Suppression of the macrophage proteasome by ethanol impairs MHC class I antigen processing and presentation.

Alcohol binge-drinking (acute ethanol consumption) is immunosuppressive and alters both the innate and adaptive arms of the immune system. Antigen presentation by macrophages (and other antigen presenting cells) represents an important function of the innate immune system that, in part, determines the outcome of the host immune response. Ethanol has been shown to suppress antigen presentation in antigen presenting cells though mechanisms of this impairment are not well understood. The constitutive and immunoproteasomes are important components of the cellular proteolytic machinery responsible for the initial steps critical to the generation of MHC Class I peptides for antigen presentation. In this study, we used an in-vitro cell culture model of acute alcohol exposure to study the effect of ethanol on the proteasome function in RAW 264.7 cells. Additionally, primary murine peritoneal macrophages obtained by peritoneal lavage from C57BL/6 mice were used to confirm our cell culture findings. We demonstrate that ethanol impairs proteasome function in peritoneal macrophages through suppression of chymotrypsin-like (Cht-L) proteasome activity as well as composition of the immunoproteasome subunit LMP7. Using primary murine peritoneal macrophages, we have further demonstrated that, ethanol-induced impairment of the proteasome function suppresses processing of antigenic proteins and peptides by the macrophage and in turn suppresses the presentation of these antigens to cells of adaptive immunity. The results of this study provide an important mechanism to explain the immunosuppressive effects of acute ethanol exposure.

Lymphocyte apoptosis in murine Pneumocystis pneumonia.

BACKGROUND: Apoptosis of lymphocytes is important in the termination of an immune response to infection but has also been shown to have detrimental effects in animal models of systemic infection and sepsis. We sought to characterize lymphocyte apoptosis in an animal model of pneumonia due to Pneumocystis murina, an infection localized to the lungs. METHODS: Control mice and mice depleted of CD4+ lymphocytes were inoculated with Pneumocystis. Apoptosis of lung and spleen lymphocytes was assayed by flow cytometry and PCR assay of apoptotic proteins. RESULTS: In control mice, apoptosis of lung lymphocytes was maximal just after the infection was cleared from lung tissue and then declined. However, in CD4-depleted mice, apoptosis was also upregulated in recruited lymphocytes in spite of progressive infection. In splenic lymphocytes, apoptosis was observed early at 1 week after inoculation and then declined. Apoptosis of lung lymphocytes in control mice was associated with a decrease in mRNA for Bcl-2 and an increase in mRNA for Bim. In CD4-depleted mice, lavaged CD8+ cells did change intracellular Bcl-2 but showed increased mRNA for Bim. CONCLUSION: Apoptosis of both pulmonary and extrapulmonary lymphocytes is part of the normal host response to Pneumocystis but is also triggered in CD4-deficient animals with progressive infection. In normal mice apoptosis of pulmonary lymphocytes may serve to terminate the immune response in lung tissue. Apoptosis of lung lymphocytes takes place via both the intrinsic and extrinsic apoptotic pathways and is associated with changes in both pro- and anti-apoptotic proteins.

Interleukin-12 and host defense against murine Pneumocystis pneumonia.

Little is known about the role of the cytokine interleukin-12 (IL-12) in Pneumocystis pneumonia or its potential use as immunotherapy. We asked whether release of IL-12 is part of the normal host response to this infection and whether local treatment with IL-12 or gene transfer of IL-12 could accelerate clearance of infection. IL-12 was assayed by enzyme-linked immunosorbent assay in normal mice and in mice deficient in IL-12 after inoculation of Pneumocystis carinii. P. carinii-infected mice were treated with local instillation of IL-12 and gene transfer of the IL-12 gene. Inoculation of P. carinii into normal mice evoked a brisk release of IL-12 into lung tissue, and IL-12 P35-deficient mice showed delayed clearance of infection measured by PCR for P. carinii rRNA. In control mice, intranasal recombinant IL-12 accelerated clearance of infection, and this was associated with increased recruitment of inflammatory cells into lavage fluid and increased release of tumor necrosis factor alpha, IL-12, and gamma interferon. Similar results were observed in infected mice depleted of CD4+ lymphocytes by using in vivo transfer of the IL-12 gene in a replication-deficient adenoviral vector. IL-12 is part of the normal host response to infection with P. carinii. IL-12 therapy can enhance host resistance to infection in both normal mice and mice depleted of CD4+ T lymphocytes. A treatment effect of IL-12 is mediated through enhanced inflammatory cell recruitment into lung tissue and increased tissue concentrations of proinflammatory cytokines.

Acute alcohol intoxication suppresses the pulmonary ELR-negative CXC chemokine response to lipopolysaccharide.

Alcohol abuse impairs the pulmonary immune response to infection and increases the morbidity and mortality of bacterial pneumonia. Acute alcohol intoxication suppresses lung expression of CXC chemokines bearing the Glu-Leu-Arg motif (ELR+) following lipopolysaccharide (LPS) challenge, but its effect on the structurally related ELR- CXC chemokines, which attract T cells, is unknown. We therefore investigated the effect of acute alcohol intoxication on the pulmonary response to intratracheal (i.t.) LPS challenge for the ELR- CXC chemokines monokine induced by gamma (MIG or CXCL9), interferon-inducible protein 10 (IP-10 or CXCL10), and interferon-inducible T cell alpha chemoattractant (I-TAC or CXCL11). Male C57BL/6 or C3H/HeN mice were given an intraperitoneal injection of ethanol (3.0 g/kg) or phosphate buffered saline 30 min before i.t. LPS challenge. Chemokine mRNA transcripts were measured at 0, 2, 6, and 16 h. Acute alcohol intoxication inhibited the lung's expression of all three chemokine genes in response to LPS. Lung IFN-gamma mRNA was also inhibited by acute intoxication over the same time course. The in vitro effect of ethanol on chemokine secretion was further studied in the MH-S alveolar macrophage cell line. IP-10, MIG, and I-TAC in response to LPS were enhanced by exogenous interferon (IFN)-gamma, and these responses were blunted by exposure to ethanol. Alcohol exposure did not affect MH-S cell nuclear factor kappa beta p65 nuclear localization during challenge, despite dose-dependent inhibition of Erk 1/2 phosphorylation. In addition, phospho-signal transduction and activator of transcription 1 was not decreased in the presence of acute ethanol, thereby indicating that acute intoxication does not affect IFN-gamma signaling in MH-S cells. Recruitment of CD3+ T cells into the alveolar space 4 days after LPS challenge was moderately impaired by acute ethanol intoxication. These results implicate acute ethanol intoxication as a significant inhibitor of lymphocyte chemoattractant expression during pulmonary inflammation.

Interleukin-23 (IL-23)-IL-17 cytokine axis in murine Pneumocystis carinii infection.

Host defense mechanisms against Pneumocystis carinii are not fully understood. Previous work in the murine model has shown that host defense against infection is critically dependent upon host CD4(+) T cells. The recently described Th17 immune response is predominantly a function of effector CD4(+) T cells stimulated by interleukin-23 (IL-23), but whether these cells are required for defense against P. carinii infection is unknown. We tested the hypothesis that P. carinii stimulates the early release of IL-23, leading to increases in IL-17 production and lung effector CD4(+) T-cell population that mediate clearance of infection. In vitro, stimulation of alveolar macrophages with P. carinii induced IL-23, and IL-23p19 mRNA was expressed in lungs of mice infected with this pathogen. To address the role of IL-23 in resistance to P. carinii, IL-23p19-/- and wild-type control C57BL/6 mice were infected and their fungal burdens and cytokine/chemokine responses were compared. IL-23p19-/- mice displayed transient but impaired clearance of infection, which was most apparent 2 weeks after inoculation. In confirmatory studies, the administration of either anti-IL-23p19 or anti-IL-17 neutralizing antibody to wild-type mice infected with P. carinii also caused increases in fungal burdens. IL-17 and the lymphocyte chemokines IP-10, MIG, MIP-1alpha, MIP-1beta, and RANTES were decreased in the lungs of infected IL-23p19-/- mice in comparison to their levels in the lungs of wild-type mice. In IL-23p19-/- mice infected with P. carinii, there were fewer effector CD4(+) T cells in the lung tissue. Collectively, these studies indicate that the IL-23-IL-17 axis participates in host defense against P. carinii.

The role of Langerhans cells in Pseudomonas aeruginosa infection.

PURPOSE: Previous experimental studies have shown that extended-wear contact lens usage results in a centripetal migration of Langerhans cells from the conjunctiva into the central cornea. To test the consequences of this, Langerhans cells were induced into the cornea before Pseudomonas aeruginosa infection in BALB/c mice that are normally resistant (the cornea heals) and in C57BL/6 mice that are susceptible (the cornea perforates) to bacterial challenge. METHODS: Mean clinical scores, slit lamp examination, adenosine diphosphatase (ADPase), and acid phosphatase staining as well as immunostaining with DEC-205, B7-1, CD4, and interleukin-2 receptor (IL-2R) antibodies and histopathologic, RT-PCR, and delayed-type hypersensitivity (DTH) analyses were used to examine the effects on bacterial disease after polystyrene bead induction of Langerhans cells into the cornea before bacterial challenge. RESULTS: No difference in disease response was observed in bead- versus sham-treated C57BL/6 mice after bacterial infection; however, significant differences leading to corneal perforation were seen in BALB/c mice that included an increased number of Langerhans cells in the central cornea at 1 and 6 days after infection, an increased number of B7-1+ (mature) Langerhans cells at 6 days after infection, CD4+ and IL-2R+ T cells at 5 days after infection, enhanced DTH, and increased mRNA levels for IFN-gamma in cornea and cervical lymph nodes. Alternately, levels of IL-4 were significantly higher in the cornea and cervical lymph nodes of sham- versus bead-treated animals. CONCLUSIONS: These data provide evidence that Langerhans cells are critical in the innate immune response to P. aeruginosa and provide new information regarding the mechanisms governing resistance versus susceptibility to bacterial infection with this opportunistic pathogen.

Role of IL-12 and IFN-gamma in Pseudomonas aeruginosa corneal infection.

PURPOSE: In Pseudomonas aeruginosa ocular infection, T-helper cell 1-responsive mouse strains are susceptible (the cornea perforates), and neutralization of IFN-gamma before infection has been shown to delay the onset of perforation. IFN-gamma is the predominant cytokine induced by IL-12, and positive regulation of IL-12 by IFN-gamma, if unchecked, leads to excessive cytokine production and toxicity. Despite its potential importance, the role of IL-12 in ocular infection with P. aeruginosa remains unexplored and was the purpose of this study. METHODS: IL-12 knockout mice, histopathology, RT/PCR and ELISA analyses, immunocytochemistry, and quantitation of viable bacteria in cornea were used to examine the role of IL-12 in IFN-gamma production and the susceptibility phenotype. RESULTS: To directly test the effect of IL-12 on IFN-gamma production, IL-12 knockout and wild-type C57BL/6 mice were used. Both groups of mice were susceptible to infection, with corneal perforation seen at 5 to 7 days after infection. RT-PCR and ELISA analyses confirmed that IL-12 message and protein levels were elevated after infection only in the wild-type mouse cornea. Other differences between the two groups were detected. Knockout versus wild-type mice showed a significant decrease in IFN-gamma mRNA levels in the cornea and cervical lymph nodes and decreased TNF-alpha protein levels in cornea. Corneas of knockout mice also had a significant increase in bacterial load at 5 days after infection when compared with wild-type mice. CONCLUSIONS: These data provide evidence that IL-12 is important in IFN-gamma production and in the absence of the cytokine, both IFN-gamma and TNF-alpha levels in cornea are significantly decreased, resulting in unchecked bacterial growth and perforation.