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The Journal of Immunology, 2002, 169: 4559-4567.
Copyright © 2002 by The American Association of Immunologists

HIV-1 Envelope gp41 Peptides Promote Migration of Human Fc{epsilon}RI+ Cells and Inhibit IL-13 Synthesis Through Interaction with Formyl Peptide Receptors1

Amato de Paulis, Giovanni Florio, Nella Prevete, Massimo Triggiani, Isabella Fiorentino, Arturo Genovese and Gianni Marone2

Division of Clinical Immunology and Allergy, University of Naples Federico II, Naples, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We evaluated the effects of synthetic peptides (2017, 2019, 2020, 2021, 2023, 2027, 2029, 2030, 2031, and 2035) encompassing the structure of HIV-1MN envelope gp41 on both chemotaxis of human basophils and the release of preformed mediators (histamine) and of cytokines (IL-13). Peptides 2019 and 2021 were potent basophil chemoattractants, whereas the other peptides examined were ineffective. Preincubation of basophils with FMLP or gp41 2019 resulted in complete desensitization to a subsequent challenge with homologous stimulus. Incubation of basophils with low concentration (5 x 10-7 M) of FMLP, which binds with high affinity to N-formyl peptide receptor (FPR), but not to FPR-like 1, did not affect the chemotactic response to a heterologous stimulus (gp41 2019). In contrast, a high concentration (10-4 M) of FMLP, which binds also to FPR-like 1, significantly reduced the chemotactic response to gp41 2019. The FPR antagonist cyclosporin H inhibited chemotaxis induced by FMLP, but not by gp41 2019. None of these peptides singly induced the release of histamine or cytokines (IL-4 and IL-13) from basophils. However, low concentrations of peptides 2019 and 2021 (10-8–10-6 M) inhibited histamine release from basophils challenged with FMLP but not the secretion caused by anti-IgE and gp120. Preincubation of basophils with peptides 2019 and 2021 inhibited the expression of both IL-13 mRNA, and the FMLP-induced release of IL-13 from basophils. These data highlight the complexity of the interactions between viral and bacterial peptides with FPR subtypes on human basophils.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The human immunodeficiency viruses HIV-1 and HIV-2 destroy CD4+ lymphocytes, thus leading to AIDS (1). Entry of HIV-1 into immune cells is mediated by the viral envelope glycoproteins, which are organized into oligomeric, probably trimeric, spikes on the surface of the virion (2). The spike surface consists of gp120 associated by noncovalent interactions with gp41 (2, 3). Entry of HIV-1 viruses into immune cells involves gp120 binding to the CD4 glycoprotein, which serves as the primary receptor (4). CD4 binding induces conformational changes in gp120 that lead to the exposure and/or formation of a binding site(s) for specific chemokine receptors (CCR5, CXCR4, and CCR3), which serve as obligate coreceptors for virus entry (5, 6, 7). Interaction with coreceptors induces further conformational changes in the envelope protein and exposure of the hydrophobic fusion domain of the transmembrane gp41 subunit, which then mediates fusion of the opposed cell and virus membranes (8).

We have demonstrated that HIV-1gp120 from different clades is a potent stimulus for histamine and cytokine (IL-4 and IL-13) release from basophils from healthy individuals seronegative for Abs to HIV-1 and HIV-2. gp120 acts as a viral superantigen, i.e., it interacts with the VH3 region of IgE to induce mediator release from human Fc{epsilon}RI+ cells (9). In addition, we have shown that by interacting with the {beta}-chemokine receptor CCR3, the HIV-1 Tat protein is a potent chemoattractant for human basophils and mast cells. Moreover, Tat protein up-regulates the level of CCR3 mRNA and surface expression of CCR3 on human basophils (10). Recent evidence suggests that a population of basophils and/or mast cells in peripheral blood can be infected in vitro by M-tropic strains of HIV-1 (11, 12). Consequently, human Fc{epsilon}RI+ cells are important in HIV-1 infection (13, 14, 15, 16).

Several lines of evidence indicate that gp41 exerts also multiple effects on the immune system. Soluble gp41 has been shown to induce the production of proinflammatory cytokines such as TNF-{alpha} and IL-1 (17, 18, 19, 20, 21) and to increase the activity of nitric oxide synthetase in mononuclear phagocytes and glial cells (22). gp41 was also reported to suppress human lymphocyte proliferation (23) and to favor a Th2-type immune response by inducing IL-10 production in monocytes (24). There is evidence that gp41 is a potent inhibitor of the activation of the receptor for FMLP (25). Moreover, it has been demonstrated that synthetic peptides of gp41 attract and activate human monocytes and neutrophils by using G-protein-coupled N-formyl peptide receptors (FPRs)3 (26, 27).

Several natural N-formyl peptides, including the prototype FMLP, have been purified from bacterial supernatants, which suggests they are biologically relevant ligands for formyl peptide receptors. FMLP binds and activates G-protein-coupled, seven transmembrane (STM) cell receptors. Three STM receptors expressed by phagocytic leukocytes have been identified and cloned: neutrophils express the high-affinity receptor (FPR) and its homologue FPR-like (FPRL) 1, whereas monocytes express FPR, FPRL1, and FPRL2 (28, 29). FPR is a high-affinity receptor for FMLP, whereas FPRL1 has a much lower affinity (30, 31). FPRL1 is a promiscuous receptor activated by serum amyloid A (32), lipoxin A4 (33), and by various synthetic peptides (34). Cyclosporin H (CsH) and spinorphin are specific antagonists at the FMLP receptor subtype FPR (35, 36, 37). No natural FPRL2 agonists have been identified. However, it has been demonstrated that Helicobacter pylori peptide Hp (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) activates the monocyte via FPRL2 (34).

Given the possible involvement of Fc{epsilon}RI+ cells in HIV-1 infection, we investigated the effects of synthetic peptides of gp41 on human basophils that express FPRs (35, 38). We found that two synthetic peptides 2019 and 2021 are potent chemoattractants for human basophils and inhibit FMLP-induced IL-13 synthesis in human Fc{epsilon}RI+ cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

The following were purchased: 60% HClO4 (Baker, Deventer, The Netherlands); human serum albumin (HSA) and PIPES (Sigma-Aldrich, St. Louis, MO); HBSS, FCS, TRIzol, and murine Moloney leukemia virus transcriptase (murine Moloney leukemia virus) (Life Technologies, Grand Island, NY); FMLP (Calbiochem, La Jolla, CA); RPMI 1640 with 25 mM HEPES buffer, Eagle’s MEM (Flow Laboratories, Irvine, Scotland); dextran 70 and Percoll (Pharmacia, Uppsala, Sweden). CsH was obtained from Drs. D. Romer and E. Rissi (Novartis, Basel, Switzerland). Recombinant gp120MN and synthetic peptides of gp41MN were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Disease, National Institutes of Health (Bethesda, MD). Rabbit anti-human-Fc{epsilon} Ab was a generous gift from Drs. T. Ishizaka and K. Ishizaka (La Jolla Institute for Allergy and Immunology, La Jolla, CA).

Buffers

The PIPES buffer used in these experiments was made up of 25 mM PIPES, pH 7.4, 110 mM NaCl, 5 mM KCl. The mixture is referred to as P. PCG contains, in addition to P, 5 mM CaCl2 and 1 g/L D-glucose (39). PACGM contains, in addition to P, 3% HSA, 1 mM CaCl2, 1 g/L dextrose, and 0.25 g/L MgCl2 · 6H2O, pH 7.4; PGMD contains 0.25 g/L MgCl2 · 6H2O, 10 mg/L DNase, and 1 g/L gelatin in addition to P, pH 7.4. PBS contains 8 g/L NaCl, 1.15 g/L Na2HPO4, 200 mg/L KCl, and 200 mg/L KH2PO4, pH 7.4.

Purification of peripheral blood basophils

Basophils were purified from peripheral blood cells of healthy volunteers, seronegative for Abs to HIV-1 and HIV-2, who ranged in age from 20 to 39 years (mean 31.9 ± 6.3 years). "Buffy coat" cell packs from healthy volunteers, provided by the Immunohematology Service (University of Naples Federico II, Naples, Italy), were reconstituted in PBS containing 0.5 g/L HSA and 3.42 g/L sodium citrate, and loaded onto a countercurrent elutriator (model J2-21; Beckman Coulter, Fullerton, CA). Several fractions were collected, and fractions containing basophils in large numbers (>20 x 106 basophils) and of good purity (>15%) were enriched by discontinuous Percoll gradients (40). Basophils were further purified to near homogeneity (> 98%) by depleting B cells, monocytes, NK cells, dendritic cells, erythrocytes, platelets, neutrophils, eosinophils, and T cells, using a mixture of hapten-conjugated CD3, CD7, CD14, CD15, CD16, CD36, CD45RA, and anti-HLA-DR Abs and MACS MicroBeads coupled to an anti-hapten mAb. The magnetically labeled cells were depleted by retaining them on a MACS column in the magnetic field of the MidiMACS (Miltenyi Biotec, Bergisch Gladbach, Germany). Yields ranged from 3 to 10 x 106 basophils with purity usually above 98%, assessed by basophil staining with Alcian Blue and counting in a Spiers-Levy eosinophil counter (Carlo Erba, Milan, Italy) (39).

Histamine release

Basophils (~6 x 104 basophils/tube) were resuspended in PCG, and 0.1 ml of the cell suspension was placed in 12 x 75 mm polyethylene tubes (Sarstedt, Princeton, NJ) and warmed to 37°C; 0.1 ml of each prewarmed releasing stimulus was added, and incubation was continued at 37°C for 45 min (9). At the end of this step, the reactions were stopped by centrifugation (1,000 x g, 22°C, 2 min), and the cell-free supernatants were stored at -20°C for subsequent assay of histamine content with an automated fluorometric technique (41). Total histamine content was assessed by lysis induced by incubating the cells with 2% HClO4 before centrifugation. To calculate histamine release as a percentage of total cellular histamine, the spontaneous release of histamine from mast cells (2% to 6% of the total cellular histamine) was subtracted from both the numerator and denominator (40). The percentage of histamine release was calculated according the following equation: ((A - B)/(T - B)) x 100, where A is the sample, B is the spontaneous histamine release, and T is the total histamine content. All values are based on the means of duplicate or triplicate determinations. Replicates differed in histamine content by <10%.

Chemotaxis assay

Basophil chemotaxis was performed using a modified Boyden chamber technique as described elsewhere (40, 42). Briefly, 25 µl of PACGM buffer or various concentrations of the chemoattractants in the same buffer were placed in triplicate in the lower compartment of a 48-well microchemotaxis chamber (NeuroProbe, Cabin John, MD). The lower compartments were covered with polycarbonate membranes with 5-µm pores (Nuclepore, Pleasanton, CA). Fifty microliters of the cell suspensions (5 x 104/well) resuspended in PACGM were pipetted into the upper compartments. The chemotactic chamber was then incubated for 1 h at 37°C in a humidified incubator with 5% CO2 (Automatic CO2 Incubator, Model 160 IR; ICN Flow, Buckinghamshire, U.K.). At the end of incubation, the membrane was removed, washed with PBS on the upper side, fixed, and stained with May-Grunwald/Giemsa, and then mounted on a microscope slide with Cytoseal (Stephens Scientific, Springfield, NJ). Basophil chemotaxis was quantitated microscopically by counting the number of cells attached to the surface of the 5-µm cellulose nitrate filter. In each experiment, 10 fields per triplicate filter were measured at x 40 magnification. The results were compared with buffer controls. Checkboard analyses were performed to discriminate between chemotaxis and nondirected migration (chemokinesis) of basophils. In these experiments, basophils were placed in the upper chambers and various concentrations of gp41 peptides, FMLP, or PACGM buffer were added to either the upper or lower wells or to both. Spontaneous migration (chemokinesis) was determined in the absence of chemoattractant or when stimuli were added to either the lower or upper chambers. The basophil migratory response to chemotactic stimuli was largely due to chemotaxis and not to chemokinesis. Indeed, a checkboard analysis, in which chemoattractants above and below the filters were varied, resulted in significant migration only when there was a gradient of the factor below the filters.

Isolation of cellular mRNA and RT-PCR

Total RNA was extracted from basophils using a single-step method with TRIzol according to the manufacturer’s instructions. One microgram of total RNA was converted into cDNA in a standard reverse transcriptase reaction, using murine Moloney leukemia virus at 200 U/µl and oligo(dT) as primer. cDNA was then titrated for {beta}-actin message, and equivalent templates of cDNAs, obtained from different cultures, were amplified for IL-13 message using the specific primers described previously (43). Amplification was performed using a 9600 Thermocycler (PerkinElmer, Monza, Italy). Amplification protocol consisted of 30 cycles as follows: denaturation, 1 min at 94°C; annealing, 1 min at 56°C; extension at 72°C for 1 min. Finally, there was an extension at 72°C for 10 min. The PCR products were loaded on 2% agarose gel and run in a submarine gel apparatus. The gel was then stained with ethidium bromide and bands were visualized on a UV source. Bands were quantified by scanning the gel in a Fluorimager (Molecular Dynamics, Sunnyvale, CA) and measuring relative fluorescence units with ImageQuant software.

IL-4 and IL-13 ELISA

IL-4 and IL-13 release in the culture supernatants of basophils was measured in duplicate determinations by a commercially available ELISA (R&D Systems, Minneapolis, MN) (44).

Lactate dehydrogenase (LDH) assay

LDH release at the end of the incubations was determined as an index of cytotoxicity. LDH was measured in cell-free supernatants using a commercially available kit (Sigma-Aldrich) (45).

Statistical analysis

The results are expressed as mean ± SEM. Statistical significance was analyzed by one-way analysis of variance and when the F value was significant, by Duncan’s multiple range test (46). Differences were considered significant when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of HIV-1 gp41 peptides on chemotaxis of human basophils

Previous studies produced apparently conflicting results about the effects of gp41 and its peptides on phagocyte chemotaxis. For instance, gp41 of the MN strains markedly reduces chemotaxis in response to FMLP (25), whereas the synthetic peptide T21of gp41, by activating FPR (26), is a potent chemotactic stimulus for human phagocytes (26). In a first series of experiments, we evaluated the effects of HIV-1 gp41 peptides (2017, 2019, 2020, 2021, 2023, 2027, 2029, 2030, 2031, and 2035) encompassing the gp41MN sequence (Table IGo) or FMLP on chemotaxis of basophils purified (>98%) from peripheral blood from healthy individuals seronegative for Abs to HIV-1 and HIV-2. Fig. 1Go shows the results of six experiments that demonstrate that gp41MN peptides 2019 and 2021 caused basophil chemotaxis, whereas the other peptides were ineffective. The maximal chemotaxis observed was slightly lower than that induced by the well-characterized and efficacious basophil chemoattractant FMLP.


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  		Table I. Peptide sequences of HIV-1MN gp41

 


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  		FIGURE 1. Effects of HIV-1 gp41 peptides or FMLP on human basophil chemotaxis. Basophils obtained from peripheral blood of donors negative for HIV-1 and HIV-2 Abs were allowed to migrate toward the indicated gp41 peptides (5 x 10-7 M) or FMLP (5 x 10-7 M) for 1 h at 37°C in a humidified incubator with 5% CO2. Values are the mean ± SEM of six experiments with different basophil preparations. *, p < 0.01 compared with basophils preincubated in the absence of chemotactic stimuli.

 
Effects of HIV-1 envelope gp41 peptides on histamine and cytokine release from human basophils

We then tested increasing concentrations (10-8–10-6 M) of the panel of synthetic gp41 peptides for their effect on histamine release from human basophils purified (> 98%) from peripheral blood from healthy individuals seronegative for Abs to HIV-1 and HIV-2. In six experiments, none of the peptides tested caused the release of histamine from basophils (data not shown). In these experiments we used, as controls, two IgE-mediated stimuli (anti-IgE and gp120) that cross-link IgE on the surface of basophils (9, 47). We also used FMLP, which activated an STM receptor on human basophils independent of the IgE receptor (38, 39). As previously shown (9, 47), anti-IgE and gp120 induced histamine release from basophils from donors seronegative for Abs to HIV-1 and HIV-2. In these experiments, FMLP also caused the release of histamine from basophils isolated from healthy individuals. The concentrations of gp41 peptides used (10-8–10-6 M) did not induce spontaneous LDH release from basophils. In a series of three experiments, the same panel of synthetic HIV-1 gp41 peptides did not induce IL-4 or IL-13 release from basophils (data not shown).

Effects of CsH on basophil chemotaxis induced by HIV-1 gp41 peptides and FMLP

We have previously demonstrated that CsH is a specific inhibitor of the release of preformed and de novo synthesized mediators induced by FMLP from basophils (35). More recent results have demonstrated that CsH is a specific FPR antagonist (29, 36). In three experiments, we examined the effects of CsH (800 nM) on basophil chemotaxis induced by FMLP (5 x 10-7 M) and by gp41 2019 (5 x 10-7 M). Preincubation (15 min at 37°C) of basophils with CsH inhibited (>70%) basophil chemotaxis induced by FMLP. In none of these experiments did CsH inhibit basophil chemotaxis induced by gp41 2019 (Fig. 2GoA). Similar results were obtained when basophil chemotaxis was induced by gp41 2021 (Fig. 2GoB). These results are compatible with the hypothesis that FMLP induced basophil chemotaxis through the activation of FPR, whereas the gp41 peptides acted through different subtypes of FMLP receptors.



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  		FIGURE 2. A, Effect of CsH on human basophil chemotaxis induced by FMLP (5 x 10-7 M) or gp41 (2019) (5 x 10-7 M). Basophils isolated from normal donors negative for HIV-1 and HIV-2 Abs were preincubated (5 min at 37°C) with CsH (800 nM) or buffer. Basophils were allowed to migrate toward FMLP (5 x 10-7 M) or gp41 (2019) (5 x 10-7 M) for 1 h at 37°C in a humidified incubator with 5% CO2. Values are the mean ± SEM obtained from three experiments with different basophil preparations. *, p < 0.01 when compared with FMLP. B, Effect of CsH on human basophil chemotaxis induced by FMLP (5 x 10-7 M) or gp41 (2021) (5 x 10-7 M). Basophils isolated from normal donors negative for HIV-1 and HIV-2 Abs were preincubated (5 min at 37°C) with CsH (800 nM) or buffer. Basophils were allowed to migrate toward FMLP (5 x 10-7 M) or gp41 (2021) (5 x 10-7 M) for 1 h at 37°C in a humidified incubator with 5% CO2. Values are the mean ± SEM obtained from three experiments with different basophil preparations. *, p < 0.01 when compared with FMLP.

 
Cross-desensitization between HIV-1 gp41 peptides and FMLP

The relationship between gp41 peptides and FMLP was further examined by evaluating the effects of cross-desensitization between these stimuli on basophil chemotaxis. Purified basophils were treated with buffer or a low concentration of FMLP (5 x 10-7 M) or gp41 2019 (5 x 10-7 M) in P-EDTA for 30 min at 37°C. At the end of incubation, cells were washed and suspended in PACGM. Fig. 3GoA shows the results of a typical experiment of three in which the response to FMLP or gp41 2019 was significantly desensitized by preincubation with the homologous stimulus. When basophils were desensitized by preincubation with a low concentration (5 x 10-7 M) of FMLP, the response to heterologous stimulus (gp41 2019) was not significantly affected. Interestingly, different results were obtained when basophils were desensitized by exposure to a high concentration (10-4 M) of FMLP, which binds also to FPRL1 (26). Fig. 3GoB shows a typical experiment of three in which the response to gp41 2019 and to gp41 2021 was significantly desensitized by preincubation with a high concentration of FMLP.



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  		FIGURE 3. A, Effects of cross-desensitization between a low concentration of FMLP (5 x 10-7 M) and gp41 (2019) (5 x 10-7 M) on basophil chemotaxis. Basophils isolated from normal donors negative for HIV-1 and HIV-2 were incubated in PIPES buffer containing EDTA (4 mM), FMLP (5 x 10-7 M), or gp41 (2019) (5 x 10-7 M) for 30 min at 37°C. At the end of incubation, cells were washed (two times), resuspended in PACGM, and challenged with the chemotactic stimuli (FMLP, 5 x 10-7 M, or gp41 (2019), 5 x 10-7 M). Basophils were allowed to migrate for 1 h at 37°C in a humidified incubator with 5% CO2. Values are the mean ± SEM from triplicate determinations. *, p < 0.01 when compared with FMLP; **, p < 0.01 when compared with gp41 (2019). B, Effects of desensitization with a high concentration of FMLP (10-4 M) on basophil chemotaxis. Basophils isolated from normal donors negative for HIV-1 and HIV-2 were incubated in PIPES buffer containing EDTA (4 mM) or FMLP (10-4 M) for 30 min at 37°C. At the end of incubation, cells were washed (two times) and resuspended in PACGM and challenged with gp41 (2019) or gp41 (2021) (5 x 10-7 M). Basophils were allowed to migrate for 1 h at 37°C in a humidified incubator with 5% CO2. Each bar represents the mean ± SEM from triplicate determinations. *, p < 0.01 when compared with gp41 (2019). **, p < 0.01 when compared with gp41 (2021).

 
Effects of HIV-1 gp41 peptides on FMLP-induced histamine release from human basophils

We next assessed the effects of increasing concentrations of overlapping sequences of gp41 on histamine release from human basophils induced by FMLP. Fig. 4Go shows the results from four experiments in which basophils were preincubated (60 min at 37°C) with optimal concentrations (10-6 M) of gp41 peptides before challenge with FMLP. Only the two synthetic peptides 2019 and 2021 (10-6 M) inhibited >90% FMLP-induced histamine release in a concentration-dependent fashion. The other peptides examined had little or no effect on basophil mediator release induced by FMLP. In a series of three experiments we evaluated the effects of various concentrations (10-8–10-6 M) of gp41 peptide 2019 and 2021 on histamine release induced by FMLP from basophils. The two synthetic peptides, 2019 and 2021, caused concentration-dependent inhibition of histamine release induced by FMLP, ranging from 10% at 10-8 M to ~90% at 10-6 M (data not shown). These experiments were performed with 60 min of preincubation before challenge with FMLP because the results obtained in three kinetic experiments showed that the maximal inhibitory effects of peptides 2019 and 2021 occurred within this time of preincubation (Fig. 5Go).



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  		FIGURE 4. Effect of an optimal concentration (10-6 M) of HIV-1 envelope gp41 peptides on histamine release induced by FMLP (10-6 M) from human basophils purified from normal donors negative for HIV-1 and HIV-2 Abs. Cells were preincubated (60 min at 37°C) with gp41 peptides and then incubated (30 min at 37°C) with an optimal concentration (10-6 M) of FMLP. Values are the means ± SEM obtained from four experiments with different basophil preparations. *, p < 0.01 when compared with FMLP.

 


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  		FIGURE 5. The effect of HIV-1 envelope gp41 peptides 2019 and 2021 in relation to the preincubation time. Cells were incubated with peptides 2019 or 2021 (10-6 M) for 15, 35, or 60 min and then challenged (30 min at 37°C) with FMLP (10-6 M). Each point represents the mean ± SEM of the percentage inhibition of three experiments in which FMLP induced the release of 40.2 ± 6.3% of total histamine content. *, p < 0.01 when compared with histamine release induced by FMLP alone.

 
Effects of HIV-1 gp41 peptides on anti-IgE-induced histamine release from human basophils

Previous studies have demonstrated that the activation of FPR and cross-linking of Fc{epsilon}RI by anti-IgE induce different biochemical steps in human basophils (48). Therefore, we examined the effects of gp41 peptides on histamine release from basophils induced by anti-IgE that cross-links the Fc{epsilon}RI receptor, which is independent of the FPR (38). Preincubation (5–60 min at 37°C) of basophils with different concentrations (10-8–10-6 M) of gp41 peptides did not inhibit the release of histamine from basophils induced by anti-IgE (Fig. 6Go).



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  		FIGURE 6. Effect of various concentrations (10-8 to 10-6 M) of HIV-1 envelope gp41 peptides 2019 and 2021 on histamine release induced by anti-IgE from human basophils purified from normal donors negative for HIV-1 and HIV-2 Abs. Cells were preincubated (60 min at 37°C) with peptides 2019 and 2021 and then incubated (30 min at 37°C) with an optimal concentration (0.1 µg/ml) of anti-IgE. Values are the mean of duplicate determinations from a typical experiment.

 
Interaction between HIV-1 gp41 peptides and FMLP-induced histamine release from basophils

In a series of experiments, we examined the interaction between gp41 peptides and FMLP on histamine release from basophils. Fig. 7GoA shows the results obtained in one of eight experiments in which preincubation (60 min at 37°C) of basophils with peptide 2019 (10-8–3 x 10-7 M) concentration-dependently inhibited FMLP-induced histamine release from basophils. Incubation of basophils with increasing concentrations of peptide 2019 depressed the maximal effect of FMLP. Similar results were obtained in three experiments in which basophils were preincubated with peptide 2021 (10-8–10-6 M} before challenge with increasing concentrations of FMLP (Fig. 7GoB).



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  		FIGURE 7. A, Effect of various concentrations (10-8 to 3 x 10-7 M) of HIV-1 envelope peptide 2019 on histamine release induced by FMLP from human basophils purified from normal donors negative for HIV-1 and HIV-2 with FMLP Abs. Cells were preincubated (60 min at 37°C) with peptide 2019 and then incubated (30 min at 37°C) with increasing concentrations of FMLP. Values are the mean of duplicate determinations from a typical experiment. B, Effect of various concentrations (10-8 to 10-6 M) of HIV-1 envelope peptide 2021 on histamine release induced by FMLP from human basophils purified from normal donors negative for HIV-1 and HIV-2 Abs. Cells were preincubated (60 min at 37°C) with peptide 2021 and then incubated (30 min at 37°C) with increasing concentrations of FMLP. Values are the means ± SEM obtained from three experiments with different basophil preparations.

 
Effects of HIV-1 gp41 peptide 2021 on IL-13 mRNA induced by FMLP in human basophils

It has been reported that FMLP stimulates de novo production of IL-13 in human basophils (49). To examine further whether gp41 peptides regulate the synthesis of cytokines in human basophils, we examined the effects of peptide 2021 on FMLP-induced IL-13 mRNA expression in human basophils. Fig. 8Go depicts the specific RT-PCR amplification products from one representative experiment of three. Adequate normalization of RNA for each sample was confirmed by the quality of RT-PCR amplification products for the constitutive marker gene of {beta}-actin. FMLP increased IL-13 mRNA expression after 6 h of incubation. Preincubation of basophils with peptide 2021 inhibited IL-13 mRNA expression.



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  		FIGURE 8. Effect of peptide 2021 on IL-13 mRNA expression induced by FMLP in human basophils. Basophils were preincubated (60 min at 37°C) with peptide 2021 (10-6 M) before FMLP (10-6 M) was added. The cells were then incubated for an additional 6 h at 37°C. mRNA transcript for IL-13 was analyzed by RT-PCR.

 
Effects of HIV-1 peptides 2019 and 2021 on FMLP-induced IL-13 release from human basophils

The above data demonstrate that gp41 peptide 2021 inhibits IL-13 synthesis induced by FMLP in basophils. We also assessed the effects of increasing concentrations of peptides 2019 and 2021 on IL-13 release from human basophils induced by FMLP. Fig. 9GoA shows the results of three experiments in which basophils were preincubated (60 min at 37°C) with different concentrations of peptide 2019 before challenge with FMLP. Peptide 2019 caused concentration-dependent inhibition of IL-13 release, ranging from 10% at 10-8 M to ~90% at 10-6 M. Similar results were obtained with peptide 2021 (Fig. 9GoB). Thus, gp41 peptides 2019 and 2021 inhibit both cytokine synthesis and release from human basophils activated by FMLP.



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  		FIGURE 9. A, Effect of various concentrations (10-8–10-6 M) of peptide 2019 on IL-13 secretion from purified human basophils challenged with FMLP (10-6 M). The cells were preincubated (60 min at 37°C) with peptide 2019 before FMLP challenge. The cells were then incubated for an additional 18 h at 37°C. Values are the means ± SEM obtained from three experiments. *, p < 0.01 when compared with FMLP. B, Effect of various concentrations (10-8 to 10-6 M) of peptide 2021 on IL-13 secretion from purified human basophils challenged with FMLP (10-6 M). The cells were preincubated (60 min at 37°C) with peptide 2021 before FMLP challenge. The cells were then incubated for an additional 18 h at 37°C. Values are the means ± SEM obtained from three experiments. *, p < 0.01 when compared with FMLP.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that two ectodomain peptides of HIV-1 envelope gp41MN, 2019 and 2021, are potent chemoattractants for human Fc{epsilon}RI+ cells. Moreover, these two peptides selectively inhibit the release of preformed mediators and of cytokines from human basophils activated by FMLP. This is the first demonstration that ectodomain peptides of HIV-1 gp41 are functionally active on human Fc{epsilon}RI+ cells presumably through interaction with FMLP receptor(s).

Increasing evidence indicates that human Fc{epsilon}RI+ cells could be involved in HIV-1 infection in a variety of ways (11, 12, 13, 14, 15, 16). In fact, we have demonstrated that gp120 from divergent HIV-1 isolates from different clades interacts with the VH3 region of IgE on human Fc{epsilon}RI+ (9, 47). The superantigenic interaction between gp120 and IgE leads to the synthesis and release of IL-4 and IL-13 from Fc{epsilon}RI+ cells (9). In addition, we found that the HIV-1 Tat protein is a potent chemoattractant for human Fc{epsilon}RI+ cells and up-regulates the expression of CCR3 on these cells (10). These results demonstrate that two distinct HIV-1 proteins, gp120 and Tat, can activate human Fc{epsilon}RI+ cells through different immunologic mechanisms.

Human basophils, but not mast cells, also express receptors for FMLP which induce their chemotaxis and the release of proinflammatory mediators (35, 38, 40). Three FPRs have been identified on different cells (28, 29). These receptor subtypes belong to the STM, G-protein-coupled Rhodopsin superfamily (50, 51). The FPR has a high affinity for FMLP and is activated by picomolar concentrations of FMLP. FPRL1 is a promiscuous receptor activated in response to higher concentrations of FMLP (50), lipoxin A4 (33), H. pylori peptide Hp (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) (34), serum amyloid A (32), and by different synthetic peptides (29). Human monocytes express FPRL2 and Hp (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) is chemotactic through the activation of this receptor subtype (29). The distribution of FPR subtypes in human basophils and their biological significance in immune responses in HIV-1 infection remain to be established.

The results of our experiments reveal that interactions between gp41 peptides and FMLP receptors expressed on human basophils are more complex than hitherto believed. CsH is known to block FPR-evoked responses (29, 35, 36). Accordingly, CsH blocked the chemotactic activity of FMLP on basophils, but had no effect on the response evoked by gp41 peptides 2019 and 2021. To try to clarify the specificity in the activation route for gp41 peptides and FMLP, we performed cross-desensitization experiments. Upon binding of FMLP to its FPR, the occupied receptor is phosphorylated (52). Cells are subsequently desensitized and unable to generate signals through the same receptor. We have demonstrated that basophils preincubated with FMLP in the absence of Ca2+ were unable to generate a chemotactic response when rechallenged with the same agonist in a Ca2+ containing buffer (homologous desensitization). Similarly, basophils preincubated with gp41 peptide 2019 or 2021 were desensitized to a subsequent challenge with the homologous stimulus. Heterologous desensitization provides interesting and apparently contrasting results. When basophils were preincubated with a low concentration (5 x 10-7 M) of FMLP, which binds with high affinity only to FPR, but not to FPRL1 (50), the chemotactic response to heterologous stimulus (gp41 2019) was not affected. In contrast, when basophils were exposed to high concentrations (10-4 M) of FMLP, which binds also to FPRL1 (50), the chemotactic response to gp41 2019 was significantly reduced. The results of these two groups of experiments are consistent with the hypothesis that gp41 peptides and FMLP act through different FPR subtypes to induce chemotaxis of human basophils: FMLP acts essentially through the interaction with FPR, whereas gp41 peptides presumably act through FPRL1. This hypothesis is supported by the different pharmacologic effect of CsH on basophil chemotaxis induced by FMLP and gp41 peptides. Interestingly in this context, recent experiments performed in our laboratory have demonstrated the presence of FPRL1 mRNA in human basophils (data not shown).

Several investigators have started to examine the complexity of interactions of HIV-1 gp41 peptides and FPRs expressed on human phagocytes. It was found that the retroviral-derived exapeptide LDLLFL is a potent inhibitor of FMLP-induced responses of human monocytes and granulocytes (53). Lawless et al. (54) reported that two synthetic peptides, designated T21 and T20, corresponding to amino acid sequences 558–595 and 643–678, respectively, in HIV-1LAI gp41, are strong inhibitors of HIV-1 viral fusion. Ueda et al. (25) reported that HIV-1 envelope gp41 is a potent inhibitor of chemotaxis induced by FMLP in human monocytes. The same group of investigators reported that T21 activates human phagocytes by using FPR and FPRL1 (26). It was also found that synthetic T20 analogs lacking N-terminal amino acids acted as FPR antagonists (27). Furthermore, a synthetic peptide corresponding to amino acid residues 414–434 down-regulates the expression of CCR5 and CXCR4 receptors by activating FPRL1 (55). Finally, a synthetic exapeptide (Trp-Lys-Tyr-Val-D-Met) that uses both FPR and FPRL1 acts as a potent agonist to stimulate human phagocytes (50, 51) with preference for FPRL1 (51). Taken together, these findings emphasize the different effects of retroviral-derived peptides on human immune cells. In addition, they indicate that natural and synthetic peptides can act as either agonist or antagonist at the FPR level.

Our findings demonstrate also that prolonged incubation with low concentrations of peptides 2019 and 2021 of HIV-1 envelope gp41 of the MN strains inhibits the release of preformed mediators and of cytokine synthesis from human basophils activated by FMLP. Although several synthetic peptides encompassing the structure of HIV-1MN envelope gp41 do not activate the release of cytokines and proinflammatory mediators from human basophils, peptides 2019 and 2021 specifically antagonize the activation of basophils induced by FMLP. These novel results are of interest for several reasons. First, they show that gp41 peptides 2019 and 2021 exert different effects on basophil chemotaxis and on mediator secretion from these cells. In fact, in the former system the peptides induce basophil chemotaxis presumably by interacting with FPRL1. In the latter system, low concentrations of peptides 2019 and 2021 are devoid of any activating property on the release of mediators, but they antagonize the releasing activity of FMLP on basophils. Interestingly, the inhibitory effect of peptides 2019 and 2021 on FMLP-induced basophil activation is not immediate and requires a long preincubation time (35–60 min). This lag could be necessary to activate/deactivate certain metabolic steps or to cause FMLP receptor internalization.

Taken together, these results might suggest that the chemotactic activity and the release of mediators from basophils induced by FMLP and gp41 peptides are mediated by activation of different FPR subtypes. Alternatively, the possibility exists that signal transduction mechanisms underlying chemotaxis and secretion are differently activated, inhibited and/or modulated by gp41 peptides and FMLP. Finally, it might be that FMLP and gp41 peptides, in addition to FPR, FPRL1 and FPRL2, activate or antagonize additional unknown receptor(s) on basophils. Whatever the mechanisms, our findings leave no doubt as to the complexity of the interactions between FMLP, gp41 peptides, and FMP subtypes in human basophils.

During HIV-1 infection, the envelope gp160 is enzymatically cleaved, yielding two mature proteins, the transmembrane gp41 and surface gp120 (56, 57). HIV-1 infection is initiated by high-affinity binding of gp120 to CD4, the primary receptor for HIV-1 (58, 59). gp41 is thought to mediate fusion between viral and cellular membranes by the insertion of hydrophobic N terminus into the plasma membranes (57, 60). Several lines of inquiry indicate that gp41 also may exert multiple effects on the host immune system. Soluble gp41 has been shown to induce the release of proinflammatory cytokines (TNF-{alpha} and IL-1) (17, 18, 19, 20, 21), to suppress lymphocyte proliferation (23) and to induce IL-10 in monocytes (24). gp41 has been detected in brain tissues of patients affected by AIDS dementia (22) and Abs recognizing various epitopes of gp41 appear at early stages of HIV infection (61). Moreover, it has been reported that monocytes and neutrophils from HIV-1-infected patients responded poorly to a variety of chemoattractants, including FMLP (62, 63, 64, 65, 66). The interaction of gp41 peptides with FPRs on human basophils might be of clinical relevance in patients with HIV-1 infection.

We previously demonstrated that gp120 directly induces the release of preformed mediators and of cytokines (IL-4 and IL-13) from human basophils purified from healthy individuals (9). In this study, we found that another envelope glycoprotein, gp41, can interfere with cytokine production by inhibiting the synthesis of cytokines induced by FMLP in human basophils. Together these findings indicate the complexity of the interaction between HIV-1 envelope peptides and human Fc{epsilon}RI+ cells. The biological significance in host defense and immune responses in HIV-1 infection of our findings requires additional investigations.

In conclusion, we provide the first evidence that two envelope gp41 peptides are potent chemoattractants for human Fc{epsilon}RI+ cells and inhibit FMLP-induced mediator release from human basophils. Because HIV-1 enters the body predominantly through mucosal surfaces and because early phases of infection are associated with high levels of viremia, basophils can be exposed to high local levels of envelope gp41 peptides. This suggests that Fc{epsilon}RI+ cells can contribute, also through this novel mechanism, to the dysregulation of the immune system in HIV-1 infection.


   Acknowledgments
 
This paper is dedicated to Rita Levi-Montalcini who first suggested an involvement of Fc{epsilon}RI+ cells in HIV-1 infection. We thank Dr. Giorgio Fratellanza for providing buffy coat cell packs and the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Disease, National Institutes of Health (Bethesda, MD) for the gift of recombinant gp120MN and synthetic peptides of gp41MN.


   Footnotes
 
1 This work was supported by grants from the Istituto Superiore Sanità (AIDS Project 40B.64 and 40.B1), Ministero della Salute "Alzheimer Project," Ministero dell’Università e della Ricerca Scientifica e Tecnologica National Project "Helicobacter pylori infection: host-pathogen interactions," and Consiglio Nazionale delle Ricerche (Target Project Biotechnology No. 01.00191. PF31 and 01.00295. PF49; Rome, Italy). Back

2 Address correspondence and reprint requests to Dr. Gianni Marone, Division of Clinical Immunology and Allergy, University of Naples Federico II, Via Sergio Pansini 5, 80131 Napoli, Italy. E-mail address: marone{at}unina.it Back

3 Abbreviations used in this paper: FPR, N-formyl peptide receptor; STM, seven transmembrane; FPRL, FPR-like; CsH, cyclosporin H; HSA, human serum albumin; LDH, lactate dehydrogenase. Back

Received for publication April 22, 2002. Accepted for publication August 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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A. de Paulis, N. Montuori, N. Prevete, I. Fiorentino, F. W. Rossi, V. Visconte, G. Rossi, G. Marone, and P. Ragno
Urokinase Induces Basophil Chemotaxis through a Urokinase Receptor Epitope That Is an Endogenous Ligand for Formyl Peptide Receptor-Like 1 and -Like 2
J. Immunol., November 1, 2004; 173(9): 5739 - 5748.
[Abstract] [Full Text] [PDF]

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