
a lipoxin by the lipoxin transport system. For example, 
n-formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate can 
enhance uptake of lipoxin by the lipoxin transport system. An enhancer of 
the lipoxin transport system can also be used to modulate lipoxin-mediated 
responses in a cell which has a lipoxin transport system by contacting the 
cell with the enhancer. For example, an enhancer of the lipoxin transport 
system can be used to up-regulate a response which is stimulated by a 
lipoxin by increasing transport of the lipoxin into a cell. Alternatively, 
an enhancer of the lipoxin transport system can be used to down-regulate a 
response which is inhibited by a lipoxin by increasing uptake of the 
lipoxin by the cell,

DETAILED DESCRIPTION OF THE INVENTION 
This invention relates to lipoxin uptake by a cell via a lipoxin transport 
system. The invention is based, at least in part, on the discovery of a 
transport system in cells which promotes uptake of lipoxin by the cells. 
The lipoxin transport system has properties of a carrier-mediated 
transport system. Influx of lipoxin into cells by the lipoxin transport 
system is sensitive to certain anionic inhibitors such as 
3,5-diiodo-salicylic acid (DISA), pentachlorophenol (PCP) and 
.alpha.-cyano-.beta.-(1-phenylindol-3-yl)acrylic acid (UK-5099), and 
certain organomercurial (and sulfhydryl-reactive) agents such as mersalyl 
and p-hydroxymercuribenzoate (pHMB). Influx of lipoxin by the transport 
system also exhibits a dependence on pH (pK'5.9) but is independent of 
membrane voltage and Na.sup.+ concentration. These properties of the 
transport system are consistent with an H.sup.+ +lipoxin cotransport 
system. Some cell types are also known to have specific receptors for 
lipoxins. Inhibitors of the lipoxin transport system do not affect binding 
of lipoxin to specific lipoxin receptors on cells. Thus, the lipoxin 
transport system of the invention represents a distinct pathway for 
lipoxin uptake which is different from and independent of binding of 
lipoxin to specific lipoxin receptors. This transport pathway can serve as 
a target for intervention in order to increase or decrease uptake of 
lipoxin by a cell. Modulating lipoxin uptake provides a means by which to 
up- or downregulate physiological responses mediated by lipoxins. 
A. Properties of the Lipoxin Transport System 
The lipoxin transport system of the invention has several characteristics 
which can be used to identify the presence of the transport system on a 
particular cell and distinguish the transport system from binding of 
lipoxin to specific receptors. The primary characteristic utilized to 
identify lipoxin transport mediated by the lipoxin transport system is the 
ability of certain inhibitors to inhibit lipoxin transport by this system. 
A lipoxin transport system can be identified on a cell by incubating the 
cell with a lipoxin (e.g. LXA.sub.4) which is labeled with a detectable 
substance (e.g. radioactively labeled), measuring the uptake of lipoxin by 
the cell land examining the effect of certain inhibitors and/or conditions 
on uptake of lipoxin by the cell to determine whether uptake is mediated 
by the lipoxin transport system. For example, a cell can be incubated with 
.sup.3 H-LXA.sub.4 and uptake of lipoxin by the cell can be determined by 
measuring the association of the radiolabel with the cell over time. 
Association of lipoxin with the cell could be due to binding of lipoxin to 
specific receptors on the cell, transporter-mediated uptake of the lipoxin 
or a combination of the two mechanisms. To determine the amount of lipoxin 
uptake which is transporter-mediated, the cell is incubated with both 
labeled lipoxin and an inhibitor of the lipoxin transport system. For 
example, the cell can be incubated with .sup.3 H-LXA.sub.4 and DISA. 
Alternatively, an inhibitor selected from PCP, UK-5099, mersalyl and pHMB 
can be used. Association of lipoxin with the cell which is due to binding 
of lipoxin to specific receptors is unaffected by the presence of these 
inhibitors and thus will still be detectable. However, association of 
lipoxin with the cell which is due to uptake of lipoxin by the lipoxin 
transport system is inhibited by these inhibitors and thus will be 
decreased or eliminated. By comparing the amount of lipoxin which 
associates with the cell in the presence and in the absence of DISA (or 
another appropriate inhibitor), uptake of lipoxin via the lipoxin 
transport system can be determined. 
The effect of certain conditions on association of lipoxin with the cell 
can also be used to identify transporter-mediated uptake of lipoxin by the 
cell. For example, transporter-mediated uptake is increased by lowering 
the extracellular pH to 6.4, whereas it is decreased by raising the 
extracellular pH to 8.4. Binding of lipoxin to specific receptors is not 
affected by such pH variations. Thus, a cell can be incubated with a 
labeled lipoxin (e.g. .sup.3 H-LXA.sub.4) and the effect of varying the 
extracellular pH on association of the lipoxin with cell can be determined 
as an indicator of the presence or absence of a lipoxin transport system 
in the cell. 
Additionally, certain compounds which enhance uptake of lipoxin by a cell 
via the lipoxin transport system have been identified. For example, 
n-formyl-methionyl-leucylphenylalanine (FMLP) and phorbol myristate 
acetate (PMA) can increase the initial rate of lipoxin influx into a cell 
by the lipoxin transport system. Thus, a cell can be incubated with a 
labeled lipoxin (e.g. .sup.3 H-LXA.sub.4) and the effect of FMLP or PMA on 
association of the lipoxin with cell can be determined. Increased uptake 
of lipoxin in the presence of FMLP or PMA can be used as an additional 
indicator of the presence of a lipoxin transport system in the cell. 
In contrast, other conditions and compounds have little or no effect on the 
lipoxin transport system. Transporter-mediated uptake is not affected by 
removing extracellular Na.sup.+ or by raising extracellular K.sup.+ (which 
depolarizes membrane voltage). Additionally, transporter-mediated uptake 
is not inhibited by probenecid or the disulfonic stilbene SITS, two 
general inhibitors of anion transport. Several sulfhydryl-reactive 
compounds also do not inhibit transporter-mediated uptake including NEM, 
iodoacetate and 2,2'-dithiobispyridine (hereinafter 2,2'-DTBP), whereas 
the sulfhydryl-reactive compounds 7-chloro-4-nitrobenz-2-oxa-1,3-diazole 
(hereinafter NBD-Cl) and eosin-5-maleimide cause only modest inhibition 
(approximately 20%). The lack of (or minimal) effect of these conditions 
or compounds on lipoxin uptake by a cell can be used as additional 
indicators that the uptake occurs by a lipoxin transport system. 
The lipoxin transport system displays other characteristics which can be 
utilized as additional indicators of lipoxin transport by this system. For 
example, the inhibitor profile of the lipoxin transport system 
distinguishes it from several other known specialized anion transport 
systems. These systems include a Cl.sup.- /HCO.sub.3.sup.- exchanger, a 
sulfate carrier and an H.sup.+ +lactate.sup.- cotransporter. For example, 
the Cl.sup.- /HCO.sub.3 .sup.31 exchanger is resistant to pHMB whereas 
the lipoxin transporter is inhibited by pHMB and, additionally, the 
Cl.sup.- /HCO.sub.3 .sup.- exchanger displays a pH-dependence opposite to 
that for lipoxin influx (Simchowitz, L. et al., (1991) Am. J. Physiol. 
261:C906-C915). The sulfate carrier is very sensitive to the disulfonic 
stilbene SITS and to probenecid (Simchowitz, L. and Davis, A. O., (1989) 
J. Gen. Physiol 94:95-124) while lipoxin uptake is not. Lactic acid fluxes 
are completely blocked by NEM and NBD-CL (Simchowitz, L. and Vogt, S. K. 
(1993) J. Membr. Biol. 131:23-34) while those of lipoxin are not. Also, 
the lipoxin transport system displays substrate specificity in that not 
all arachidonic acid derivatives are transported by the system. For 
example, arachidonic acid, prostaglandin E.sub.2 (hereinafter PGE.sub.2), 
15-HETE, and the leukotrienes B.sub.4, C.sub.4 and D.sub.4 are not 
transported by the lipoxin transport system. Furthermore, methyl ester 
derivatives of lipoxins can enter cells by non-ionic diffusion, thus 
bypassing the lipoxin transport system. Because of the ester linkage 
through the carboxyl group, the lipoxin methyl ester derivative is 
uncharged and therefore lipophilic. Thus, the methyl ester derivative can 
permeate cells by simple diffusion whereas the lipoxin free acid cannot. 
Inhibitors which block transporter-mediated uptake of lipoxins do not 
affect entry of methyl ester derivatives of lipoxins into the cell. 
When assaying a cell for the presence of a lipoxin transport system, 
conditions can be chosen which promote measurement of transporter-mediated 
lipoxin uptake while reducing the contribution of lipoxin receptor binding 
to the observed association of lipoxin with the cell. That is, a 
concentration of labeled lipoxin can be chosen which rapidly saturates 
binding of the lipoxin to specific lipoxin receptors. Under this 
condition, lipoxin uptake by the cell which occurs subsequent to 
saturation of the specific receptors should be exclusively due to 
transporter-mediated uptake, which can be confirmed by examining the 
sensitivity of this uptake to DISA or other inhibitors. A concentration of 
lipoxin can be chosen based upon the K.sub.d of receptor binding. For 
example, the binding of .sup.3 H-LXA.sub.4 to specific receptors has been 
found to have a K.sub.d of 0.5.+-.0.3 nM. A concentration of at least 
about 10-fold greater than the K.sub.d, e.g., 5 nM, can be used to 
saturate receptor binding. Preferably, a concentration of at least about 
100-fold greater than the K.sub.d, e.g. 50 nM, is used to saturate 
receptor binding. At these concentrations, binding of the lipoxin to 
specific receptors is rapidly saturated, i.e., is saturated within about 1 
minute after incubating the lipoxin with a cell expressing lipoxin 
receptors. One can continue to measure the association of lipoxin with the 
cell over time, e.g., at 10 min., 20 min. and 30 min. post-incubation. 
Uptake of lipoxin by the transport system is not saturated under these 
conditions and thus additional accumulation of lipoxin within the cell due 
to transporter-mediated uptake can be measured. 
In addition to identifying a cell that has a lipoxin transport system, by 
the aforementioned procedures, the rate of uptake of lipoxin by the cell 
via the lipoxin transport system can be determined by measuring the uptake 
of lipoxin by the cell over time, wherein the uptake is inhibitable by 
DISA or another inhibitor (e.g., PCP, UK-5099, mersalyl or pHMB). This 
rate determination is described in detail in the Examples. Influx of 
LXA.sub.4 into neutrophils by the lipoxin transport system has been found 
to occur at a rate of about 0.6 fmol/10.sup.6 cells/min. 
B. Identification of Molecules that Inhibit or Enhance Lipoxin Transport 
A cell which has a lipoxin transport system can be used to screen molecules 
for their ability to inhibit or enhance lipoxin uptake by the lipoxin 
transport system. The invention provides a method for identifying a 
molecule which is an inhibitor of a lipoxin transport system. In the first 
step of the method, a cell, or membrane vesicle thereof, which has a 
lipoxin transport system is provided. A whole intact cell can be used or a 
membrane vesicle which contains the lipoxin transport system can be used. 
The cell or membrane vesicle containing the lipoxin transport system allow 
for the transfer of a lipoxin from an extracellular site to an 
intracellular or intravesicular site, which can be measured. Membrane 
vesicles can be prepared from intact cells by standard procedures known in 
the art. The term "lipoxin transport system" is used herein to describe a 
system in which transport of a lipoxin by the system is inhibitable by a 
compound selected from a group consisting of DISA, PCP, UK-5099, mersalyl 
and pHMB. The cell (or membrane vesicle) which is provided may also have 
specific lipoxin receptors. However, binding of lipoxin to these receptors 
is not inhibitable by DISA, PCP, UK-509, mersalyl or pHMB. 
The cell, or membrane vesicle, thereof, is then contacted with a labeled 
lipoxin in the presence of a molecule to be tested. The term "labeled 
lipoxin" is used herein to describe a lipoxin which is labeled with a 
detectable substance. Preferably, the detectable substance is a 
radioactive isotope. For example, a lipoxin can be labeled with .sup.3 H. 
Alternatively, a lipoxin could be labeled with .sup.14 C. Alternative 
detectable substances include fluorescent and luminescent materials. The 
requirements for the detectable substance include that it must not 
interfere with uptake of the lipoxin by the lipoxin transport system and 
it must allow measurement of association of the labeled lipoxin with the 
cell. 
After contacting the cell, or membrane vesicle thereof, with the labeled 
lipoxin, uptake of the labeled lipoxin is measured. This can be 
accomplished by measuring the amount of the detectable substance (e.g., 
the radiolabel) which is specifically associated with the cell. For 
example, at various intervals post-incubation, an aliquot of the cells can 
be separated from the incubation medium and the cell-associated label 
(e.g. amount of radiolabel present in the cell pellet) can be measured. 
A molecule which is an inhibitor of a lipoxin transport system can be 
identified by its ability to inhibit uptake of a labeled lipoxin by a 
cell, wherein the uptake is also inhibitable by DISA, PCP, UK-5099, 
mersalyl or pHMB. The lipoxin uptake defined in this way excludes lipoxin 
uptake by a cell which is due to binding of lipoxin to specific receptors. 
The amount and/or rate of lipoxin transport which is inhibitable by DISA 
or another inhibitor can be predetermined for the cell to be used in the 
method by the procedures described earlier. For example, in a preferred 
embodiment the cell used in the method is a neutrophil, which has both 
lipoxin specific receptors and a lipoxin transport system. In the presence 
of 5 nM LXA.sub.4, about 3 fmol LXA.sub.4 /10.sup.6 cells becomes 
associated with neutrophils within 1 minute due to binding to specific 
lipoxin receptors, whereupon the receptors become saturated. 
Transport-mediated uptake of LXA.sub.4 by neutrophils continues to occur 
at a rate of about 0.6 fmol/10.sup.6 cells/minute above that due to 
specific binding to receptors. When incubated in the presence of 0.5 mM 
DISA, this additional (i.e., 0.6 fmol/10.sup.6 cells/minute) LXA.sub.4 
influx is blocked, whereas the initial association of LXA.sub.4 due to 
receptor binding (3 fmol/10.sup.6 cells) is still detectable. An inhibitor 
of a lipoxin transport system can therefore be identified using 
neutrophils by identifying a molecule which blocks the additional 0.6 
fmol/10.sup.6 cells/minute influx of lipoxin into the cells above that due 
to receptor binding. Thus, this method provides a way to identify 
molecules which specifically inhibit only one of two possible routes of 
entry of lipoxin into a cell. 
In another embodiment, the cell used in the method is a differentiated 
HL-60 cell (or membrane vesicle thereof). HL-60 cells which have been 
stimulated to differentiate by exposure to retinoic acid (which induces a 
neutrophil-like morphology) have been found to express the lipoxin 
transport system upon differentiation. Other stimuli which induce HL-60 
cells to differentiate to a neutrophil-like morphology include 
dibutyryl-cAMP and dimethyl sulfoxide and it is likely that these stimuli 
can also be used to induce the lipoxin transport system on HL-60 cells. 
The lipoxin transport system has not been found to be present on 
unstimulated human erythrocytes, lymphocytes and platelets and thus these 
cell types are not preferred for use in this method. However, acquisition 
of such a transport system could arise after exposure of resting cells to 
appropriate stimuli or activating agents. Other cell types which possess 
the lipoxin transport system or which, upon stimulation, are induced to 
express the lipoxin transport system can be identified by procedures 
described earlier based upon the properties of the transport system. Such 
cells can then be used to identify inhibitors of lipoxin uptake by the 
lipoxin transport system. 
The specificity of an inhibitor identified according to the method of the 
invention can be further determined by utilizing the known properties of 
the lipoxin transport system. For example, an inhibitor specific for the 
transport system would not affect entry of molecules into the cell by 
non-ionic diff-usion. Accordingly, the inhibitor would not affect uptake 
of a lipoxin methyl ester derivative by the cell. The inhibitor might 
inhibit uptake of other molecules by other transport systems (e.g., the 
anionic inhibitors DISA, PCP, UK-5099 and mersalyl are known to also block 
other transport systems such as the Cl.sup.- /HCO.sub.3- and the H.sup.+ 
+lactate.sup.- cotransporter). Alternatively, an inhibitor might be 
specific for the lipoxin transport system and not affect the activity of 
other anion transport systems having a different substrate specificity. In 
this case, for example, the inhibitor would not affect transport by a 
Cl.sup.- /HCO.sub.3- exchanger, a sulfate carrier or an H.sup.+ 
+lactate.sup.- cotransporter present in the cell. 
Similar to the method for identifying a molecule which is an inhibitor of a 
lipoxin transport system, the invention provides a method for identifying 
a molecule which enhances transport of lipoxin by a lipoxin transport 
system or which induces the expression of the transport system on cells 
which do not constitutively possess it. The method involves providing a 
cell (or membrane vesicle thereof) which has a lipoxin transport system 
(e.g., neutrophils) or which does not have a lipoxin transport system 
constitutively but in which the transport system can be induced (e.g., 
HL-60 cells). The cell is contacted with a labeled lipoxin in the presence 
of a molecule to be tested, uptake of the lipoxin by the cell is measured 
and the ability of the molecule to enhance or induce uptake of lipoxin by 
the cell is determined. A molecule which enhances or induces uptake of 
lipoxin by the cell by the lipoxin transport system can be identified 
based upon this ability. As described earlier, lipoxin uptake by a lipoxin 
transport system is defined herein as lipoxin transport which is 
inhibitable by DISA, PCP, UK-5099, mersalyl or pHMB, thereby excluding 
lipoxin uptake mediated by specific receptors. 
The ability of a molecule to induce or enhance lipoxin uptake by the 
lipoxin transport system is determined by comparing the amount and/or rate 
of uptake of lipoxin in the presence of the molecule tested to a 
predetermined amount and/or rate of lipoxin influx into the cell by the 
lipoxin transport system in the absence of the molecule tested. For 
example, in a preferred embodiment the cell used in the method is a 
neutrophil, which has a lipoxin influx rate due to transporter-mediated 
uptake of about 0.6 fmol/10.sup.6 cells/minute. In the presence of a 
molecule which enhances transport of lipoxin by a lipoxin transport 
system, the transporter-mediated influx of lipoxin into neutrophils would 
be greater than 0.6 fmol/10.sup.6 cells/minute. Alternatively, a cell 
which does not transport lipoxin in the absence of a molecule to be tested 
can be used to identify an inducer of lipoxin transport. In this case, the 
rate of transport of lipoxin by the lipoxin transport system in the 
absence of an inducer molecule would be 0 fmol/10.sup.6 cells/minute and 
in the presence of an inducer molecule would be greater than 0 
fmol/10.sup.6 cells/minute. 
In another embodiment, the cell used to identify a molecule which induces 
or enhances lipoxin transport by a lipoxin transport system is an HL-60 
cell (or membrane vesicle thereof). For example, when HL-60 cells are 
differentiated (e.g., by treatment with retinoic acid) they express the 
lipoxin transport system. Thus, differentiated HL-60 cells can be used to 
identify an enhancer of the lipoxin transport system. Alternatively, in an 
undifferentiated state, HL-60 cells do not express the lipoxin transport 
system but can be induced to express the system. Thus, undifferentiated 
HL-60 cells can be used to identify an inducer of a lipoxin transport 
system. The lipoxin transport system has not been found to be present on 
unstimulated human erythrocytes, lymphocytes and platelets and thus these 
cell types are not preferred for identifying an enhancer of the transport 
system. However, these or other cell types can be stimulated with a 
molecule to determine whether the moelcule induces lipoxin transport by 
the lipoxin transport system. Other cell types which possess the lipoxin 
transport system can be identified by procedures described earlier based 
upon the properties of the transport system and used to identify enhancers 
of lipoxin transport. 
Lipoxin uptake by mechanisms other than the lipoxin transport system, e.g. 
binding of lipoxin to specific lipoxin receptors and non-ionic diffusion 
of methyl ester lipoxin derivatives into a cell, would not be affected by 
molecules which specifically induce or enhance lipoxin transport by the 
lipoxin transport system. The specificity of an inducer or enhancer of the 
lipoxin transport system can be determined by measuring the binding of 
labeled lipoxin to specific receptors (i.e., the DISA-insensitive 
component of lipoxin uptake by the cell) or the uptake of labeled lipoxin 
methyl ester by the cell in the presence and absence of the molecule. 
C. Inhibiting or Enhancing Lipoxin Transport by a Lipoxin Transport System 
The lipoxin transport system of the invention provides a pathway for 
lipoxin entry into a cell which can be targeted in order to increase or 
decrease lipoxin uptake by the cell. Accordingly, the invention provides a 
method for inhibiting uptake of a lipoxin by a cell which has a lipoxin 
transport system comprising contacting the cell with a molecule which is 
an inhibitor of the lipoxin transport system. In one embodiment, the 
inhibitor of the lipoxin transport system is a weak organic acid (pK'4-6) 
so that at physiological pH it exists predominantly as an anion. Examples 
of anionic compounds which can inhibit uptake of lipoxin by the lipoxin 
transport system include DISA, PCP and UK-5099. Certain anionic compounds 
do not inhibit lipoxin transport by the lipoxin transport system. These 
compounds include probenecid and disulfonic stilbene SITS. In another 
embodiment, the inhibitor of the lipoxin transport system is an 
organomercurial agent. Examples of organomercurial agents which can 
inhibit uptake of lipoxin by the lipoxin transport system include mersalyl 
and pHMB. Certain other sulfhydryl-reactive compounds (which includes the 
organomercurial agents) do not inhibit transporter-mediated uptake. These 
compounds include NEM, iodoacetate and 2,2'-DTBP. Additional compounds 
which can inhibit lipoxin uptake by the lipoxin transport system are 
diphenylamine-2-carboxylate and niflumate. Other compounds which can 
inhibit uptake of lipoxin by the lipoxin transport system can be 
identified by the aforementioned method for identifying such molecules. 
Once identified, a molecule which is an inhibitor of a lipoxin transport 
system can be used to inhibit lipoxin uptake by a cell by contacting the 
molecule with a cell which has a lipoxin transport system. The term 
"contacting" as used herein is intended to include incubating a cell with 
a molecule in vitro, e.g., adding the molecule to a medium containing the 
cell in vitro, and exposing the cell to the molecule in vivo, e.g., 
administering the molecule in vivo by a route such that the cell will be 
contacted by the molecule. The term "contacting" is also intended to 
include other possible methods of introducing a molecule into a cell, such 
as by transfection (e.g., of nucleic acid molecules), microinjection, 
liposome-mediated transfer etc. which result in uptake of the molecule by 
the cell. Non-limiting inhibitory concentrations for known inhibitor 
molecules are as follows (expressed as K.sub.0.5, the concentration at 
which 50% of lipoxin transport is inhibited): DISA-12 .mu.M; PCP-25 .mu.M; 
and mersalyl-110 .mu.M. 
A cell which has both a lipoxin transport system and specific lipoxin 
receptors can be contacted with a molecule which inhibits lipoxin uptake 
by the lipoxin transport system to specifically inhibit entry of lipoxin 
into the cell via this pathway while not affecting entry of lipoxin into 
the cell via specific receptors. Blocking of the transport-mediated 
pathway for lipoxin uptake while preserving the function of the 
receptor-mediated pathway for lipoxin uptake can be useful for 
specifically interfering with lipoxin-mediated responses which are 
stimulated through the transport pathway while maintaining 
lipoxin-mediated responses which are stimulated through binding of lipoxin 
to specific receptors. Alternatively, it may be desirable to inhibit 
lipoxin entry into cells by both mechanisms (i.e., transporter-mediated 
and receptor-mediated) in order to interfere with lipoxin-mediated 
responses involving both pathways. Accordingly, a cell can be contacted 
both with a molecule which inhibits lipoxin uptake by the lipoxin 
transport system and with a molecule which inhibits binding of lipoxin to 
specific lipoxin receptors. 
The invention further provides a method for inducing or enhancing transport 
of a lipoxin into a cell which has a lipoxin transport system comprising 
contacting the cell with a molecule which induces or enhances transport of 
a lipoxin by the lipoxin transport system. For example, certain molecules 
have been identified which increase the initial rate of uptake of lipoxin 
by neutrophils by the lipoxin transport system. These molecules, which can 
thus function as enhancers of lipoxin transport, include 
n-formyl-methionyl-leucyl-phenylalanine (FMLP), phorbol myristate acetate 
(PMA) and the cationic ionophore A23187. Accordingly, a cell (e.g., a 
neutrophil) can be contacted with FMLP, PMA or A23187 to enhance uptake of 
lipoxin by the cell by the lipoxin transport system. Other compounds which 
can induce or enhance uptake of lipoxin by the lipoxin transport system 
can be identified by the aforementioned method for identifying such 
molecules. Once identified, a molecule which is an inducer or enhancer of 
a lipoxin transport system can be used to induce or enhance lipoxin uptake 
by a cell by contacting the molecule with a cell which has a lipoxin 
transport system or in which a lipoxin transport system can be induced. 
D. Other Substrates for the Lipoxin Transport System 
The lipoxin transport system of the invention exhibits substrate 
specificity in that it does not transport certain other arachidonic acid 
derivatives such as PGE.sub.2, 15-HETE, LTB.sub.4, LTC.sub.4 or LTD.sub.4 
to the same degree as it transports lipoxins (see Example 6). There is 
also evidence that the lipoxin transport system exhibits stereospecificity 
in that the 11-transcontaining isomer of LXA.sub.4 is transported at about 
a 3-fold lower rate relative to the native 11-cis-containing LXA.sub.4. 
The ability of a compound to be transported by the lipoxin transport system 
(i.e., to be a substrate for the system) can be determined directly by 
labeling a molecule to be tested with a detectable substance, contacting a 
cell having a lipoxin transport system with the labeled molecule and 
measuring uptake of the labeled molecule by the lipoxin transport system. 
That the molecule is transported by the lipoxin transport system is 
determined based upon the known properties of the lipoxin transport 
system, such as inhibition of transport by DISA, PCP, UK-5099, mersalyl or 
pHMB, and/or other additional distinguishing characteristics as described 
in Section A. 
The ability of a compound to be transported by the lipoxin transport system 
(i.e., to be a substrate for the system) can also be determined based upon 
its ability to competitively inhibit uptake of a labeled lipoxin by the 
lipoxin transport system (i.e., can act as a competing substrate), thereby 
reducing transport of the labeled lipoxin by the transport system when 
both the test compound and the labeled lipoxin are incubated with a cell 
having a lipoxin transport system. For example, a test compound and a 
radiolabeled LXA.sub.4 can be incubated with neutrophils and the amount 
and/or rate of uptake of the radiolabeled LXA.sub.4 by the cells in the 
presence of the test compound can be compared with the amount and/or rate 
of uptake of the radiolabeled LXA.sub.4 by the cells in the absence of the 
test compound. A compound which is also transported by the lipoxin 
transport system will compete with the radiolabeled LXA.sub.4 for 
transporter-mediated uptake, thereby reducing the uptake of the 
radiolabeled LXA.sub.4. 
In a preferred embodiment, a compound which can act as a substrate for the 
lipoxin transport system is a lipoxin analog of a natural lipoxin. The 
term "lipoxin analog" as used herein is intended to include any compound 
which binds a lipoxin receptor recognition site or binds a macromolecule 
or complex of macromolecules, including an enzyme and its cofactor, which 
is bound by a lipoxin. Lipoxin analogs include compounds which are 
structurally similar to a natural lipoxin, compounds which share the same 
receptor recognition site, compounds which share the same or similar 
lipoxin metabolic transformation region as lipoxin, and compounds which 
are art-recognized as being analogs of lipoxin. Lipoxin analogs include 
lipoxin analog metabolites. Lipoxin analogs include compounds such as 
those described in U.S. patent application Ser. No. 08/077,300 by C. N. 
Serhan, which is hereby incorporated by reference. The term "lipoxin 
analog" is further understood to encompass compounds containing 
radioactive isotopes, such as but not limited to tritium (.sup.3 H), 
deuterium (.sup.2 H), carbon (.sup.14 C), or otherwise. The lipoxin analog 
can be radiolabeled or derivatized, for example to determine their uptake 
by a lipoxin transport system. Lipoxin analogs can also be labeled with 
other detectable substances, such as fluorescent labels. The term "natural 
lipoxin" as used herein is intended to refer to a naturally-occurring 
lipoxin or lipoxin metabolite. Where an analog has activity for a 
lipoxin-specific receptor, the natural lipoxin is the normal ligand for 
that receptor. For example, where an analog is a LXA.sub.4 analog having 
specific activity for a LXA.sub.4 specific receptor on differentiated 
HL-60 cells, the corresponding lipoxin is LXA.sub.4. Where an analog has 
activity as an antagonist to another compound (such as a leukotriene), 
which is antagonized by a naturally-occurring lipoxin, that lipoxin is the 
corresponding natural lipoxin. 
Nonlimiting examples of the structures and syntheses of both lipoxins and 
lipoxin analogs are illustrated in the following patents and publications: 
Nicolaou, K. C. et al. (1989). Biochim. Biophys. Acta 1003:44-53; 
Nicolaou, K. C. et al. (1989). J. Org. Chem. 54: 5527-5535; Nicolaou, K. 
C. et al.(1991). Angew. Chem. Int. Ed. Engl. 30: 1100-1116; U.S. Pat. Nos. 
4,576,758; 4,560,514; 5,079261; and 5,049,681; and JP Patent Nos. 
3,227,922; 63,088,153; 62,198,677; and 1,228,994. Methods of making 
lipoxin analogs which have longer tissue half-lives than the corresponding 
natural lipoxin are illustrated in the Serhan, C. N., U.S. patent 
application entitled "Lipoxin Analogs," Ser. No. 08/077,300 (cited supra). 
Analogs may also be synthesized by a person of ordinary skill using the 
well-known methods of eicosanoid synthesis illustrated in the cited 
references. 
The invention provides a method for identifying a lipoxin analog which is 
transported by a lipoxin transport system. The method involves providing a 
cell (or membrane vesicle thereof) which has a lipoxin transport system, 
contacting the cell with a lipoxin analog to be tested together with a 
labeled natural lipoxin which is transported by the lipoxin transport 
system, measuring the rate of uptake of the labeled natural lipoxin by the 
cell, determining the ability of the natural lipoxin to competitively 
inhibit uptake of the natural lipoxin, said uptake being inhibitable by 
DISA, PCP, UK-5099, mersalyl or pHMB, and identifying a lipoxin analog 
which is transported by a lipoxin transport system by the ability of the 
lipoxin analog to competitively inhibit uptake of the natural lipoxin by 
the lipoxin transport system. Preferred cells, or membrane vesicles 
thereof, for use in the method are neutrophils and differentiated HL-60 
cells, or membrane vesicles thereof. For example, neutrophils are 
incubated with a lipoxin analog and a radiolabeled natural lipoxin (e.g. 
.sup.3 H-LXA.sub.4) and uptake of the labeled natural lipoxin by the 
neutrophils is assessed by measuring the cell-associated radiolabel over 
time. The amount and/or rate of uptake of the labeled natural lipoxin in 
the presence of the lipoxin analog is compared to a predetermined amount 
and/or rate of uptake of the natural lipoxin by the cells in the absence 
of the lipoxin analog (determined by procedures described earlier). A 
lipoxin analog which is transported by a lipoxin transport system is 
identified based upon the ability of the lipoxin analog to competitively 
inhibit (i.e., decrease) uptake of the natural lipoxin by the lipoxin 
transport system (i.e., uptake which is inhibitable by DISA, PCP, UK-5099, 
mersalyl or pHMB). For example, transporter-mediated uptake of the natural 
lipoxin LXA.sub.4 by neutrophils is known to occur at a rate of about 0.6 
fmol/10.sup.6 cells/minute above that due to specific receptor binding in 
the absence of a lipoxin analog. In the presence of a lipoxin analog which 
is transported by a lipoxin transport system, transporter-mediated uptake 
of natural lipoxin A.sub.4 by neutrophils is at a rate lower than about 
0.6 fmol/10.sup.6 cells/minute. 
Additionally, uptake of a lipoxin analog by the lipoxin transport system 
can be assessed directly using a labeled lipoxin analog. Accordingly, an 
alternative method for identifying a lipoxin analog which is transported 
by a lipoxin transport system involves providing a cell with a lipoxin 
transport system, contacting the cell with a lipoxin analog which is 
labeled (e.g., radiolabeled), measuring uptake of the labeled lipoxin 
analog by the lipoxin transport system, wherein said uptake is inhibitable 
by DISA, PCP, UK-5099, mersalyl or pHMB, and identifying a lipoxin analog 
which is transported by a lipoxin transport system by the uptake of the 
lipoxin analog. The rate of uptake of a lipoxin analog can be determined 
(as described in the Examples) and a lipoxin analog which is transported 
at a faster rate than a natural lipoxin by a lipoxin transport system can 
be identified. For example, a lipoxin analog which is transported at a 
faster rate than a natural lipoxin by neutrophils can be identified by the 
uptake of the lipoxin analog by neutrophils at a rate greater than about 
0.6 fmol/10.sup.6 cells/minute. 
E. Modulating Lipoxin-Mediated Responses 
Lipoxins are known to regulate a variety of biochemical and physiological 
events, including events involved in cell activation, signal transduction 
and stimulus-response coupling (see for example Samuelsson, et al. (1987) 
Science 237:1171-1176). Modulating the function of the lipoxin transport 
system of the invention provides a means by which to upregulate or 
downregulate lipoxin-mediated responses through increasing or decreasing 
uptake of lipoxins by a cell via the lipoxin transport system. 
Molecules which inhibit, enhance or induce uptake of lipoxins by the 
lipoxin transport system can be used to modulate lipoxin-mediated 
responses. For example, an inhibitor of a lipoxin transport system can be 
used to increase or decrease a physiological response, depending upon 
whether uptake of a lipoxin by the transport system stimulates or inhibits 
the particular response. For example, a physiological response by a cell 
which is increased by uptake of a lipoxin by the lipoxin transport system 
can be decreased by contacting the cell with an inhibitor of the lipoxin 
transport system to decrease lipoxin uptake by the cell. Alternatively, a 
physiological response which is decreased by uptake of a lipoxin by the 
lipoxin transport system can be increased by contacting the cell with an 
inhibitor of the lipoxin transport system to decrease lipoxin uptake by 
the cell. Likewise, an inducer or an enhancer of a lipoxin transport 
system can be used to increase or decrease physiological responses, 
depending upon whether uptake of a lipoxin by the transport system 
stimulates or inhibits a particular response. For example, a physiological 
response by a cell which is increased by uptake of a lipoxin by the 
lipoxin transport system can be increased by contacting the cell with an 
enhancer of the lipoxin transport system to increase lipoxin uptake by the 
cell. Alternatively, a physiological response which is decreased by uptake 
of a lipoxin by the lipoxin transport system can be decreased by 
contacting the cell with an enhancer of the lipoxin transport system to 
increase lipoxin uptake by the cell. 
One type of physiological response which can be modulated by lipoxins is 
phagocytosis by neutrophils. LXA.sub.4 and LXB.sub.4 can inhibit 
phagocytosis by neutrophils which have been stimulated to be phagocytic 
(e.g., by exposure to particulate agents, such as red blood cells see 
Example 9!, microorganisms or crystals; while not required, the level of 
phagocytosis can be further enhanced by soluble stimuli such as FMLP). The 
inhibition of phagocytosis mediated by LXA.sub.4 displays a pH dependence 
which is consistent with LXA.sub.4 being taken up by the neutrophils by 
the lipoxin transport system. Accordingly, it is likely that neutrophil 
phagocytosis can be increased by decreasing lipoxin uptake by the lipoxin 
transport system (e.g., by raising the extracellular pH or by contacting 
the cell with an inhibitor of the lipoxin transport system). 
Alternatively, it is likely that neutrophil phagocytosis can be decreased 
by increasing lipoxin uptake by the lipoxin transport system (e.g., by 
lowering the extracellular pH or by contacting the cell with an enhancer 
of the lipoxin transport system). 
Other neutrophil-mediated physiological responses may also be stimulated or 
inhibited by uptake of lipoxins by the lipoxin transport system. For 
example, it has been shown that LXA.sub.4 can inhibit chemotaxis of 
polymorphonuclear leukocytes (Lee, T. H., et al. (1991) Biochem. Biophys. 
Res. Commun. 180:1416). Many neutrophil-mediated responses can be measured 
by standard in vitro assays. For example, neutrophil chemotaxis and 
granule enzyme release can be assayed as described in Simchowitz, L. and 
Cragoe, E. J. (1986) J. Biol. Chem. 261:6492-6500. The generation of 
superoxide radicals by neutrophils can be measured as described in 
Simchowitz, L. (1985) J. Clin. Invest. 76:1079-1089. These assays can be 
performed in the presence and absence of lipoxins (e.g., LXA.sub.4 and/or 
LXB.sub.4) to determine the effect of lipoxins (e.g., stimulation or 
inhibition) on these neutrophil responses. It can then be determined 
whether a physiological response by a neutrophil which is modulated (e.g., 
up- or down-regulated) by a lipoxin involves uptake of the lipoxin by the 
lipoxin transport system on the neutrophil. For example, the response can 
be measured in the presence of the lipoxin together with an inhibitor or 
enhancer of the lipoxin transport system (e.g., pH 8.4 can be used to 
inhibit the activity of the lipoxin transport system or an inhibitory 
compound can be used; alternatively, pH 6.4 can be used to enhance the 
activity of the lipoxin transport system or an enhancer compound can be 
used). 
Additionally, many responses by other cell types (in addition to 
neutrophils) and many systemic responses are known to be stimulated or 
inhibited by lipoxins. For example, LXA.sub.4 has vasodilatory effects and 
inhibits leukotriene-mediated vasoconstriction and leukotriene-mediated 
inflammation (see for example Badr, K. F. et al (1989) Proc. Natl. Acad. 
Sci. USA 86:3438; Hedqvist, P. et al. (1989) Acta Physiol. Scand. 137:571; 
Katoh, T. et al. (1992) Am. J.Physiol. 263 (Renal Fluid Electrolyte 
Physiol. 32) F436). LXA.sub.4 can also stimulate myeloid bone marrow 
colony formation (see for example Stenke, L., et al. (1991) Biochem. 
Biophys. Res. Commun. 180:255). The involvement of the lipoxin transport 
system in uptake of lipoxins by cells, resulting in stimulation or 
inhibition of such responses, can be determined based upon the 
characteristics of the lipoxin transport system provided by the invention. 
Physiological responses in which lipoxin uptake is mediated by the lipoxin 
transport system can then be modulated using inhibitors, enhancers and 
inducers of the lipoxin transport system as described herein. 
A lipoxin analog which maintains the ability to mediate physiological 
responses but which is transported at a faster rate than a natural lipoxin 
by a lipoxin transport system can also be used to modulate 
lipoxin-mediated responses. The invention provides a method for 
stimulating a lipoxin-mediated response by a cell which has a lipoxin 
transport system comprising contacting the cell with a lipoxin analog 
which is transported into the cell by the lipoxin transport system at a 
faster rate than a natural lipoxin. Such a lipoxin analog can be 
identified by procedures described earlier. 
The present invention is further illustrated by the following examples 
which should in no way be construed as being further limiting. The 
contents of all references, issued patents and published patent 
applications cited throughout all portions of this application including 
the background are expressly incorporated by reference. 
The following methodology was used in the Examples. 
Incubation Media 
The standard medium, Dulbecco's phosphate-buffered saline (PBS) with 
Ca.sup.2+ and Mg.sup.2+ (Bio-Whittaker Walkersville, Md.!), used in this 
study had the following composition: 138 mM NaCl, 8.1 mM Na.sub.2 
HPO.sub.4, 2.7 mM KCl, 1.1 mM KH.sub.2 PO.sub.4, 0.9 mM CaCl.sub.2, and 
0.5 mM MgCl.sub.2. The medium was supplemented with 5.6 mM glucose and the 
pH brought to 7.40 with NaOH. The cation composition of the media was 
manipulated by substituting equimolar amounts of either K.sup.+ or 
N-methyl-D-glucamine for Na.sup.+. For experiments in which the 
extracellular pH (pH.sub.o) of the media was varied between 5.0 and 8.4, 
the solutions were buffered with MES (pK'6.0), HEPES (pK'7.3), or Tricine 
(pK'7.8) as appropriate. 
Cell Isolation Procedures 
Neutrophils: Human peripheral neutrophils were isolated from heparinized 
blood by sequential Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, 
N.J.) gradient centrifugation and dextran sedimentation at room 
temperature (Boyum, A. (1968) Scand. J. Clin. Lab. Invest. 21 (suppl. 
97):77-89). Contaminating erythrocytes were removed by hypotonic lysis in 
0.22% saline for 30 seconds. The neutrophils were washed twice and then 
counted. The purity of the neutrophil suspensions averaged 98%. Cell 
viability (&gt;99%), as assessed by eosin Y exclusion, was not affected by 
any of the agents or incubation conditions tested. Cells were kept in the 
standard medium for 1 hour at 37.degree. C. prior to experimentation. All 
assays were carried out at 37.degree. C. 
HL-60 Cells: HL-60 cells were seeded into RPMI medium supplemented with 100 
U/ml penicillin, 100 .mu.g/ml streptomycin, and 10% fetal calf serum 
(Hyclone, Logan, Utah) and grown at 37.degree. C. in 250 ml flasks in a 5% 
CO.sub.2 /95% air atmosphere. Cells were induced to differentiate toward a 
neutrophil-like phenotype by exposure to 1 .mu.M retinoic acid for 5 days. 
Red Blood Cells (RBC): RBC were obtained directly from the heparinized 
blood of normal human donors. During repeated (four) washing steps, the 
uppermost portion of the cell pellets was aspirated and discarded to 
remove residual minor contamination of buffy coat cells. 
Lymphocvtes: The lymphocyte-rich interface layer was taken at the 
Ficoll-Hypaque gradient step of the neutrophil purification procedure. 
Platelets were removed by slow-speed centrifugation (see below) and 
contaminating monocytes and neutrophils by adherence to plastic Petri 
dishes for 1 hour at 37 .degree. C. 
Platelets: Platelets were purified to homogeneity from acid citrate 
dextrose-treated blood according to established protocols (Romano, M. and 
Serhan, C. N. (1992) Biochemistry 31:8269-8277). 
Radiolabeled Compounds 
11,12-.sup.3 H!LXA.sub.4, henceforth designated .sup.3 H!LXA.sub.4, and 
its methyl ester derivative, .sup.3 H!LXA.sub.4 ME, were prepared from 
11,12-acetylenic LXA.sub.4 ME (Cascade) by a custom tritiation performed 
at the New England Nuclear/Dupont Tritiation Laboratory (Boston, Mass.). 
The products were isolated by RP-HPLC as reported previously (Fiore, S. et 
al., (1992) J. Biol. Chem. 267:16168-16176) after LiOH saponification. The 
specific activity was 40.5 Ci/mmole. 
Reagents and Chemicals 
Inorganic salts were obtained from Fisher Scientific, St. Louis, Mo. The 
following reagents were purchased from Sigma Chemical Company, St. Louis, 
Mo.: N-methyl-D-glucamine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic 
acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 
N-tris(hydroxymethyl)methylglycine (Tricine), Nethylmaleimide (NEM), 
D-glucose, 3,5-diiodosalicylic acid (DISA), mersalyl, pentachlorophenol 
(PCP), probenecid, sodium p-hydroxymercuribenzoate (pHMB), niflumic acid, 
and iodoacetic acid. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic 
acid (SITS) was bought from Pierce Chemical Co., Rockford, Ill.; 
diphenylamine-2-carboxylic acid from Fluka; and arachidonic acid, 
prostaglandin E.sub.2 (PGE.sub.2), leukotriene B.sub.4 (LTB.sub.4), 
leukotriene C.sub.4 (LTC.sub.4), leukotriene D.sub.4 (LTD.sub.4), and 
15-HETE from BIOMOL Research Laboratories, Plymouth Meeting, Pa. 
.alpha.-Cyano-.beta.-(1-phenylindol-3-yl)acrylic acid (UK-5099) was 
graciously provided by Pfizer Central Research Laboratories, Sandwich, 
Kent, UK. 
Unidirectional Tracer Flux Measurements 
Incubations were performed at 37.degree. C. in capped, plastic tubes 
(Falcon Plastics, Oxnard, Calif.) under various experimental conditions 
(neutrophils 15-20.times.10.sup.6 /ml). Influx experiments were performed 
in the presence of .sup.3 H!LXA.sub.4 or .sup.3 H!LXA.sub.4 ME (0.1 
.mu.Ci/ml) at a final concentration of 5 nM. At stated intervals, 
duplicate aliquots of the cell suspensions were layered onto 0.5 ml of 
silicone oil (Versilube F-5, General Electric Corp., Waterford, N.Y.) 
contained in 1.5 ml plastic tubes and centrifuged for 1 min at 8,000 g in 
a microcentrifuge (Beckman Instruments, Fullerton, Calif.). Cell 
separation occurred in &lt;5 s. 
The aqueous and oil phases were aspirated and discarded. The neutrophil 
pellets were excised and counted in a liquid scintillation counter (Wallac 
1409, Pharmacia LKB Nuclear, Gaithersburg, Md.). Influx is expressed here 
as fmol/10.sup.6 cells/min whereas flux rates in all previous work from 
our laboratory (L.S.) have been reported in meq/liter of cell water.min. 
For ease of comparison, the unit of fmol/10.sup.6 cells/min can be 
converted to nmol/liter of cell water.min by dividing by 0.274 based on a 
cell water volume of 0.274 .mu.l/10.sup.6 cells (Simchowitz, L., et al. 
(1992) J. Gen. Physiol. 22:453-479). For efflux studies, neutrophils were 
loaded with .sup.3 H!LXA.sub.4 (0.1 .mu.Ci/ml) for 15 min at 37.degree. 
C. in the standard medium. 
Thereafter, the cells were spun down and then resuspended in the various 
experimental solutions at 37.degree. C. Aliquots were taken at stated 
intervals for measurement of the amount of residual radioactivity that 
remained associated with the cell pellet. Samples were spun over silicone 
oil and handled as described for influx studies. 
Ligand Binding Assays 
Binding experiments were performed at 4.degree. C. in an ice-water bath as 
described above for the tracer flux determinations except that the fmal 
concentration of .sup.3 H!LXA.sub.4 was reduced to 0.3 nM. This value 
approximates the K.sub.d for binding (0.5.+-.0.3 nM, Fiore (1992) J. Biol. 
Chem. 267:16168-16176). Unlabeled LXA.sub.4 was added to a parallel set of 
tubes in 1000-fold excess to determine total and nonspecific binding, 
respectively. 
Data Analysis 
Transport system-mediated influx of .sup.3 H!LXA.sub.4, corrected for the 
very rapid (complete within 1 min), inhibitor-resistant specific binding, 
followed equations of the form: 
EQU C.sub.t =C.sub..infin. 1=exp (-kt)! (1) 
where C.sub.t is the cell label at time t, C.sub..infin. is the cell label 
at steady-state, and k is the rate coefficient. Equation 1 was fit to the 
data by a nonlinear least-squares program, and the initial influx rate 
computed from the product kC.sub..infin.. The change in some of the 
measured variables often appeared to be linear over the period of study; 
in those cases, the influx rate was computed from the slope of the linear 
regression line. Trapping of label within the extracellular space of the 
cell pellet, determined using .sup.3 H!H.sub.2 O and .sup.14 C!inulin, 
was negligibly small. The efflux rate coefficients were computed by 
least-squares fitting the time course data to a single exponential 
equation. 
Abbreviations 
The abbreviations used are: lipoxin A.sub.4 (LXA.sub.4), 
(5S,6R,15S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid; lipoxin 
A.sub.4 methyl ester (LXA.sub.4 ME); 3,5-diiodo-salicylic acid (DISA); 
pentachlorophenol (PCP); .alpha.-cyano-.beta.-(1-phenylindol-3-yl)acrylic 
acid (UK-5099); p-hydroxymercuribenzoate (pHMB); N-ethylmaleimide (NEM); 
4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS); 
prostaglandin E.sub.2 (PGE.sub.2); leukotriene B.sub.4 (LTB.sub.4), 
(5S,12R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid; leukotriene 
C.sub.4 (LTC.sub.4), 
(5S)-hydroxy-(6R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic 
acid; leukotriene D.sub.4 (LTD.sub.4), 
(5S)-hydroxy-(6R)-S-cysteinylglycyl-7,9-trans-11,14-ciseicosatetraenoic 
acid; 15-HETE, (15S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid; 
reverse phase-high pressure liquid chromatography (RP-HPLC). 
EXAMPLE 1 
Effect of Inhibitors and pH on LXA.sub.4 Influx into Neutrophils 
The time course of influx of .sup.3 H!LXA.sub.4 into isolated human 
neutrophils is presented in FIG. 1. Also shown is the effect, or lack 
thereof, of different extracellular ionic conditions and drugs. At 
zero-time, cells were resuspended in media containing labeled LXA.sub.4 at 
a total concentration of 5 nM. At stated times, aliquots of the neutrophil 
suspensions were taken and the cell pellets isolated by rapid 
centrifugation through a cushion of silicone oil and counted for 
radioactivity. Cell-associated .sup.3 H!LXA.sub.4 is expressed as 
fmol/10.sup.6 cells. Results represent the means.+-.SEM of three to seven 
separate experiments, each performed in duplicate. 
The studies shown in the left panel were conducted in the presence of 
medium alone at pH.sub.o 7.40 (Control), 1 mM probenecid, 0.4 mM SITS, 
Na.sup.+ -free medium (equimolar replacement by N-methyl-D-glucamine), 
high K.sup.+ medium (120 mM K.sup.+, 25 mM Na.sup.+), medium at pH.sub.o 
6.40, and medium at 8.40. The upper and middle curves are single 
exponential fits of the data points starting at 2.88 fmol/10.sup.6 cells: 
for pH.sub.o 6.40 (upper curve), initial influx rate=5.5.+-.1.0 
fmol/10.sup.6 cells/min and fmal uptake=15.1.+-.0.6 fmol/110.sup.6 cells; 
for Control, SITS, probenecid, Na.sup.+ -free medium, and high K.sup.+ 
medium (combined data, middle curve), initial influx rate=0.57.+-.0.09 
fmol/10.sup.6 cells/min and final uptake=7.26.+-.0.26 fmol/10.sup.6 cells. 
The pH.sub.o 8.40 data were fit to a straight line with a 
slope=-0.002.+-.0.013 fmol/10.sup.6 cells/min. 
The studies shown in the right panel were conducted in the presence of 
medium alone, 0.5 mM DISA, 0.5 mM mersalyl, 0.4 mM PCP, and 0.2 mM 
UK-5099. The Control data set is the same as that shown in the left-hand 
panel. Note, however, that the Y-axis scale is different in the two 
panels. The curves for medium (Control), mersalyl, UK-5099, and PCP are 
single exponential fits with initial influx rates of 0.57.+-.0.12, 
0.10.+-.0.01, 0.030.+-.0.009, and 0.015.+-.0.004 fmol/10.sup.6 cells/min. 
A horizontal line has been drawn at 2.88, the average of all of the DISA 
data points (a fit of the DISA data set to a straight line gave a slope of 
0.0046.+-.0.0051 fmol/10.sup.6 cells/min, which could not be distinguished 
from zero). 
From an external concentration of 5 nM, uptake under control conditions 
appears to be divisible into at least two components: (1) a rapid portion 
that is complete by 1 min, equivalent to .about.3 fmol/10.sup.6 cells and 
(2) a more gradual and prolonged phase that takes place over the next 30 
min. This impression was confirmed through the use of a variety of 
different inhibitors and extracellular pH. Inspection of data in the left 
and right panels reveals that DISA, PCP, UK-5099, and mersalyl (each at 
0.2-0.5 mM) all inhibited the uptake of .sup.3 H!LXA.sub.4. These 
compounds have previously been observed to block several different anion 
transport processes in human neutrophils including Cl.sup.- /HCO.sub.3 
-exchange (Simchowitz, L., et al. (1988) in Cell Physiology of Blood 
(Gunn, R. B. and Parker, J. C., eds.) pp. 193-208, Rockefeller University 
Press, New York; Simchowitz, L., et al. (1991) Am. J. Physiol. 
261:C906-C915), H.sup.+ +lactate.sup.- cotransport (Simchowitz, L. and 
Textor, J. A. (1992) J. Gen. Physiol. 100:341-367), and cell 
swelling-induced Cl.sup.- channels (Stoddard, J. S., et al., (1993) Am. J. 
Physiol. 26:C156-C165). Of interest, two general inhibitors of anion 
transport in these and other cells (Simchowitz, L. and Davis, A. O. (1989) 
J. Gen. Physiol. 94:95-124), probenecid (1 mM) and the disulfonic stilbene 
SITS (0.4 mM), had no apparent impact on .sup.3 H!LXA.sub.4 influx. All 
of these drugs are weak organic acids (pK'4-6) so that at physiologic pH 
they exist predominantly in the form of anions. This should also be the 
case for LXA.sub.4 and on this basis, we speculated that at least one of 
the two components of .sup.3 H!LXA.sub.4 uptake might represent a 
specialized transport system for LXA.sub.4 anion. The analysis to follow 
demonstrates that the initial, rapid portion of LXA.sub.4 uptake can be 
largely ascribed to specific binding to putative receptors while the 
second, more gradual phase of uptake indeed represents transport 
system-mediated influx into the cell. 
In the presence of 0.5 mM DISA, 2.9 fmol/10.sup.6 cells of uptake occurs by 
1 min of incubation, but no additional increase in cell-associated counts 
can be detected over the next 30 min. We propose that the uptake observed 
at 1 min constitutes binding to receptors and that this concentration of 
DISA completely blocks the second component of uptake referable to 
transport system-mediated transport. Note that the second phase of .sup.3 
H!LXA.sub.4 influx with all of the other drugs also appears to originate 
at the same starting value, namely, .about.3 fmol/10.sup.6 cells at 1 min. 
If this indeed represents receptor binding, this level signifies 
.about.1700 sites/cell which agrees with the finding of .about.1830 
sites/cell previously reported (Fiore, S., et al., (1992) J. Biol. Chem. 
26:16168-16176). 
Considering the DISA-resistant uptake as background, influx into control 
cells proceeded at a rate of 0.57.+-.0.12 fmol/10.sup.6 cells/min. In the 
presence of 0.4 mM PCP, 0.2 mM UK-5099, and 0.5 mM mersalyl, influx rates 
of 0.015.+-.0.004, 0.030.+-.0.009, and 0.10.+-.0.01 fmol/10.sup.6 
cells/min were observed, corresponding to inhibitions of 97, 95, and 82%. 
Marked inhibition (&gt;85%) was also observed with 0.5 mM 
diphenylamine-2-carboxylate and 0.2 mM niflumate. Reverse phase-high 
pressure liquid chromatography analysis using tandem electrochemical 
detection and ultraviolet monitoring indicated preservation of the 
integrity of LXA.sub.4 after 30 min of exposure to DISA, PCP, UK-5099, and 
mersalyl. These findings indicate that lack of appreciable uptake of 
.sup.3 H!LXA.sub.4 in the presence of inhibitors was not the result of 
either chemical degradation or the interaction of .sup.3 H!LXA.sub.4 with 
the drugs. 
EXAMPLE 2 
The LXA.sub.4 Transport System Is Consistent With an Anion Cotransport 
System 
The possible ionic basis of this presumed DISA-inhibitable LXA.sub.4 
transport was investigated next. The data of FIG. 1 (left panel) indicate 
that complete removal of extracellular Na.sup.+ (equimolar replacement by 
N-methyl-D-glucamine.sup.+) had no significant effect on total uptake, 
thereby negating a Na.sup.+ -dependent process. Likewise, raising 
extracellular K.sup.+ from 4 to 120 mM, thereby depolarizing membrane 
voltage from .about.-60 to .about.0 mV, caused no change in the time 
course of .sup.3 H!LXA.sub.4 entry. This finding lends credence to an 
electroneutral mechanism. In contrast, varying extracellular pH had a 
profound effect: lowering pH.sub.o to 6.40 dramatically increased both the 
initial rate and the final steady-state level of uptake, while raising 
pH.sub.o to 8.40 essentially abolished uptake, reducing influx to levels 
indistinguishable from that with DISA. In fact, at pH.sub.o 8.40, there 
was no measurable DISA-sensitive influx. The enhancement of .sup.3 
H!LXA.sub.4 influx by acidification and suppression by alkalinization are 
compatible with an H.sup.+ +LXA.sub.4 anion cotransport system. These 
results, however, are equally consistent with (a) entry of LXA.sub.4 by 
non-ionic diffusion as the undissociated free acid, (b) pH-induced changes 
in binding to or recruitment of surface receptors, and (c) allosteric 
effects of pH on the hypothetical transporter. 
EXAMPLE 3 
LXA.sub.4 Transport Is Not By Ionic Diffusion or by Specific Receptor 
Binding 
Three lines of evidence provide strong arguments against an appreciable 
role for simple diffusion of LXA.sub.4 as the free acid. First, the pH 
dependence of .sup.3 H!LXA.sub.4 influx into neutrophils was measured 
(FIG. 2). The studies were conducted as in FIG. 1 in media wherein the 
pH.sub.o was varied between 5.0 and 8.3. The uptake of .sup.3 H!LXA.sub.4 
was determined at three different time points that were relevant to the 
wide range of influx rates observed (e.g., 0.25, 0.5, and 0.75 min in the 
case of pH.sub.o 5.0-6.5; 1, 2.5, and 5 min for pH.sub.o 6.8-7.1; and 5, 
10, and 20 min for pH.sub.o 7.4-8.3). Influx rates were calculated after 
subtraction of the DISA-resistant, carrier-independent component that most 
likely represents binding to receptors. 
The data points have been fit to a titration curve which yielded a pK' of 
5.9.+-.0.1. Results have been taken from four experiments. The data of 
FIG. 2, which displays the rate of LXA.sub.4 influx as a function of 
pH.sub.o, yields an apparent pK of 5.9.+-.0.1. This value is about 2 pH 
units more alkaline than the pK' (.about.3.8) of the carboxylic acid 
moiety of LXA.sub.4 . If entry were predominantly through non-ionic 
diffusion then an effective pK of .about.4 would be expected. Second, one 
would not anticipate an LXA.sub.4 influx pathway such as non-ionic 
diffusion to exhibit the striking sensitivity to DISA, PCP, UK-5099, and 
mersalyl. The final piece of supporting evidence for the mechanism 
underlying LXA.sub.4 influx being other than that of non-ionic diffusion 
is shown in Table I. Here, the effect of various drugs on the uptake of 
.sup.3 H!LXA.sub.4 methyl ester was measured. This compound is uncharged 
by virtue of the ester linkage through the carboxyl group and is very 
lipophilic. As such, LXA.sub.4 ME should permeate via simple diffusion, a 
process one would predict to be very rapid and not sensitive to the drugs 
which block influx of LXA.sub.4 free acid. All of these expectations were 
verified by the data of Table I. .sup.3 H!LXA.sub.4 ME influx rates in 
medium alone averaged 535.+-.136 fmol/10.sup.6 cells/min, 1,000-fold 
faster than for LXA.sub.4 free acid and were completely resistant to 
concentrations of DISA, PCP, and mersalyl which inhibit .sup.3 
H!LXA.sub.4 influx by &gt;80%. Moreover, given the lack of a titratable 
carboxylic acid group, LXA.sub.4 ME uptake did not display the marked 
pH-dependence characteristic of LXA.sub.4 influx. 
TABLE I 
______________________________________ 
.sup.3 H!LXA.sub.4 Methyl Ester Influx and .sup.3 H!LXA.sub.4 
Binding to Human Neutrophils: Lack of Effect of 
Drugs and Extracellular pH 
Unlabeled 
.sup.3 H!LXA.sub.4 
.sup.3 H!LXA.sub.4 ME Influx 
LXA.sub.4 
Binding 
Conditions 
(fmol/10.sup.6 cells/min) 
0.3 .mu.M 
(fmol/10.sup.6 cells) 
______________________________________ 
Medium 535 .+-. 136 - 1.05 .+-. 0.03 
Medium + 0.40 .+-. 0.08 
DISA 0.5 mM 
481 .+-. 170 - 1.23 .+-. 0.08 
DISA 0.5 mM + 0.41 .+-. 0.05 
PCP 0.4 mM 
439 .+-. 63 - 1.18 .+-. 0.04 
PCP 0.4 mM + 0.36 .+-. 0.04 
Mersalyl 0.5 mM 
500 .+-. 184 - 1.13 .+-. 0.05 
Mersalyl 0.5 mM + 0.39 .+-. 0.03 
pH.sub.O 6.40 
465 .+-. 45 - 1.02 .+-. 0.09 
pH.sub.O 6.40 + 0.39 .+-. 0.04 
pH.sub.O 8.40 
553 .+-. 129 - 1.19 .+-. 0.12 
pH.sub.O 8.40 + 0.42 .+-. 0.09 
______________________________________ 
Neutrophils were resuspended in the various media containing .sup.3 
H!LXA.sub.4 ME at a concentration of 5 nM. Uptake was measured at 
37.degree. C. as in FIG. 1 though at 15 s intervals through 1 min due to 
the very rapid kinetics. Influx rates were calculated by fitting the data 
points to a single exponential equation (Eq. 1). Results have been taken 
from three experiments based upon duplicate determinations. The binding of 
.sup.3 H!LXA.sub.4 to human neutrophils was assessed at 4.degree. C. at a 
final concentration of 0.3 nM. Cells were exposed to the drugs and 
different pH.sub.o media simultaneously along with LXA.sub.4. Total 
binding was determined after 5 min of incubation and is given on the line 
denoted by the "-" sign under the heading "unlabeled LXA.sub.4 0.3 .mu.M". 
Non-specific binding was measured, also at 5 min, in the presence of a 
1,000-fold excess of unlabeled LXA.sub.4 (0.3 .mu.M) and is given on the 
line where the "+" sign appears. Specific binding was taken as the 
difference between total and non-specific binding. There were no 
significant differences in any of these parameters under any of the 
treatment conditions. Results are from three separate experiments, each 
performed in triplicate. 
Recently, binding of .sup.3 H!LXA.sub.4 to specific binding sites has been 
demonstrated and specific binding has been correlated with functional 
responses in both neutrophils and HL-60 cells (Fiore, S., et al. (1992) J. 
Biol. Chem. 27:16168-16176; Fiore, S., et al. (1993) Blood 81:3395-3403). 
Specific binding, defined as the cell-associated label which could be 
displaced in the presence of a 1,000-fold excess of unlabeled LXA.sub.4, 
amounted to .about.70% of total binding (Table I) and was observed at both 
4.degree. C. and 37.degree. C. Further investigations at 4.degree. C. 
indicated a K.sub.d of 0.5.+-.0.3 nM with .about.1830 binding sites/cell 
and a half-time for binding of .15 s. Conceivably, changes in receptor 
binding at 37.degree. C. as compared to 4.degree. C. where most of the 
previous studies were conducted, could account for the progressive 
increase in cell-associated LXA.sub.4 counts between 1 and 30 min (FIG. 
1). Up-regulation, recycling, or recruitment of new receptors and changes 
in affinity are only a few of the many possible examples. As given in 
Table I, however, none of the experimental maneuvers, including drugs 
(DISA, PCP, and mersalyl) or varying pH.sub.o (6.4-8.4) had any 
significant effect on specific (or non-specific) association of .sup.3 
H!LXA.sub.4 with its surface receptors. These findings indicate that 
specific binding to cell surface receptors cannot account for the present 
results. 
The above mentioned results essentially rule out non-ionic diffusion and 
receptor binding as likely possibilities to explain the second phase of 
LXA.sub.4 influx. This process is in all likelihood due to a 
carrier-mediated H.sup.+ +LXA4-cotransport system or some other formal 
equivalent such as LXA.sub.4.sup.- /OH.sup.- (or HCO.sub.3.sup.-) 
exchange. 
EXAMPLE 4 
Inhibitor Profile of the LXA.sub.4 Transport System 
The dose-dependencies of inhibition of LXA.sub.4 influx by DISA, PCP, and 
mersalyl are depicted in FIG. 3. Studies were performed as in FIG. 1 in 
the presence of varying concentrations of DISA, PCP, and mersalyl (0-500 
uM). Uptake was measured at 10 and 20 min and the data points fit to 
single exponentials as described in the legend to FIG. 1. Transport influx 
rates were determined after subtraction of non-transport system-mediated 
uptake, the latter taken as that occurring at pH.sub.o 8.40 or in the 
presence of 1 mM DISA. The initial influx rates have been plotted against 
the added drug concentration. Results have been taken from three 
experiments for each condition. 
In these studies, the receptor-bound component, taken as non-transport 
system-mediated uptake, was determined by performing studies at pH.sub.o 
8.4 or in the presence of 1000 .mu.M DISA. This "transport 
system-independent background" was then subtracted from the total uptake 
at any given time interval in order to derive the transport 
system-mediated influx. The data sets for the three drugs followed simple 
Michaelis-Menten inhibition kinetics, the curves yielding K.sub.0.5 values 
of 12.+-.2 .mu.M for DISA, 25.+-.5 .mu.M for PCP, and 112.+-.33 .mu.M for 
mersalyl. As remarked above, all of these agents behave as anions which 
presumably compete, although this remains to be proven conclusively, with 
LXA.sub.4.sup.- for binding to the external translocation site of the 
LXA.sub.4.sup.- carrier. 
In addition to its being an anion, mersalyl, an organomercurial, is also a 
sulfhydryl-reactive agent, suggesting the possibility that a critical 
thiol group on the transport protein may be implicated. In view of this, a 
few other SH-reactive compounds were evaluated for activity. However, 1 mM 
NEM and 0.4 mM iodoacetate lacked efficacy in this system. Guided by prior 
experience in our identification of a mersalyl-sensitive H.sup.+ 
+lactateco-transport system in these cells (Simchowitz, L. and Textor, J. 
A. (1992) J. Gen Physiol 100:341-367; Simchowitz, L. and Vogt, S. K. 
(1993) J. Membr. Biol. 131:23-34), we examined the question of whether any 
of a number of SH-reactive compounds might cause irreversible inhibition 
of .sup.3 H!LXA.sub.4 influx when preincubated with neutrophils. For 
these studies (Table II), cells were pretreated with drugs for 30 min at 
4.degree. C. in order to prevent metabolic production of lactic acid and 
its intracellular accumulation and resultant fall in intracellular pH 
consequent to a block of the lactate carrier (Simchowitz, L. and Textor, 
J. A. (1992) J. Gen Physiol 100:341-367). As shown, 0.25 mM mersalyl and 
pHMB, a related organomercurial, led to .about.65% irreversible inhibition 
when cells were subsequently washed and assayed for their ability to 
transport .sup.3 H!LXA.sub.4 at 37.degree. C. NBD-CL and 
eosin-5-maleimide caused more modest inhibition (.about.20%) while 
2,2'-DTBP was without appreciable effect. 
TABLE II 
______________________________________ 
Ability of Sulfhydryl-Reactive Agents to Cause 
Irreversible Inhibition of .sup.3 H!LXA.sub.4 Influx 
Concentration 
% Inhibition 
Compound (mM) of Transport 
______________________________________ 
Mersalyl 0.25 67 .+-. 5 
pHMB 0.25 66 .+-. 4 
NBD-Cl 0.25 22 .+-. 7 
2,2'-DTBP 0.50 5 .+-. 3 
Eosin-5-maleimide 
0.50 19 .+-. 6 
______________________________________ 
Aliquots of a neutrophil suspension were pretreated with drugs at the 
stated concentrations for 30 min at 4.degree. C. Thereafter, the cells 
were washed once to get rid of excess free drug and finally resuspended in 
medium containing .sup.3 H!LXA.sub.4 at 5 rM. Uptake was measured at 1, 
5, and 10 min and the initial influx rates determined as detailed in the 
legends to FIGS. 1 and 2. Influx rates after preincubation with the 
various thiol reagents were compared to those with medium alone (0% 
inhibition) and with 0.5 mM DISA (taken as 100% inhibition) in order to 
calculate the % inhibition of transport system-mediated transport. Results 
represent the means+SEM of three separate experiments for each condition. 
NBD-Cl=7-chloro-4-nitrobenz-2-oxa-1,3-diazole; 
2,2'-DTBP=2,2'-dithiobispyridine. 
EXAMPLE 5 
Enhancers of LXA.sub.4 Transport by the Transport System 
Certain compounds were found to enhance rather than inhibit uptake of 
LXA.sub.4 by neutrophils via the lipoxin transport system. .sup.3 
H!LXA.sub.4 influx experiments were performed as described in the previous 
examples in the presence or absence of 100 nM 
n-formylmethionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate 
(PMA), a phorbol diester. Exposure of neutrophils to 100 nM FMLP led to a 
.about.2-fold increase in the initial rate of .sup.3 H!LXA.sub.4 influx, 
as shown in FIG. 4. The enhanced uptake was completely sensitive to 
inhibition by DISA, characteristic of uptake by the lipoxin transport 
system. Exposure of neutrophils to 100 nM PMA caused a .about.4-fold 
increase in the initial rate of .sup.3 H!LXA.sub.4 entry into cells (see 
FIG. 4). (The apparent decrease in uptake seen after 10 minutes most 
likely represents degradation of the radiolabeled probe). In contrast, 
4-.alpha.-PMA, a biologically inactive isomer of PMA, had no effect on 
lipoxin uptake, thereby ruling out non-specific detergent-like actions of 
PMA on the neutrophil membrane. The PMA-induced enhancement of .sup.3 
H!LXA.sub.4 influx was likewise competely sensitive to inhibition by DISA. 
The divalent cation ionophore A23187 (at 1 .mu.M concentration) also had a 
modest stimulatory effect (.about.1.5-fold enhancement) on lipoxin uptake. 
EXAMPLE 6 
Additional Properties of the LXA.sub.4 Transport System 
Substrate Saturation 
Carrier-mediated LXA.sub.4 transport would be expected to display the 
general property of substrate saturation and so we monitored the rate of 
.sup.3 H!LXA.sub.4 influx as a function of added LXA.sub.4 concentration 
between 0.6 nM and 5 .mu.M (FIG. 5). Experiments were carried out as in 
FIG. 1 in the presence of a constant amount of .sup.3 H!LXA.sub.4 . 
Increasing quantities of unlabeled LXA.sub.4 were added to achieve total 
concentrations of 0.6 nM to 5 .mu.M. Uptake was measured at 5 and 10 min 
and the influx rates calculated as described for FIG. 2. As shown, a 
linear relationship applies when the .sup.3 H!MLXA.sub.4 influx rates are 
plotted against the LXA.sub.4 concentration: slope=0.116.+-.0.003. Results 
are from three experiments. Contrary to expectation, over a 4 log dose 
range, no tendency towards saturation was evident and influx remained 
strictly proportional to the LXA.sub.4 concentration. It is important to 
point out that in these experiments .sup.3 H!LXA.sub.4 uptake was always 
markedly sensitive to 0.5 mM DISA. As 5 .mu.M LXA.sub.4 approaches or 
exceeds the upper limit of the physiologically relevant range, higher 
concentrations were not tested. 
Temperature-Dependence 
To assess the temperature-dependence of the transport reaction, we also 
determined the rate of DISA-sensitive .sup.3 H!LXA.sub.4 influx as a 
function of temperature between 4.degree. and 37.degree. C. Uptake of 
.sup.3 H!LXA.sub.4 was measured at three different points along the 
entire time course (1-30 min) and influx rates were determined as for FIG. 
2. Results are from three experiments. The data are graphed in FIG. 6 in 
the form of a conventional Arrhenius plot. The results convey a linear 
relationship exhibiting a rather shallow slope that translates into a 
relatively low activation energy of 15.0.+-.0.8 cal/mole. 
Effect of Different Eicosanoids 
The effect of a number of other eicosanoids on the influx rate of .sup.3 
H!LXA.sub.4 was also evaluated (Table III). The studies were based on the 
rationale that these agents might behave as competing substrates and 
therefore reduce LXA.sub.4 transport were they to share the same carrier 
and bind with high affinity. The data reveal, however, that over the 
concentration range 10-100 nM, neither arachidonic acid, PGE.sub.2, 
15-HETE, LTB.sub.4, nor the cysteinyl leukotrienes LTC.sub.4 and LTD.sub.4 
perturbed the rate of .sup.3 H!LXA.sub.4 uptake. These results provide 
evidence that the LXA.sub.4 carrier system is distinct and probably does 
not represent a common, more generalized transport pathway for a wide 
variety of arachidonate-derived products. 
TABLE III 
______________________________________ 
Lack of Effect of a Variety of Eicosanoids on .sup.3 H!LXA.sub.4 
Influx into Neutrophils 
Concentration 
.sup.3 H!LXA.sub.4 Influx 
Conditions (nM) (fmol/10.sup.6 cells/min) 
______________________________________ 
Control 0.66 .+-. 0.04 
Arachidonic Acid 
100 0.70 .+-. 0.02 
Arachidonic Acid 
30 0.66 .+-. 0.02 
Arachidonic Acid 
10 0.69 .+-. 0.03 
PGE.sub.2 100 0.60 .+-. 0.03 
PGE.sub.2 30 0.59 .+-. 0.03 
PGE.sub.2 10 0.58 .+-. 0.03 
15-HETE 100 0.71 .+-. 0.06 
15-HETE 30 0.69 .+-. 0.05 
15-HETE 10 0.73 .+-. 0.05 
LTB.sub.4 100 0.70 .+-. 0.06 
LTB.sub.4 30 0.64 .+-. 0.04 
LTB.sub.4 10 0.67 .+-. 0.04 
LTC.sub.4 100 0.78 .+-. 0.07 
LTC.sub.4 30 0.76 .+-. 0.05 
LTC.sub.4 10 0.70 .+-. 0.05 
LTD.sub.4 100 0.61 .+-. 0.03 
LTD.sub.4 30 0.59 .+-. 0.04 
LTD.sub.4 10 0.63 .+-. 0.03 
______________________________________ 
Experiments were performed as described for FIG. 3 in the presence of 
stated concentrations of eicosanoids. The neutrophils were exposed to the 
compounds at zero-time and uptake of .sup.3 H1!LXA.sub.4 was assessed at 
10 and 20 min. Results are from three experiments for each condition. 
EXAMPLE 7 
Presence of the LXA.sub.4 Transport System in Other Blood Cells 
It was next determined whether or not a similar lipoxin transport activity 
might be present in other blood cells (Table IV). For these studies, 
purified suspensions of unstimulated human erythrocytes, lymphocytes, and 
platelets that were isolated from peripheral blood were used as well as 
undifferentiated HL-60 cells, a stable human promyelocytic leukemia cell 
line, and retinoic acid-induced HL-60 cells which display many of the 
phenotypic features of normal mature neutrophils (Collins, S., et al., 
(1978) Proc. Natl. Acad. Sci. USA 75:2458-2462). As shown in Table IV, 
there was no DISA-sensitive component of .sup.3 H!LXA.sub.4 uptake into 
unstimulated human red cells, lymphocytes, platelets, or undifferentiated 
HL-60 cells. The results imply that these cell types in a resting state do 
not express an LXA.sub.4 carrier on their plasma membranes. In contrast, 
HL-60 cells that had been terminally differentiated along a 
neutrophil-like pathway by a 5-day exposure to 1 .mu.M retinoic acid 
acquired a new DISA-sensitive influx route for LXA.sub.4 that strongly 
resembled that of normal neutrophils in time course and magnitude. 
TABLE IV 
______________________________________ 
Presence of an LXA.sub.4 Transport System in Other Blood Cells 
.sup.3 H!LXA.sub.4 Uptake Rates 
(fmol/10.sup.6 cells/min) 
in the presence of: 
Cell Type Medium DISA 
______________________________________ 
Erythrocytes 0.0014 .+-. 0.0006 
0.0019 .+-. 0.0008 
Lymphocytes 0.0019 .+-. 0.0048 
0.0053 .+-. 0.0036 
Platelets -0.0003 .+-. 0.0015 
-0.0005 .+-. 0.0015 
HL-60 (Undifferentiated) 
0.11 .+-. 0.02 
0.12 .+-. 0.02 
HL-60 (Retinoic 
0.73 .+-. 0.15 
0.08 .+-. 0.02 
acid-induced) 
______________________________________ 
Human red cells, lymphocytes, and platelets were isolated from peripheral 
blood as described under Methods. Undifferentiated HL-60 cells were grown 
in culture according to standard protocols. Differentiated HL-60 cells 
were obtained by exposing cells to 1 .mu.M retinoic acid for 5 days in 
culture. After harvesting and isolation, all cells were kept in the 
standard medium for 1 hour at 37.degree. C. prior to assay. The uptake of 
.sup.3 H!LXA.sub.4 was determined as in FIG. 1 (concentration=5 nM, 
time=1-30 min) and the influx rates in the presence and absence of 0.5 mM 
DISA were calculated as for FIG. 1. Results represent the means.+-.SEM of 
three-four separate experiments for each cell type. 
EXAMPLE 8 
Release of LXA.sub.4 
"Efflux" kinetics are presented in FIG. 7. Cells were first labeled with 
.sup.3 H!LXA.sub.4 by incubating them with 5 nM LXA.sub.4 for 15 min at 
37.degree. C. The cells were then pelleted and resuspended in the various 
experimental media, each in the absence of LXA.sub.4. The loss of counts 
from the neutrophil pellet was followed over time. Results of three-four 
experiments are expressed as relative cell content, defined as 
cell-associated cpm at a given time divided by the starting cpm at 
zero-time. The curves represent declining single exponential fits to the 
individual sets of data. The parameters of the exponentials were as 
follows: for Control, rate coefficient=0.097.+-.0.013 min-.sup.1 and final 
value=0.54.+-.0.03; for 0.5 mM DISA, 0.4 mM PCP, and pH.sub.o 8.40 
(combined data set), rate coefficient 0.098.+-.0.008 min.sup.-1 and final 
value=0.46.+-.0.02; and for 0.5 mM mersalyl and pH.sub.o 6.40 (combined 
data), rate coefficient 0.079.+-.0.012 min.sup.-1 and final 
value=0.56.+-.0.03. 
It is immediately apparent that the principal mechanism underlying the loss 
of cell-associated LXA.sub.4 is distinctly different from that for uptake. 
While at most only slight effects could be observed, none of the drugs 
tested or extremes of pH.sub.o (6.4-8.4) caused substantial changes in the 
off-rate. This implies that uptake and "efflux" signify two different 
phenomena. Conceivably, since none of the experimental maneuvers noted 
above altered receptor binding to any significant extent, it is quite 
possible that the loss of cell label actually represents dissociation from 
cell surface receptors. Moreover, LXA.sub.4 that is translocated inward 
via the carrier to enter the cytosol may then preferentially partition 
into various membrane domains and organelles or become sequestered within 
hydrophobic compartments, thereby making it inaccessible to the plasma 
membrane-localized LXA.sub.4 carrier. Some evidence for binding to nuclear 
and other cytoplasmic constituents has already been provided by 
subcellular fractionation studies (Fiore, S., et al., (1992) J. Biol. 
Chem. 267:16168-16176). One point, however, is clear: degradation of 
labeled LXA.sub.4 cannot account for these findings as LXA.sub.4 is not 
subject to metabolic transformation in neutrophils even at 37.degree. C. 
(Fiore, S., et al., (1992) J. Biol. Chem. 267:116168-16176). 
EXAMPLE 9 
Effect of Lipoxins on Neutrophil Phagocytosis 
When exposed to particulate material (e.g., red blood cell, microorganisms, 
crytals etc.), neutrophils ingest this particulate matter, a process 
termed phagocytosis. In this example, the ability of neutrophils to 
phagocytose opsonized sheep red blood cells was measured in the presence 
and in the absence of either LXA.sub.4 or LXB.sub.4. 
Neutrophils were prepared as described in the general methodology and 
suspended at a concentration of 10.times.10.sup.6 -15.times.10.sup.6 
cells/ml in Hepes Hanks buffer, pH 7.4, supplemented with 1 mg/ml BSA, 1 
mg/ml glucose, 0.5 mM MgCl.sub.2 and 1.0 mM CaCl.sub.2 --2H.sub.2 O. 
The sheep red blood cells (SRBCs) were prepared by washing the cells with 
1.times. veronal-buffered saline (VBS), incubating the cells with a 1:500 
dilution of rabbit anti-SRBC IgG antibody at 37.degree. C. for 15 minutes, 
centrifuging the cells at 2000 rpm for 5 minutes at 4.degree. C. and 
washing the cells twice with 1.times.VBS. SRBCs were resuspended at a 
final concentration of 10.times.10.sup.8 cells/ml. 
For each phagocytosis reaction, a mixture was prepared which contained 25 
.mu.l of neutrophils (at 10.times.10.sup.6 -15.times.10.sup.6 cells/ml), 
an agent to be tested (e.g., LXA.sub.4 or LXB.sub.4 at final 
concentrations between 1 and 1000 nM) and media to a final volume of 100 
.mu.l. For control reactions, the agent to be tested (e.g., LXA.sub.4 or 
LXB.sub.4) was omitted. Each component was pipetted separately to the 
bottom of a 12.times.75 mm test tube to allow thorough mixing of the 
components. 
To each 100 .mu.l mixture was added 15 .mu.l of opsonized SRBCs to start 
the phagocytosis reaction. The tubes were vortexed and placed in a 
37.degree. C. incubator for 30 minutes. After 30 minutes, the tubes were 
placed on ice to stop the reaction. 
Non-phagocytosed SRBCs were lysed by adding 1.0 ml of 0.83% NH.sub.4 Cl to 
each tube, with shaking, and centrifuging the tubes quickly at 1500 rpm 
for 3 minutes. The supernatant was removed and the pellets were 
resuspended in 30 gl of PBS. The 30 .mu.l solution was placed onto a 
polylysine-coated microscope slide and after 15 minutes the excess 
solution was removed from the slide. The cells on the slide were fixed 
with 1% glutaraldehyde for 5 minutes and then the excess glutaraldehyde 
was removed. The cells were stained with Giemsa Stain by immersing the 
slide in the stain for 25 minutes, rinsing the slide with water and 
allowing the slide to dry overnight. 
The slides were examined with a light microscope at 100.times.power. SRBCs 
which were phagocytosed by neutrophils were visible as yellow cells within 
blue-stained neutrophils. The results for each reaction were expressed as 
a phagocytic index, representing the number of SRBCs phagocytosed per 100 
neutrophils. FIG. 8 shows a graph of the phagocytic index plotted against 
time for neutrophils incubated with media alone (control) or with 100 nM 
or 1000 nM LXA.sub.4 or LXB.sub.4. The results demonstrate that increasing 
amounts of lipoxin inhibit the phagocytic activity of neutrophils as 
measured by this assay. LXA.sub.4 was more effective at inhibiting 
phagocytosis than LXB.sub.4. 
To examine whether the inhibitory effect of LXA.sub.4 on neutrophil 
phagocytosis was mediated by uptake of LXA.sub.4 by the lipoxin transport 
system present on neutrophils, the effect of altering the pH on the 
ability of LXA.sub.4 to inhibit phagocytosis was determined. Phagocytosis 
reactions similar to those described above were performed, except that in 
addition to performing the reactions at an extracellular pH of 7.4 (as 
described above), the extracellular pH was either decreased to pH 6.4 or 
increased to pH 8.4. The results are shown in FIG. 9, which depicts a 
graph of the phagocytic index plotted against lipoxin concentration. The 
results demonstrate that the ability of LXA.sub.4 to inhibit neutrophil 
phagocytosis increases as the extracellular pH is decreased (i.e., as the 
acidity of the extracellular media is increased) and decreases as the 
extracellular pH is increased (i.e., as the acidity of the extracellular 
media is decreased). Thus, LXA.sub.4 -mediated inhibition of neutrophil 
phagocytosis exhibits pH-dependence. This pH dependence qualitatively 
resembles the pH-dependence of the lipoxin transport system, which also 
exhibits increased activity at pH 6.4 and decreased activity at pH 8.4. 
Therefore, the inhibition of neutrophil phagocytosis by LXA.sub.4 is 
consistent with LXA.sub.4 being taken up by the neutrophils via the 
lipoxin transport system. 
EQUIVALENTS 
Those skilled in the art will recognize, or be able to ascertain using no 
more than routine experimentation, numerous equivalents to the specific 
procedures described herein. Such equivalents are considered to be within 
the scope of this invention and are covered by the following claims.