HIV gp120 Induces the Release of Proinflammatory, Angiogenic, and Lymphangiogenic Factors from Human Lung Mast Cells

Human lung mast cells (HLMCs) express the high-affinity receptor FcεRI for IgE and are involved in chronic pulmonary diseases occurring at high frequency among HIV-infected individuals. Immunoglobulin superantigens bind to the variable regions of either the heavy or light chain of immunoglobulins (Igs). Glycoprotein 120 (gp120) of HIV-1 is a typical immunoglobulin superantigen interacting with the heavy chain, variable 3 (VH3) region of human Igs. The present study investigated whether immunoglobulin superantigen gp120 caused the release of different classes of proinflammatory and immunoregulatory mediators from HLMCs. The results show that gp120 from different clades induced the rapid (30 min) release of preformed mediators (histamine and tryptase) from HLMCs. gp120 also caused the de novo synthesis of cysteinyl leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) from HLMCs. Incubation (6 h) of HLMC with gp120 induced the release of angiogenic (VEGF-A) and lymphangiogenic (VEGF-C) factors from HLMCs. The activating property of gp120 was mediated through the interaction with IgE VH3+ bound to FcεRI. Our data indicate that HIV gp120 is a viral superantigen, which induces the release of different proinflammatory, angiogenic, and lymphangiogenic factors from HLMCs. These observations could contribute to understanding, at least in part, the pathophysiology of chronic pulmonary diseases in HIV-infected individuals.


Introduction
The human immunodeficiency virus (HIV-1) affects more than 36 million people worldwide [Unaids. UNAIDS Data 2018]. Although the combined antiretroviral therapy (ART) can successfully suppress HIV viremia and delays the progression of disease [1], the chronic infection

Human IgG Anti-IgE
Human IgG anti-IgE (H-aIgE) was purified from the serum of a patient with severe atopic dermatitis as previously described [81,82]. The specificity and activity of IgG anti-IgE were tested as described elsewhere [81].

Human Monoclonal IgM and Human Polyclonal IgG
Monoclonal IgM were purified from the serum of patients with Waldenström's macroglobulinemia as described elsewhere [83]. Variable regions of these monoclonal IgM were determined using a panel of primary sequence-dependent V H family specific reagents that identify framework regions [84]. Human polyclonal IgG were purified from the serum of healthy donors [85].

Isolation of HLMCs
The study was approved by the Ethics Committee of the University of Naples Federico II (Protocol: Human MC No 7/19, 16/01/2019). The lung tissue was obtained from patients seronegative for HIV-1, HCV, and HBV undergoing thoracic surgery, mostly for lung cancer. HLMC were purified from human lung tissue by a modification of the method previously described [33]. The enzymatic dispersion tissue yields ≈ 5 × 10 5 mast cells per gram of lung tissue. The purity of these populations ranged from 3% to 18%. HLMCs were partially purified by flotation through a discontinuous Percoll gradient [83]. Mast cell purity using this technique ranged from 47% to 79% and was assessed by alcian blue staining.

Statistical Analysis
Values were expressed as means ± SEM (standard error of the mean). The one-way repeated measures analysis of variance (ANOVA) with Greenhouse-Geisser corrections was used to examine the variations of continuous variables at different experimental conditions. Results were analyzed with GraphPad Prism software (version 8.01; GraphPad Software, La Jolla, CA, USA), and p values of less than 0.05 were considered significant.

Effect of Human IgG Anti-IgE on Mediator Release from HLMCs
IgG anti-IgE (H-aIgE), purified from a small percentage of atopic dermatitis patients, induces histamine and LTC 4 release from human basophils and mast cells [81]. The activating property of H-aIgE is mediated by the interaction with IgE on basophils and mast cells [82,91]. We used this human autoantibody to activate HLMCs in vitro. H-aIgE (10 −2 to 3 µg/mL) caused a concentration-dependent histamine secretion from four different preparations of HLMCs isolated from HIV-1-negative subjects ( Figure 1). As a control, we used IgG (10 −2 to 3 µg/mL) purified from four normal donors which did not induce histamine release from HLMCs. These results indicate that mast cells purified from human lung have FcεRI-bound IgE. Values were expressed as means ± SEM (standard error of the mean). The one-way repeated measures analysis of variance (ANOVA) with Greenhouse-Geisser corrections was used to examine the variations of continuous variables at different experimental conditions. Results were analyzed with GraphPad Prism software (version 8.01; GraphPad Software, La Jolla, CA, USA), and p values of less than 0.05 were considered significant.

Effect of Human IgG Anti-IgE on Mediator Release from HLMCs
IgG anti-IgE (H-aIgE), purified from a small percentage of atopic dermatitis patients, induces histamine and LTC4 release from human basophils and mast cells [81]. The activating property of H-aIgE is mediated by the interaction with IgE on basophils and mast cells [82,91]. We used this human autoantibody to activate HLMCs in vitro. H-aIgE (10− 2 to 3 μg/mL) caused a concentration-dependent histamine secretion from four different preparations of HLMCs isolated from HIV-1-negative subjects ( Figure 1). As a control, we used IgG (10− 2 to 3 μg/mL) purified from four normal donors which did not induce histamine release from HLMCs. These results indicate that mast cells purified from human lung have FcεRI-bound IgE.

Effects of gp120 from Divergent HIV Isolates from Different Clades on Mediator Release from HLMCs
In a group of experiments we compared the effects of four recombinant gp120 (gp120MN, gp120SF2, gp120LAV, and gp120CM) derived from divergent HIV-1 isolates from different viral clades (B and E) of various geographical origins (United States, France, and Thailand) [74] (Table 1) on mediator release from HLMCs. These divergent samples of gp120 concentration-dependently (3 to 60 nM) induced histamine release from HLMCs ( Figure 2). These results imply that the capacity to induce mediator release from HLMCs is a general feature of gp120 which has maintained throughout the evolution of the virus.

Effects of gp120 from Divergent HIV Isolates from Different Clades on Mediator Release from HLMCs
In a group of experiments we compared the effects of four recombinant gp120 (gp120 MN , gp120 SF2 , gp120 LAV , and gp120 CM ) derived from divergent HIV-1 isolates from different viral clades (B and E) of various geographical origins (United States, France, and Thailand) [74] (Table 1) on mediator release from HLMCs. These divergent samples of gp120 concentration-dependently (3 to 60 nM) induced histamine release from HLMCs ( Figure 2). These results imply that the capacity to induce mediator release from HLMCs is a general feature of gp120 which has maintained throughout the evolution of the virus.

Correlation between Histamine and Tryptase Release Induced by gp120 from HLMCs
Tryptase is a neutral protease that is a selective marker for mast cells [92,93]. Large quantities of tryptase reside in the secretory granules of all mature human mast cells [92,94]. Activation of HLMCs with gp120 caused the release of tryptase as well as of histamine. Figure 3 shows that there was a positive correlation between the percentage of histamine and tryptase release induced by gp120 (r = 0.66; p < 0.001). These data demonstrate that tryptase, contained in secretory granules of mast cells, is released in parallel with histamine, implying that these cells are the source of both mediators found in supernatants of gp120-activated HLMCs.

Figure 2.
Effects of increasing concentrations of gp120 from four different isolates (gp120 MN , gp120 SF2 , gp120 LAV , gp120 CM ) on histamine secretion from HLMCs obtained from four different preparations of HLMCs obtained from donors negative for HIV-1 antibodies. HLMCs were incubated (30 min at 37 • C) with the indicated concentrations of gp120. Each point shows the mean ± SEM obtained from four different experiments.

Correlation between Histamine and Tryptase Release Induced by gp120 from HLMCs
Tryptase is a neutral protease that is a selective marker for mast cells [92,93]. Large quantities of tryptase reside in the secretory granules of all mature human mast cells [92,94]. Activation of HLMCs with gp120 caused the release of tryptase as well as of histamine. Figure 3 shows that there was a positive correlation between the percentage of histamine and tryptase release induced by gp120 (r = 0.66; p < 0.001). These data demonstrate that tryptase, contained in secretory granules of mast cells, is released in parallel with histamine, implying that these cells are the source of both mediators found in supernatants of gp120-activated HLMCs.

Effect of Lactic Acid on gp120-induced Histamine Release from HLMCs
Brief exposure to lactic acid removes IgE bound on FcεRI + cells, thus inhibiting the activating properties of IgE-mediated stimuli [95]. Lactic acid exposure completely blocked the activating effect of both gp120 and by H-aIgE on histamine release from HLMCs ( Figure 4). By contrast, the activating property of the mAb cross-linking the α-chain of FcεRI [84] was not modified by this treatment. These results suggest that gp120 induces histamine release from HLMCs through the interaction with IgE bound on mast cells.

Effect of Lactic Acid on gp120-Induced Histamine Release from HLMCs
Brief exposure to lactic acid removes IgE bound on FcεRI + cells, thus inhibiting the activating properties of IgE-mediated stimuli [95]. Lactic acid exposure completely blocked the activating effect of both gp120 and by H-aIgE on histamine release from HLMCs ( Figure 4). By contrast, the activating property of the mAb cross-linking the α-chain of FcεRI [84] was not modified by this treatment. These results suggest that gp120 induces histamine release from HLMCs through the interaction with IgE bound on mast cells.

Effects of Different IgM Myeloma Proteins on gp120-induced Mediator Release from HLMCs
To evaluate the mechanism whereby gp120 activates HLMCs, gp120 was incubated with monoclonal IgM of different VH families following the procedure previously described [84]. gp120 (10 nM) was preincubated (15 min, 37°C) with increasing concentrations (0.1 to 10 μg/ml) of a preparation of monoclonal IgM VH3 + or monoclonal IgM VH6 + . HLMCs were then added, and the incubation was continued for 30 min at 37°C. At the end of this incubation, histamine was measured in the supernatants. Preincubation with a preparation of monoclonal IgM which has the VH3 domain, concentration-dependently inhibited the effect of gp120 on histamine release from HLMCs ( Figure  5). By contrast, a monoclonal IgM which has a VH6 domain, had no effect. These results are compatible with the hypothesis that binding of gp120 to the VH3 domain of human monoclonal IgM inhibits the interaction with IgE bound to FcεRI on HLMCs.

Effects of Different IgM Myeloma Proteins on gp120-Induced Mediator Release from HLMCs
To evaluate the mechanism whereby gp120 activates HLMCs, gp120 was incubated with monoclonal IgM of different V H families following the procedure previously described [84]. gp120 (10 nM) was preincubated (15 min, 37 • C) with increasing concentrations (0.1 to 10 µg/ml) of a preparation of monoclonal IgM V H 3 + or monoclonal IgM V H 6 + . HLMCs were then added, and the incubation was continued for 30 min at 37 • C. At the end of this incubation, histamine was measured in the supernatants. Preincubation with a preparation of monoclonal IgM which has the V H 3 domain, concentration-dependently inhibited the effect of gp120 on histamine release from HLMCs ( Figure 5). By contrast, a monoclonal IgM which has a V H 6 domain, had no effect. These results are compatible with the hypothesis that binding of gp120 to the V H 3 domain of human monoclonal IgM inhibits the interaction with IgE bound to FcεRI on HLMCs.

Effects of gp120 on the de novo Synthesis of PGD2 and LTC4 from HLMCs
Activated human mast cells can rapidly synthesize several lipid mediators [23,89]. Prostaglandin D2 (PGD2) is the main cyclooxygenase metabolite synthesized de novo by human mast cells [78,96]. Activated human mast cells also synthesize cysteinyl leukotriene C4 (LTC4) through the 5lipoxygenase pathway [96][97][98]. Both lipid mediators exert a variety of proinflammatory and vasoactive effects [99,100]. In four experiments, we evaluated the production of PGD2 and LTC4 from HLMCs in response to increasing concentrations of gp120. Figure 6 shows that gp120 (3 to 60 nM) caused the de novo synthesis of both PGD2 ( Figure 6A) and LTC4 ( Figure 6B). Figure 6C shows that there was a significant correlation between the production of PGD2 and of LTC4 caused by gp120 from HLMCs (r =0.55; p < 0.001).

Figure 5.
Effects of human monoclonal IgMs on the activation of HLMCs induced by gp120. gp120 (10 nM) was preincubated (15 min at 37 • C) with increasing concentrations (1 to 10 µg/mL) of human monoclonal IgM V H 3 + or IgM V H 6 + . HLMCs were then added and incubation continued for 30 min at 37 • C. Each bar shows the mean ± SEM of histamine release from triplicate incubations. *** p < 0.001 when compared to controls.
In four experiments, we evaluated the production of PGD 2 and LTC 4 from HLMCs in response to increasing concentrations of gp120. Figure 6 shows that gp120 (3 to 60 nM) caused the de novo synthesis of both PGD 2 ( Figure 6A) and LTC 4 ( Figure 6B). Figure 6C shows that there was a significant correlation between the production of PGD 2 and of LTC 4 caused by gp120 from HLMCs (r =0.55; p < 0.001).

Effects of gp120 on the Release of Angiogenic and Lymphangiogenic Factors from HLMCs
Vascular endothelial growth factors (VEGFs) are essential regulators of the development and functions of blood and lymphatic vessels [101,102]. VEGFs play a major role in new vessel formation and in pulmonary pathophysiology [33,103]. VEGF-A is the most potent proangiogenic molecule [34,104], whereas VEGF-C plays an essential role in inflammatory and tumor lymphangiogenesis [101,102]. Therefore, we investigated the effects of increasing concentrations (10 to 60 nM) of gp120 on the release of angiogenic (VEGF-A) and lymphangiogenic factors (VEGF-C) from HLMCs. HLMCs were cultured (6 h at 37 • C) with gp120 and, at the end of incubation, the release of VEGF-A and VEGF-C was assayed in the supernatants of mast cells. Figure 7 shows that gp120 concentration-dependently caused the release of VEGF-A and VEGF-C from four different preparations of HLMCs.

Effects of gp120 on the Release of Angiogenic and Lymphangiogenic Factors from HLMCs
Vascular endothelial growth factors (VEGFs) are essential regulators of the development and functions of blood and lymphatic vessels [101,102]. VEGFs play a major role in new vessel formation and in pulmonary pathophysiology [33,103]. VEGF-A is the most potent proangiogenic molecule [34,104], whereas VEGF-C plays an essential role in inflammatory and tumor lymphangiogenesis [101,102]. Therefore, we investigated the effects of increasing concentrations (10 to 60 nM) of gp120 on the release of angiogenic (VEGF-A) and lymphangiogenic factors (VEGF-C) from HLMCs. HLMCs were cultured (6 h at 37 °C) with gp120 and, at the end of incubation, the release of VEGF-A and VEGF-C was assayed in the supernatants of mast cells. Figure 7 shows that gp120 concentrationdependently caused the release of VEGF-A and VEGF-C from four different preparations of HLMCs.

Discussion
Primary mast cells isolated from human lung parenchyma of HIV-1 negative subjects can be activated by a human IgG anti-IgE isolated from a patient with atopic dermatitis. These results suggest that HLMCs bind IgE which has a role in allergic diseases [18,105], in pulmonary disorders [22,31] and also in HIV-1 infection [106][107][108][109][110][111]. Viral (gp120) superantigen activates HLMCs to release preformed (histamine and tryptase) and de novo synthesized proinflammatory mediators (LTC4 and PGD2), angiogenic (VEGF-A), and lymphangiogenic (VEGF-C) factors. The activating property of gp120 appears to be mediated by interaction with IgE VH3 + on HLMCs.
Mast cells, present in strategic locations of human lung [19,20,26,32], are involved in several pulmonary diseases [22,31], lung remodeling [112,113], COPD [9,12,32], lung cancer [114][115][116], and asthma [17,19]. In recent years, the widespread use of ART has improved the survival of people with HIV [1], leading to the emergence of chronic inflammatory lung diseases as a noteworthy concern in this population [7,9]. COPD [9,11,12,117], lung cancer [10,118], pulmonary hypertension [13], and bronchial asthma [7,119] occur at high frequency among HIV-infected individuals. HIV infection induces a state of chronic inflammation characterized by persistent immune dysregulation [120], which likely induces the release of proinflammatory cytokines. Although our results were obtained in an experimental model in vitro, the release of several proinflammatory mediators from gp120- Figure 7. Effects of increasing concentrations of gp120 on the release of VEGF-A and VEGF-C from HLMCs from four different preparations of HLMCs. HLMCs were incubated (6 h at 37 • C) in the presence of the indicated concentrations of gp120. Each bar is the mean ± SEM obtained from four different experiments. ** p < 0.01; *** p < 0.001.

Discussion
Primary mast cells isolated from human lung parenchyma of HIV-1 negative subjects can be activated by a human IgG anti-IgE isolated from a patient with atopic dermatitis. These results suggest that HLMCs bind IgE which has a role in allergic diseases [18,105], in pulmonary disorders [22,31] and also in HIV-1 infection [106][107][108][109][110][111]. Viral (gp120) superantigen activates HLMCs to release preformed (histamine and tryptase) and de novo synthesized proinflammatory mediators (LTC 4 and PGD 2 ), angiogenic (VEGF-A), and lymphangiogenic (VEGF-C) factors. The activating property of gp120 appears to be mediated by interaction with IgE V H 3 + on HLMCs.
Mast cells, present in strategic locations of human lung [19,20,26,32], are involved in several pulmonary diseases [22,31], lung remodeling [112,113], COPD [9,12,32], lung cancer [114][115][116], and asthma [17,19]. In recent years, the widespread use of ART has improved the survival of people with HIV [1], leading to the emergence of chronic inflammatory lung diseases as a noteworthy concern in this population [7,9]. COPD [9,11,12,117], lung cancer [10,118], pulmonary hypertension [13], and bronchial asthma [7,119] occur at high frequency among HIV-infected individuals. HIV infection induces a state of chronic inflammation characterized by persistent immune dysregulation [120], which likely induces the release of proinflammatory cytokines. Although our results were obtained in an experimental model in vitro, the release of several proinflammatory mediators from gp120-activated human lung mast cells may contribute to the pathophysiology of chronic pulmonary diseases in HIV-infected patients.
Angiogenesis [129] plays a role in pulmonary pathophysiology [38,130,131]. VEGF-A is a major mediator of angiogenesis and can be produced by several immune cells [33,55,56,104,[132][133][134]. To our knowledge, this is the first evidence that gp120 can induce the release of angiogenic factors from HLMCs raising the possibility that these cells can contribute to angiogenesis, a process of pivotal relevance in lung pathophysiology [38,130,131]. Further studies are needed to comprehensively define the contributive role of lung mast cells to angiogenesis in pulmonary diseases (e.g., COPD, asthma, etc.) [7,[9][10][11][12][13] associated with HIV infection.
The mammalian lung is rich of lymphatic vessels [135] which are increased in human lung following infections [136][137][138][139]. We provide the first evidence that a superantigenic viral activation of HLMCs leads to the production of VEGF-C, a major mediator of lymphangiogenesis [140]. Lymphangiogenesis is canonically considered pivotal for the diffusion of metastasis to draining lymph nodes [101,102]. However, recent evidences indicate that VEGF-C can potentially exert protective effects, since inflammation-associated lymphangiogenesis can improve the resolution of inflammation [141,142]. Therefore, the contribution of lung mast cells to lymphangiogenesis during HIV infection commands additional investigations.

Conclusions
We have previously shown that gp120 causes cytokines (IL-4 and IL-13) release from human basophils [73]. Together with the data of the present study, it is possible to conclude that gp120 [143] can function as a viral superantigen activating HLMCs and basophils to release proinflammatory mediators (histamine, tryptase, PGD 2 , LTC 4 ), cytokines (IL-4 and IL-13), and angiogenic/lymphangiogenic factors (VEGF-A and VEGF-C). The latter results could contribute to immune dysfunction in patients with HIV.
Our study has a limitation that should be pointed out. The in vitro experiments were performed using primary human lung mast cells purified from HIVpatients undergoing thoracic surgery for lung cancer. Several independent investigators have demonstrated that human mast cells can be infected by HIV [47][48][49]52]. Importantly, human mast cells represent a long-lived reservoir of persistent HIV infection even in patients treated with ART [52,53]. There is also some evidence that circulating levels of histamine, a major mast cell mediator [17], are increased in HIV infected patients [151]. Future studies should compare the activating property of gp120 on lung mast cells isolated from HIVand HIV + subjects to support the clinical significance of our findings.
In conclusion, our results indicate that immunoglobulin superantigen gp120 can interact with IgE V H 3 + bound to FcεRI to induce the release of proinflammatory, angiogenic, and lymphangiogenic mediators from human lung mast cells. Future studies are needed to investigate whether these observations in vitro can help to understand the pathophysiology of chronic lung diseases among HIV patients. Funding: This research was funded in part by grants from Regione Campania CISI-Lab Project, CRèME Project, and TIMING Project.

Conflicts of Interest:
The authors declare no conflict of interest.

APCs
Antigen