Chang-Jiang Guo

S-nitrosylation of surfactant protein-D controls inflammatory function.

The pulmonary collectins, surfactant proteins A and D (SP-A and SP-D) have been implicated in the regulation of the innate immune system within the lung. In particular, SP-D appears to have both pro- and anti-inflammatory signaling functions. At present, the molecular mechanisms involved in switching between these functions remain unclear. SP-D differs in its quaternary structure from SP-A and the other members of the collectin family, such as C1q, in that it forms large multimers held together by the N-terminal domain, rather than aligning the triple helix domains in the traditional “bunch of flowers” arrangement. There are two cysteine residues within the hydrophobic N terminus of SP-D that are critical for multimer assembly and have been proposed to be involved in stabilizing disulfide bonds. Here we show that these cysteines exist within the reduced state in dodecameric SP-D and form a specific target for S-nitrosylation both in vitro and by endogenous, pulmonary derived nitric oxide (NO) within a rodent acute lung injury model. S-nitrosylation is becoming increasingly recognized as an important post-translational modification with signaling consequences. The formation of S-nitrosothiol (SNO)-SP-D both in vivo and in vitro results in a disruption of SP-D multimers such that trimers become evident. SNO-SP-D but not SP-D, either dodecameric or trimeric, is chemoattractive for macrophages and induces p38 MAPK phosphorylation. The signaling capacity of SNO-SP-D appears to be mediated by binding to calreticulin/CD91. We propose that NO controls the dichotomous nature of this pulmonary collectin and that posttranslational modification by S-nitrosylation causes quaternary structural alterations in SP-D, causing it to switch its inflammatory signaling role. This represents new insight into both the regulation of protein function by S-nitrosylation and NOs role in innate immunity.

Methadone Enhances Human Immunodeficiency Virus Infection of Human Immune Cells

Opiate abuse has been postulated to be a cofactor in the immunopathogenesis of acquired immunodeficiency syndrome (AIDS). This study evaluated whether methadone, a drug widely prescribed for the treatment of drug abusers with opioid dependence, affects human immunodeficiency virus (HIV) infection of human immune cells. When added to human fetal microglia and blood monocyte-derived macrophage cultures, methadone significantly enhanced HIV infection of these cells. This enhancement was associated with the up-regulation of expression of CCR5, a primary coreceptor for macrophage-tropic HIV entry into macrophages. Most importantly, the addition of methadone to the cultures of latently infected peripheral blood mononuclear cells from HIV-infected patients enhanced viral activation and replication. Although the in vivo relevance of these findings remains to be determined, the data underscore the necessity of further studies to define the role of opioids, including methadone, in the immunopathogenesis of HIV infection and AIDS.

Morphine Enhances HIV Infection of Human Blood Mononuclear Phagocytes through Modulation of β-Chemokines and CCR5 Receptor

Background Injection drug use remains a significant risk for acquiring HIV infection. The mechanisms by which morphine enhances HIV infection of human immune cells are largely unknown. Objective In this study, we sought to determine the possible mechanisms by which morphine upregulates HIV infection of human blood monocyte-derived macrophages (MDM). Methods In this study, MDM were infected with the R5, X4, and R5X4 HIV strains. HIV replication was determined by performing reverse transcriptase activity assays. HIV receptors were determined by performing reverse transcriptase polymerase chain reactions and flow cytometry assays. β-chemokines were analyzed by performing enzyme-linked immunosorbent assays. In addition, HIV R5 strain and murine leukemia virus envelope-pseudotyped HIV infection was performed to determine whether morphine affects HIV infection of macrophages at entry level. Results Morphine significantly enhanced HIV R5 strain infection of MDM but had little effect on X4 strain infection. The macrophage-tropic R5 strain envelope-pseudotyped HIV infection was markedly increased by morphine, whereas murine leukemia virus envelope-pseudotyped HIV infection was not significantly affected. Furthermore, morphine significantly upregulated CCR5 receptor expression and inhibited the endogenous production of β-chemokines in MDM. The opioid receptor antagonist naltrexone blocked the effects of morphine on the production of β-chemokines. Conclusion Opiates enhance HIV R5 strain infection of macrophages through the downregulation of β-chemokine production and upregulation of CCR5 receptor expression and may have an important role in HIV immunopathogenesis.

Interleukin‐1β upregulates functional expression of neurokinin‐1 receptor (NK‐1R) via NF‐κB in astrocytes

Cytokines and neuropeptides are modulators of neuroimmunoregulation in the central nervous system (CNS). The interaction of these modulators may have important implications in CNS diseases. We investigated whether interleukin‐1β (IL‐1β) modulates the expression of neurokinin‐1 receptor (NK‐1R), the primary receptor for substance P (SP), a potent neuropeptide in the CNS. IL‐1β upregulated NK‐1R expression in human astroglioma cells (U87 MG) and primary rat astrocytes at both mRNA and protein levels. IL‐1β treatment of U87 MG cells and primary rat astrocytes led to an increase in cytosolic Ca2+ in response to SP stimulation, indicating that IL‐1β‐induced NK‐1R is functional. CP‐96,345, a specific non‐peptide NK‐1R antagonist, inhibited SP‐induced rise of [Ca2+]i in the astroglioma cells. Investigation of the mechanism responsible for IL‐1β action revealed that IL‐1β has the ability of activating nuclear factor‐κb (NF‐κB). Caffeic acid phenethyl ester (CAPE), a specific inhibitor of NF‐κB activation, not only abrogated IL‐1β‐induced NF‐κB promoter activation, but also blocked IL‐1β‐mediated induction of NK‐1R gene expression. These findings provide additional evidence that there is a biological interaction between IL‐1β and the neuropeptide SP in the CNS, which may have important implications in the inflammatory diseases in the CNS.

Morphine enhances HIV infection of neonatal macrophages.

Perinatal transmission of HIV accounts for almost all new HIV infections in children. There is an increased risk of perinatal transmission of HIV with maternal illicit substance abuse. Little is known about neonatal immune system alteration and subsequent susceptibility to HIV infection after morphine exposure. We investigated the effects of morphine on HIV infection of neonatal monocyte-derived macrophages (MDM). Morphine significantly enhanced HIV infection of neonatal MDM. Morphine-induced HIV replication in neonatal MDM was completely suppressed by naltrexone, the opioid receptor antagonist. Morphine significantly up-regulated CCR5 receptor expression and inhibited the endogenous production of macrophage inflammatory protein-1β in neonatal MDM. Thus, morphine, most likely through alteration of β-chemokines and CCR5 receptor expression, enhances the susceptibility of neonatal MDM to HIV infection, and may have a cofactor role in perinatal HIV transmission and infection.

Alcohol Potentiates HIV‐1 Infection of Human Blood Mononuclear Phagocytes

BACKGROUND Acute and chronic alcohol abuse impairs various functions of the immune system and thus has been implicated as a cofactor in HIV infection. The mechanisms by which alcohol affects the function of human immune cells that are the targets for HIV are unknown. METHODS Human blood monocyte-derived macrophages (MDM) were incubated with or without alcohol (10-40 mM) for 24 hr and then infected with HIV for 24 hr. Culture supernatants were harvested for HIV reverse transcription assay. HIV entry receptor (CCR5, CD4, and CXCR4) expression was determined by reverse transcription-polymerase chain reaction and flow cytometry assays. Beta-chemokines were analyzed using enzyme-linked immunosorbent assay. Different HIV strains (Bal, SF-162, 89.6, and UG024) were used for infection experiments. In addition, ADA (macrophage-tropic strain) and murine leukemia virus envelope-pseudotyped HIV infection was carried out. RESULTS Although alcohol had little effect on HIV T-lymphocyte-tropic strain infection, it significantly enhanced HIV R5 strain infection in MDM. The enhancing effect of alcohol on the HIV R5 strain was further evidenced by the observation that the R5 (ADA) strain envelope-pseudotyped HIV infection is markedly increased by alcohol, whereas murine leukemia virus envelope-pseudotyped HIV infection was not affected. Alcohol significantly up-regulated CCR5 receptor expression and inhibited the endogenous production of beta-chemokines by MDM. CONCLUSION Alcohol, through the down-regulation of beta-chemokine production and the up-regulation of CCR5 receptor expression, enhances HIV R5 strain infection of MDM and may have an important role as a cofactor in the progression of HIV disease.

Alpha-defensins inhibit HIV infection of macrophages through upregulation of CC-chemokines

The possible involvement of α-defensins in CD8 T-cell-mediated anti-HIV activities has been the subject of recent investigations [1–3]. HIV host defence mechanisms are partly mediated by CD8 T-cell non-cytotoxic antiviral responses [4]. Walker et al. [5] first demonstrated that this anti-HIV activity involves a soluble factor(s) designated as CD8 cell antiviral factor (CAF) whose identity remains unknown [4]. Zhang et al. [1] proposed that α-defensins are produced by CD8 T cells and contribute to CAF-mediated anti-HIV activities. In contrast, the recent studies by Mackewicz et al. [2] and Chang et al. [3] demonstrated that the α-defensins are not produced by CD8 T cells but unexpectedly were found to be expressed by monocytes [2]. As CAF-mediated anti-HIV activity is also observed for macrophages [6,7] and monocytes express α-defensins [2], we investigated the capacity of α-defensins to suppress HIV infection of macrophages. The addition of α-defensins to peripheral blood monocyte-derived macrophage cultures markedly suppressed HIV Bal replication (Fig. 1a) [8,9]. In order to determine the mechanism(s) responsible for α-defensin-mediated HIV inhibition in macrophages, we investigated whether α-defensins regulate the expression of CC-chemokines. CC-chemokines [macrophage inflammatory protein (MIP)-1α, MIP-1β and Rantes] inhibit infection by competing with HIV M-tropic strains for the CCR5 receptor on macrophages [10,11]. Our experiments demonstrated that α-defensins dramatically enhance expression (as much as a 25-fold increase) of MIP-1α and MIP-1β messenger RNA in macrophages (Fig. 1b) [12]. This increased CC-chemokine gene expression by α-defensins was further confirmed by the demonstration of increased production (as much as a 57-fold increase) of MIP-1α and MIP-1β proteins in α-defensin-treated macrophage cultures (Fig. 1c). In addition, the antibodies to CC-chemokines completely abrogated α-defensin-mediated HIV inhibition in macrophages (Fig. 1d). Our data, therefore, indicate that the α-defensin-mediated inhibition of HIV infection of macrophages is mediated through the upregulation of CC-chemokines. This pathway is distinct from the anti-HIV activity of CAF in macrophages, because CC-chemokines are not responsible for the ability of CAF to suppress HIV infection of these cells [6,7]. Fig. 1 Effect of α-defensins on HIV infection and β-chemokine expression in macrophages The biological interaction of defensins with chemokines and chemokine receptors has been documented. Defensins functionally overlap with chemokines in microbicidal activity [13]. The treatment of dendritic cells with β-defensin-2 upregulated the expression of CC-chemokines (MIP-1α and MIP-1β) and down-regulated CCR5 expression [14]. By utilizing chemokine receptors on immune cells, defensins may contribute to the regulation of host adaptive immunity against microbial invasion [15]. Taken together, our data provide evidence that α-defensins could play a role in host defence against HIV infection of macrophages. The biological interaction of α-defensins with CC-chemokines may constitute a unique mechanism of innate immunity against HIV disease.

Immune Reconstitution during Pneumocystis Lung Infection: Disruption of Surfactant Component Expression and Function by S-Nitrosylation

Pneumocystis pneumonia (PCP), the most common opportunistic pulmonary infection associated with HIV infection, is marked by impaired gas exchange and significant hypoxemia. Immune reconstitution disease (IRD) represents a syndrome of paradoxical respiratory failure in patients with active or recently treated PCP subjected to immune reconstitution. To model IRD, C57BL/6 mice were selectively depleted of CD4+ T cells using mAb GK1.5. Following inoculation with Pneumocystis murina cysts, infection was allowed to progress for 2 wk, GK1.5 was withdrawn, and mice were followed for another 2 or 4 wk. Flow cytometry of spleen cells demonstrated recovery of CD4+ cells to >65% of nondepleted controls. Lung tissue and bronchoalveolar lavage fluid harvested from IRD mice were analyzed in tandem with samples from CD4-depleted mice that manifested progressive PCP for 6 wks. Despite significantly decreased pathogen burdens, IRD mice had persistent parenchymal lung inflammation, increased bronchoalveolar lavage fluid cellularity, markedly impaired surfactant biophysical function, and decreased amounts of surfactant phospholipid and surfactant protein (SP)-B. Paradoxically, IRD mice also had substantial increases in the lung collectin SP-D, including significant amounts of an S-nitrosylated form. By native PAGE, formation of S-nitrosylated SP-D in vivo resulted in disruption of SP-D multimers. Bronchoalveolar lavage fluid from IRD mice selectively enhanced macrophage chemotaxis in vitro, an effect that was blocked by ascorbate treatment. We conclude that while PCP impairs pulmonary function and produces abnormalities in surfactant components and biophysics, these responses are exacerbated by IRD. This worsening of pulmonary inflammation, in response to persistent Pneumocystis Ags, is mediated by recruitment of effector cells modulated by S-nitrosylated SP-D.

Early Alveolar Epithelial Dysfunction Promotes Lung Inflammation in a Mouse Model of Hermansky-Pudlak Syndrome

RATIONALE The pulmonary phenotype of Hermansky-Pudlak syndrome (HPS) in adults includes foamy alveolar type 2 cells, inflammation, and lung remodeling, but there is no information about ontogeny or early disease mediators. OBJECTIVES To establish the ontogeny of HPS lung disease in an animal model, examine disease mediators, and relate them to patients with HPS1. METHODS Mice with mutations in both HPS1/pale ear and HPS2/AP3B1/pearl (EPPE mice) were studied longitudinally. Total lung homogenate, lung tissue sections, and bronchoalveolar lavage (BAL) were examined for phospholipid, collagen, histology, cell counts, chemokines, surfactant protein D (SP-D), and S-nitrosylated SP-D. Isolated alveolar epithelial cells were examined for expression of inflammatory mediators, and chemotaxis assays were used to assess their importance. Pulmonary function test results and BAL from patients with HPS1 and normal volunteers were examined for clinical correlation. MEASUREMENTS AND MAIN RESULTS EPPE mice develop increased total lung phospholipid, followed by a macrophage-predominant pulmonary inflammation, and lung remodeling including fibrosis. BAL fluid from EPPE animals exhibited early accumulation of both SP-D and S-nitrosylated SP-D. BAL fluid from patients with HPS1 exhibited similar changes in SP-D that correlated inversely with pulmonary function. Alveolar epithelial cells demonstrated expression of both monocyte chemotactic protein (MCP)-1 and inducible nitric oxide synthase in juvenile EPPE mice. Last, BAL from EPPE mice and patients with HPS1 enhanced migration of RAW267.4 cells, which was attenuated by immunodepletion of SP-D and MCP-1. CONCLUSIONS Inflammation is initiated from the abnormal alveolar epithelial cells in HPS, and S-nitrosylated SP-D plays a significant role in amplifying pulmonary inflammation.

Interleukin-1β stimulates macrophage inflammatory protein-1α and -1β expression in human neuronal cells (NT2-N)

Chemokines are important mediators in immune responses and inflammatory processes of neuroimmunologic and infectious diseases. Although chemokines are expressed predominantly by cells of the immune system, neurons also express chemokines and chemokine receptors. We report herein that human neuronal cells (NT2‐N) produce macrophage inflammatory protein‐1α and ‐1β (MIP‐1α and MIP‐1β), which could be enhanced by interleukin (IL)‐1β at both mRNA and protein levels. The addition of supernatants from human peripheral blood monocyte‐derived macrophage (MDM) cultures induced MIP‐1β mRNA expression in NT2‐N cells. Anti‐IL‐1β antibody removed most, but not all, of the MDM culture supernatant‐induced MIP‐1β mRNA expression in NT2‐N cells, suggesting that IL‐1β in the MDM culture supernatants is a major factor in the induction of MIP‐1β expression. Investigation of the mechanism(s) responsible for IL‐1β‐induced MIP‐1α and ‐1β expression demonstrated that IL‐1β activated nuclear factor kappa B (NF‐κB) promoter‐directed luciferase activity in NT2‐N cells. Caffeic acid phenethyl ester, a potent and specific inhibitor of activation of NF‐κB, not only blocked IL‐1β‐induced activation of the NF‐κB promoter but also decreased IL‐1β‐induced MIP‐1α and ‐1β expression in NT2‐N cells. These data suggest that NF‐κB is at least partially involved in the IL‐1β‐mediated action on MIP‐1α and ‐1β in NT2‐N cells. IL‐1β‐mediated up‐regulation of β‐chemokine expression may have important implications in the immunopathogenesis of inflammatory diseases in the CNS.