T21/DP107, A Synthetic Leucine Zipper-Like Domain of the HIV-1 Envelope gp41, Attracts and Activates Human Phagocytes by Using G-Protein-Coupled Formyl Peptide Receptors1,2 Shao Bo Su,* Ji-liang Gao,‡ Wang-hua Gong,† Nancy M. Dunlop,* Philip M. Murphy,‡ Joost J. Oppenheim,* and Ji Ming Wang3* A leucine zipper-like domain, T21/DP107, located in the amino terminus of the ectodomain of gp41, is crucial to the formation of fusogenic configuration of the HIV-1 envelope protein gp41. We report that the synthetic T21/DP107 segment is a potent stimulant of migration and calcium mobilization in human monocytes and neutrophils. The activity of T21/DP107 on phagocytes was pertussis toxin-sensitive, suggesting this peptide uses Gi-coupled seven-transmembrane receptor(s). Since the bacterial chemotactic peptide fMLP partially desensitized the calcium-mobilizing activity of T21/DP107 in phagocytes, we postulated that T21/DP107 might preferentially use a lower affinity fMLP receptor. By using cells transfected to express cloned prototype chemotactic N-formyl peptide receptor (FPR) or its variant, FPR-like 1 (FPRL1), we demonstrate that T21/DP107 activates both receptors but has a much higher efficacy for FPRL1. In addition, T21/DP107 at nM concentrations induced migration of FPRL1-transfected human embryonic kidney 293 cells. In contrast, fMLP did not induce significant chemotaxis of the same cells at a concentration as high as 50 mM. Although a lipid metabolite, lipoxin A4, was a high-affinity ligand for FPRL1, it was not reported to induce Ca21 mobilization or chemotaxis in FPRL1-transfected cells. Therefore, T21/DP107 is a first chemotactic peptide agonist identified thus far for FPRL1. Our results suggest that this peptide domain of the HIV-1 gp41 may have the potential to activate host innate immune response by interacting with FPR and FPRL1 on phagocytes. The Journal of Immunology, 1999, 162: 5924 –5930.
T
he envelope proteins of HIV-1 are synthesized in the form of a precursor, gp160, which is subsequently cleaved by proteinases to yield mature proteins gp120 and gp41 (1). gp120 is noncovalently bound to the extracellular domain of gp41 and mediates viral binding to host cells through high-affinity interaction with CD4 receptors, followed by interaction with chemokine receptors that have recently been identified as HIV-1 fusion cofactors (2, 3). The viral envelope gp41 plays a critical role in fusion of HIV-1 and host cell membranes (2, 3). Structural analysis predicts the gp41 ectodomain to contain two segments as extended helices (4). One segment termed T21/DP107 in the NH2
*Laboratory of Molecular Immunoregulation, Division of Basic Sciences; †Intramural Research Support Program, Science Applications International Corporation-Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702; and ‡Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 Received for publication December 10, 1998. Accepted for publication February 17, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 S.B.S. is supported in part by a fellowship from the Office of the International Affairs, National Cancer Institute, National Institutes of Health. 2 The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The publisher or recipient acknowledges right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. 3 Address correspondence and reprint requests to Ji Ming Wang, Laboratory of Molecular Immunoregulation, Division of Basic Sciences, National Cancer Institute-Frederick Cancer Research and Development Center, Building 560, Room 31-19, Frederick, MD 21702-1201. E-mail address:
[email protected]
Copyright © 1999 by The American Association of Immunologists
terminus has a leucine zipper-like motif, whereas another segment, T20/DP178, is located in the carboxyl terminus of the gp41 ectodomain. In the absence of gp120 and the N-terminal fusion domain, the ectodomain of gp41 forms a soluble a-helical rod-like oligomer (4, 5). Synthetic analogues of both T21/DP178 and T20/ DP178 have been shown to inhibit virus-mediated cell-cell fusion and to reduce the infectious titer of cell-free virus (5–11). The anti-HIV-1 activity of these peptides is presumably due to their competitive association with the native segments on gp41, thus blocking the conversion to the fusogenic configuration of the virus. In the course of investigating the basis for HIV-1-associated suppression of monocyte functions, we found that preexposure of human monocytes to either HIV-1 envelope proteins gp120 or gp41 inhibited their chemotactic responses to a wide variety of chemoattractants, including the bacterial chemotactic peptide fMLP and a number of recently defined chemokines, through a mechanism resembling heterologous “desensitization” (12, 13). The inactivation of monocyte chemotactic responses by HIV-1 envelope proteins may be responsible for the reduced migratory response of monocytes from AIDS patients to a variety of chemoattractants in vitro (14). To further define the structural basis for the capacity of HIV-1 envelope proteins to “desensitize” host cells, we evaluated the effects of selected peptide segments of gp41 on human immune cells. We initially found that synthetic C-terminal peptide segment, T20/DP178, was a potent agonist of FPR, a seven-transmembrane, G-protein-coupled receptor on human phagocytic cells used by chemotactic N-formyl peptides (Refs. 15 and 16, and Su et al., manuscript in preparation). This led us to further investigate the effect of synthetic T21/DP107. Here, we report that T21/DP107 activates human monocytes and neutrophils by using 0022-1767/99/$02.00
The Journal of Immunology two N-formyl peptide receptors (FPR4), the prototype FPR and its variant FPR-like 1 (FPRL1), and exhibits much higher efficacy on FPRL1.
Materials and Methods Reagents and cells The T21/DP107 was synthesized and purified by the Department of Biochemistry, Colorado State University (Fort Collins, CO), according to the published sequence (aa 558 –595 of gp41) (5): Ac-NNLLRAIEAQ QHLLQLTVWGIKQLQARILAVERYLKDQ-NH2. The purity was 90% or more, and the amino acid composition was verified by mass-spectrometer. The endotoxin levels in dissolved peptide were undetectable. The synthetic formyl peptide fMLP was purchased from Sigma (St. Louis, MO). The human PBMC were isolated from leukopacks through the courtesy of Transfusion Medicine Department (National Institute of Health Clinical Center, Bethesda, MD). Monocytes were further purified by ellutriation to yield .90% purity. Human neutrophils were purified from the same leukopacks by 3% dextran sedimentation with a purity of .98%. Rat basophilic leukemia cells stably transfected with epitope-tagged FPR (ETFR) were a kind gift of Drs. H. Ali and R. Snyderman (Duke University, Durham, NC). The cells were designated ETFR and were grown in DMEM, 10% FCS, and 0.8 mg/ml geneticin (G418) to maintain selection pressure. The FPRL1 cDNA was cloned and transfected into human embryonic kidney cells 293 (designated FPRL1/293 cells), as reported previously (17). The cells were maintained in DMEM, 10% FCS, and 2 mg/ml geneticin (G418).
Chemotaxis Leukocyte, ETFR, and FPRL1/293 cell migration was assessed using a 48-well microchemotaxis chamber technique, as previously described (18 – 20). Different concentrations of stimulants were placed in wells of the lower compartment of the chamber (Neuro Probe, Cabin John, MA), and the cell suspension was seeded in wells of the upper compartment, which was separated from the lower compartment by a polycarbonate filter (Osmonics, Livermore, CA; 5-mm pore size for leukocytes, 10-mm pore size for ETFR and FPRL1/293 cells). For CD31 T lymphocytes, the filters were precoated with 20 mg/ml bovine fibronectin (Sigma). The filters for ETFR and FPRL1/293 cell migration were precoated with 50 mg/ml collagen type I (Collaborative Biomedical Products, Bedford, MA) to favor the attachment of the cells. After incubation at 37°C (90 min for monocytes, 60 min for neutrophils, 180 min for T cells, and 300 min for ETFR or FPRL1/293 cells), the filters were removed, stained, and the cells migrating across the filter were counted by light microscopy after coding the samples. The experiments were performed at least five times with each cell type, and the results are presented as the chemotaxis indexes (CI) representing the fold increase in the number of migrating cells in response to stimuli, over the spontaneous cell migration (in response to control medium). The significance of the increase in cell migration was determined using Student’s t test, and CI $ 2 was statistically significant compared with medium control (at least p , 0.05).
Calcium mobilization Calcium mobilization was assayed by incubating 107 cells/ml of monocytes, neutrophils, FPRL1, or ETFR transfectants in loading buffer containing 138 mM NaCl, 6 mM KCl, 1 mM CaCl2, 10 mM HEPES (pH 7.4), 5 mM glucose, and 0.1% BSA with 5 mM fura-2 (Sigma) at 37°C for 30 min. The dye-loaded cells were washed and resuspended in fresh loading buffer. The cells were then transferred into quartz cuvettes (106 cells in 2 ml), which were placed in a luminescence spectrometer LS50 B (PerkinElmer Limited, Beaconsfield, U.K.). Stimulants at different concentrations were added in a volume of 20 ml to the cuvettes at indicated time points. The ratio of fluorescence at 340- and 380-nm wavelength was calculated using the FL WinLab program (Perkin-Elmer). Unless specified, all experiments were performed at least five times with similar results, and data shown in this study were from representative experiments.
Results We first tested whether synthetic T21/DP107 could induce human leukocyte migration, a crucial step for cell homing and accumu4 Abbreviations used in this paper: FPR, formyl peptide receptor; FPRL1, FPR-like 1; ETFR, epitope-tagged FPR; CI, chemotaxis index; SAA, serum amyloid A.
5925 lation at sites of inflammation or injury. As shown in Fig. 1, A and B, human PBMC and neutrophils migrated in a dose-dependent manner in response to a concentration gradient of T21/DP107. The chemotactic activity of T21/DP107 was already significant at nM concentrations for both monocytes and neutrophils, and the cell response remained high with only slight reduction when T21/ DP107 was used at 1025-1024 M (Fig. 1B). In contrast to phagocytes, human CD31 T lymphocytes showed a marginally significant migration (CI 5 2) in response to high concentrations (5 3 1026 M and higher, Fig. 1B), indicating the effect of T21/DP107 is mainly on phagocytic cells. We next examined whether phagocyte migration induced by T21/DP107 was dependent upon the effect of chemotaxis or chemokinesis. Checkerboard analyses showed that monocytes migrated when higher concentrations of T21/DP107 were present in the lower wells of the chemotaxis chamber (Table I). There was no enhanced cell migration when higher concentrations of T21/DP107 were present in the upper wells. With equal concentrations of T21/DP107 in both upper and lower wells, a slightly increased monocyte migration was observed. These results suggest that the cell migration induced by T21/DP107 was due mainly to a chemotactic effect with minor contribution of chemokinesis. The migration of monocytes and neutrophils to T21/DP107 was completely inhibited by pretreatment of the cells with pertussis toxin, but not by cholera toxin or herbimycin A (Fig. 1C, and data not shown), suggesting that a G-protein of the Gi type-coupled receptor was involved (15, 16, 21–24). This was supported by the potent induction of a dosedependent, and pertussis toxin-sensitive, calcium (Ca21) mobilization in monocytes and neutrophils by T21/DP107 (Fig. 2, A and C). The Ca21-mobilizing activity of T21/DP107 was also significant at nM concentrations, indicating that this synthetic peptide is a potent activator of human phagocytic cells. The possibility that byproduct(s) formed during peptide synthesis/purification might account for the activity of T21/DP107 was unlikely, since a fusion peptide (aa 517–532 of gp41) synthesized during the same period as T21/DP107 did not posses any chemotactic or Ca21-mobilizing activity in phagocytes (data not shown). To characterize the molecular nature of the receptor(s) possibly used by T21/DP107 on phagocytic cells, a series of cross-desensitization experiments were performed by using a variety of chemoattractants. T21/DP107 did not desensitize the Ca21 flux in monocytes or neutrophils induced by chemokines, such as monocyte chemoattractant protein (MCP)-1, RANTES, MCP-3, macrophage-inflammatory protein-1a, IL-8, and stromal cell-derived factor-1a (data not shown). Therefore, T21/DP107 does not share a receptor with any of these chemokines. However, high concentrations ($1 mM) of the bacterial chemotactic N-formylated peptide fMLP had a partial desensitizing effect on T21/DP107-induced Ca21 mobilization in both monocytes and neutrophils (Fig. 2, B and D). In contrast, T21/DP107 did not significantly desensitize the effect of fMLP (Fig. 2, B and D). These results suggest that T21/DP107 may share receptor(s) with fMLP on human phagocytic cells and may have preference on a receptor for which fMLP has lower affinity. Since fMLP was known to induce Ca21 in phagocytes through at least two seven-transmembrane, G-protein-coupled receptors, FPR and FPRL1 (15–17), we tested the effect of T21/DP107 on these two receptors transfected and overexpressed in human cells that originally were not responsive to fMLP stimulation. fMLP over a wide range of concentrations induced Ca21 mobilization in FPR-transfected rat basophil leukemia cell line (ETFR cells), with minimal effective dose at low pM concentration range (Fig. 3A). In
5926
ACTIVATION OF FPR/FPRL1 BY T21/DP107
FIGURE 1. Induction of phagocyte migration by T21/DP107. Different concentrations of T21/DP107 were placed in the lower wells of the chemotaxis chamber; cell suspension was placed in the upper wells. The upper and lower wells were separated by polycarbonate filters. After incubation, the cells migrated across the filters were stained and counted. A, Visualization of monocyte and neutrophil migration in response to T21/ DP107 (1 mM) and fMLP (100 nM). B, Fold increase of leukocyte migration in response to T21/DP107 over control medium. C, Inhibition of monocyte migration in response to T21/DP107 by pretreatment of the cells with pertussis toxin (PT; 100 ng/ml, 30 min at 37°C).
contrast, the minimal effective concentration for fMLP to induce Ca21 mobilization in FPRL1-transfected cells (FPRL1/293 cells) was at low mM range (Fig. 3D). These results confirmed the pre-
vious observation that FPR is a high-affinity receptor for fMLP, whereas FPRL1 has much lower affinity (15–17). The synthetic T21/DP107 also induced Ca21 mobilization in cells transfected
Table I. Checkerboard analysis of monocyte migration in response to T21/DP107a Number of Migrated Cells in 1HPF (Mean 6 SE) T21 in upper wells (M)
T21 in Lower Wells (M)
Medium
Medium 1028 1027 1026
20 6 1 40 6 4b 64 6 3b 132 6 6b
10
28
11 6 2 28 6 3 43 6 2b 110 6 3b
1027
1026
761 762 25 6 4 89 6 6b
962 10 6 3 26 6 3 57 6 4b
a Different concentrations of T21/DP107 were placed in the upper and/or lower wells of the chemotaxis chamber; monocytes at 2 3 106/ml were placed in the upper wells. The upper and lower wells were separated by a polycarbonate filter. After incubation, the nonmigrating cells were removed, and the filter was fixed, stained, and the cells migrated across the filter were counted in three high-powered fields (HPF; 4003). The results are expressed as the mean value (6SE) of the cells in 1 HPF. Similar results were obtained in two separate experiments. b A value of p , 0.01 compared with migration in the presence of medium alone in both upper and lower wells, as determined by Student’s t test.
The Journal of Immunology
5927
FIGURE 2. Calcium (Ca21) mobilization in phagocytes induced by T21/DP107. Human monocytes (A) or neutrophils (C) were loaded with fura-2 and then were stimulated with various concentrations of T21/DP107. The ratio of fluorescence at 340 and 380 nm wave length was recorded and calculated using the FL WinLab program. B and D, Desensitization of T21/DP107 (1 mM) induced Ca21 flux by fMLP (1 mM) in monocytes (B) or neutrophils (D).
with either of these receptors (Fig. 3, B and E). However, the minimal effective dose for T21/DP107 to activate FPRL1 was at nM range as compared with low mM range on FPR, suggesting that T21/DP107 activates FPRL1 with higher efficacy. A comparison for the efficacy between T21/DP107 and fMLP on two receptors could be better illustrated by the requirement of the concentrations to elicit an equal response by these two agents. In FPR-expressing cells, to induce a change in the ratio of 2 at 340/380 nm wavelength fluorescence, 10210 fMLP vs 1025 T21/DP107 were required; whereas in FPRL1/293 cells, 5 3 1025 fMLP vs 5 3 1027 T21/DP107 induced a change in the ratio of 0.6. Such comparison indeed indicates T21 to be a more potent agonist for FPRL1 compared with fMLP. This was further supported by results of cross-
desensitization of Ca21 flux between T21/DP107 and fMLP in both receptor transfectants. As shown in Fig. 3, C and F, although sequential stimulation of the cells expressing FPR or FPRL1 with T21/DP107 and fMLP resulted in bidirectional desensitization, a 1000-fold excess of fMLP was required to desensitize the effect of T21/DP107 in FPRL1/293 cells. Conversely, a much higher concentration of T21/DP107 was necessary to completely abolish the subsequent response to fMLP of the cells transfected with FPR. In control experiments, T21/DP107 and fMLP did not induce any Ca21 mobilization in parental or mock-transfected rat basophil cell line and human embryonic kidney 293 cells (data not shown). These results further support the notion that FPR and its variant, FPRL1, are differentially activated by fMLP and T21/DP107.
FIGURE 3. Calcium mobilization in ETFR and FPRL1/293 cells induced by T21/DP107. The FPR-transfected rat basophil cell line (RBL), ETFR cells (A and B), and FPRL1/293 transfectants (D and E) were used to evaluate Ca21 flux induced by T21/DP107 or fMLP. C and F, Cross-desensitization of cell signaling between T21/DP107 and fMLP.
5928
ACTIVATION OF FPR/FPRL1 BY T21/DP107
FIGURE 4. Migration of ETFR cells and FPRL1/ 293 cells in response to T21/DP107. Different concentrations of T21/DP107 were placed in the lower wells of the chemotaxis chamber; cell suspension was placed in the upper wells. The upper and lower wells were separated by polycarbonate filters precoated with mouse collagen type I. After incubation, the cells migrated across the filters were stained and counted (A). CI represents the fold increase of leukocyte migration in response to T21/DP107 over control medium. B, Migration of FPRL1/293 cells. C, Migration of ETFR cells.
The chemotactic response of the cells transfected with FPR or FPRL1 was tested as another sensitive parameter to assess the receptor targets of T21/DP107 (18 –20). FPRL1/293 cells showed a marked migratory response to T21/DP107 with an EC50 of 50 nM (Fig. 4, A and B), but these cells failed to migrate in response to a wide range of concentrations of fMLP (Fig. 4B). In contrast, ETFR cells were induced to migrate by fMLP at nM range concentrations, but much higher concentrations of T21/DP178 were required to induce the migration of the same cells (Fig. 4C). The chemotaxis experiments indicate that fMLP is only a partial agonist for FPRL1, since it did not induce FPRL1-expressing cells to migrate. T21/DP107, on the other hand, appears to be an efficient agonist on both FPR and FPRL1, although the efficacy for FPRexpressing cells was much lower than for FPRL1-expressing cells.
Discussion In this study, we observed that T21/DP107, a synthetic peptide domain of the HIV-1 gp41, potently attracts and activates human phagocytic cells by using two seven-transmembrane, G-proteincoupled receptors, FPR and FPRL1, with higher efficacy on FPRL1. Although these receptors were identified and cloned several years ago, their precise role in host defense has not been fully elucidated (15, 16). Leukocyte infiltration at the sites of inflammation in vivo is considered to be based on migration of cells toward a gradient of chemoattractant(s), either derived from microorganisms or the local tissue. The discovery of synthetic Nformyl oligopeptide chemoattractants for phagocytes represented a major advance in the study of leukocyte locomotion (25). Several natural N-formyl peptide chemoattractants, including the prototype N-formyl peptide, fMLP, have since been purified from bacterial
supernatants, providing evidence in support of them being biologically relevant ligands for FPR on phagocytic cells. Mitochondrial proteins are also N-formylated and are chemotactic for neutrophils (26), thus constituting an endogenous source of chemotactic peptides released by damaged tissue. Although early studies indicated that the N-formyl group was essential for optimal agonist potency (27), recent studies have shown that nonformylated peptides may also attract and activate phagocytes (15, 16). The prototype receptor for formyl peptides designated FPR is expressed by neutrophils and monocytes and was cloned several years ago (15, 16). The FPR was subsequently shown to have a much broader spectrum of agonists than initially expected. In fact, the synthetic pentapeptide Met-Nle-Leu-Phe-Phe-OH, either N-formylated or N-acetylated, is more potent than the parental prototype fMLP in the induction of Ca21 flux in human neutrophils (24). Amino terminal urea-substituted and carbonate-modified peptides have also been shown to be potent agonists for FPR (28, 29). Structural analysis of FPR suggests that the binding pocket of this receptor is able to accommodate an amino-terminal group larger than a formyl group (28, 29), and this may explain the capacity of this receptor to interact with a great variety of endogenously derived, as well as exogenous, ligands. FPRL1 was identified and molecularly cloned from human phagocytic cells by low-stringency hybridization of the cDNA library with the FPR sequence and was initially defined as an orphan receptor (17, 30 –32). The cloning of the same receptor, termed FPRH2, from a genomic library was also described (33). FPRL1 possesses 69% identity at the amino acid level to FPR (15, 16), and both receptors are expressed by monocytes and neutrophils and are clustered on human chromosome 19q13 (33, 34). While fMLP is a
The Journal of Immunology high-affinity agonist for FPR, it interacts with and induces Ca21 flux in FPRL1 only at high concentrations (Fig. 3D, and Refs. 17, 31, and 34). In our study, fMLP did not induce significant migration of FPRL1/293 cells at a concentration as high as 50 mM (5 3 1025 M; Fig. 4B), suggesting that fMLP is not a full agonist for FPRL1. In contrast, T21/DP107, although also activating both receptors, showed a much higher efficacy on FPRL1 and induces migration of FPRL1/293 cells at nM concentrations. Thus, compared with fMLP, T21/DP107 is a functionally more relevant agonist for FPRL1. Although FPRL1 is mainly expressed in monocytes and neutrophils, cells other than phagocytes, such as hepatocytes, have also been shown to express FPRL1 (15). Recently, the expression of this receptor has been reported to be highly inducible in epithelial cells by specific cytokines, such as IL-13 and IFN-g (35). Therefore, FPRL1 may play an important role in inflammatory and immunological responses in human cells. In support of this notion, we recently identified FPRL1 to be a functional receptor for a normal serum protein, serum amyloid A (SAA) (36), which increases its concentration by up to several hundred-fold during acute phase responses and is a potent phagocyte chemoattractant and activator (37, 38). SAA and T21/DP107 attenuated each other’s Ca21-mobilizing activity in FPRL1/293 cells, indicating that these two chemoattractants share FPRL1 as their functional receptor (Su et al., data not shown). It should be noted that T21/DP107 does not bear any significant sequence homology to either fMLP or SAA. Therefore, FPRL1, like its prototype FPR, is also capable of hosting a broad spectrum of ligands. It should be pointed out that in our study, human CD31 T lymphocytes showed a weak migration in response to high concentrations of T21/DP107. Whether this low level migration is also mediated by the presence of FPRL1 in T cells is under investigation. In addition to peptide and protein agonists, a lipid metabolite lipoxin A4 (LXA4) has been reported to be a high-affinity ligand and potent agonist for FPRL1 (also termed LXA4R) (39). LXA4 is an eicosanoid generated during a number of host reactions, such as inflammation, thrombosis, and atherosclerosis, and was initially discovered as an inhibitor of immune responses (reviewed in Ref. 40). LXA4 was subsequently reported to inhibit neutrophil chemotaxis (41) and transepithelial migration induced by chemotactic agents (42). LXA4 bound to Chinese hamster ovary cells transfected with FPRL1(LXA4R) with high affinity and increased GTPase activity and the release of esterified arachidonate (39). Thus, LXA4 has been proposed to be an endogenously produced ligand for FPRL1 (39, 43). Although LXA4 has not been documented to induce Ca21 mobilization in neutrophils or FPRL1transfected cells (39), it was reported to induce Ca21 flux and chemotaxis in monocytes, presumably through FPRL1 (44, 45). Based on these observations, differential activation of second messengers in monocytes vs neutrophils by LXA4 was postulated. In our study, we did not detect significant induction of Ca21 flux or chemotaxis in FPRL1/293 cells by a commercially available LXA4 (Biomol, Plymouth Meeting, PA), nor did we observe inhibition of T21/DP107 signaling by this LXA4 in either phagocytes or FPRL1/293 cells. Since LXA4 is a highly unstable lipid metabolite that could rapidly convert to biologically inactive form, we cannot exclude the possibility that the LXA4 we used might have lost its activity during experiments. Further study is needed to better preserve the activity of LXA4 and to compare the interaction of FPRL1 with its protein/peptide ligands, such as T21/DP107, SAA, and fMLP, vs its lipid ligand LXA4 to clarify these results. Although the signal transduction pathways mediated by FPRL1 have not been extensively studied, the high level of homology to FPR, sensitivity to pertussis toxin, and mediation of potent phago-
5929 cyte migration and activation by its agonists suggest that FPRL1 and FPR may share many signal transduction steps following activation. The binding of FPR by agonists results in a G-proteinmediated signaling cascade leading to cell adhesion, chemotaxis, release of oxygen intermediates, enhanced phagocytosis, and bacterial killing, as well as mitogen-activated protein kinase activation leading to gene transcription (15, 16). Activation by fMLP can also lead to heterologous desensitization of the subsequent cell response to other G-protein receptor ligands (46, 47), including chemokines. In our previous study, incubation of human phagocytes with FPRL1 agonist SAA resulted in a reduction of cell responses to a number of chemoattractants (38), suggesting that activation of FPRL1 may also activate signaling events that cause desensitization of other G-protein-coupled chemotactic receptors. The relevance of our current findings to the course of disease effects on host and benefits, if any, to HIV-1 infection remains to be established. However, our observations do suggest some speculation possibilities. It has been reported that monocytes and neutrophils isolated from HIV-1-infected patients responded poorly to a variety of chemoattractants, including fMLP (14, 48 –51) in vitro. We have found that recombinant soluble gp41 of the HIV-1 is able to potently down-regulate the expression and function of fMLP receptor and the receptors for a variety of chemokines on monocytes, including CCR5 and CXCR4, two major HIV-1 fusion cofactors (13). Intriguingly, the effect of gp41 on monocytes is dependent on the presence of cellular CD4, another fusion receptor of HIV-1 (13). It is not clear whether soluble gp41 itself is capable of interacting with FPR or, alternatively, conjugation with CD4 may cause exposure of its domains to interact with these receptors. Also, further study is needed to examine whether HIV-1 envelope proteins undergo proteolytic cleavage in vivo to yield peptide fragments that interact with FPR and/or FPRL1. In a parallel study, we found that synthetic peptide T20/DP178 of the gp41 C-terminal domain was a potent and selective FPR agonist, while its analogues lacking several amino acids were FPR antagonists (Su et al., manuscript in preparation). This, together with the present observation of T21/DP107, suggests that gp41 may possess multiple domains that could potentially interact with cellular receptors, thus affecting the immune responses. It has been reported that gp41 Ag could be detected in brain tissues of AIDS dementia (52), and Abs recognizing various epitopes of gp41 appear at early stages of HIV-1 infection (53). In fact, we found that both synthetic T21/ DP107 and T20/DP178 epitopes of gp41 were recognized by sera from AIDS patients by immunoblotting (data not shown), suggesting that gp41 and its epitopes are accessible to host cells, including APCs. Therefore, although the receptors for formylated peptides such as FPR and FPRL1 are not used by HIV-1 for fusion, they may participate in the regulation of host innate immune responses seen in AIDS patients characterized by an initial stimulation of immune system in the early stage of the disease followed by progressive immunosuppression.
Acknowledgments We thank Drs. H. Ali and R. Snyderman (Duke University, Durham, NC) for providing ETFR cells, and C. Fogle for secretarial assistance.
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