The inflammasome regulates release of caspase activation-dependent cytokines, including IL-1β, IL-18, and high-mobility group box 1 (HMGB1)1-5. During the course of studying HMGB1 release mechanisms, we discovered an important role of double-stranded RNA dependent protein kinase (PKR) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminum, rotenone, live E. coli, anthrax lethal toxin, DNA transfection, and S. Typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1beta, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with multiple inflammasome components, including NLR family pyrin domain-containing 3 (NLRP3), NLR family pyrin domain-containing 1 (NLRP1), NLR family CARD domain-containing protein 4 (NLRC4), Absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell free system with recombinant NLRP3, ASC and pro-casapse-1 reconstitutes inflammasome activity. These results reveal a critical role of PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation.
Chloroquine and its analogue hydroxychloroquine (HCQ) have been shown to inhibit a variety of viral infections including influenza and adenovirus through blockade of viral entry via inhibition of endosomal acidification. We have extended these observations to human immunodeficiency virus type 1 (HIV-1) infection utilizing primary T cells and monocytes, a T cell line (CEM), and a monocytic cell line (U-937). HCQ inhibited HIV-1 replication (> 75%), as measured by reverse transcriptase activity, in the primary T cells and monocytes as well as the T cell and monocytic cell lines. HCQ itself had no anti-reverse transcriptase activity and was not toxic to the cells at concentrations inhibitory to viral replication. Intracytoplasmic staining with an anti-p24 antibody, 24 h after infection, revealed the presence of intracytoplasmic virus, suggesting that the drug does not block viral entry. The production of steady-state HIV-1 mRNA was not affected by HCQ in that comparable levels of HIV-1 mRNA could be detected by Northern blot analysis and by in situ hybridization in both the HCQ-treated and untreated cells. However, HCQ does appear to affect production of infectious HIV-1 virions because viral isolates from HCQ-treated cells could not infect target CEM cells. These data suggest that HCQ may be useful adjunctive therapy in the treatment of HIV-1 infection.
Expression of major histocompatibility complex (MHC) class II antigens is a requirement for accessory cell function in antigen presentation. Recent reports have demonstrated the presence of class II antigens on human bronchial epithelial cells. In the present study, immunohistochemical staining revealed HLA-DR on human airway epithelial cells obtained from two different mucosal sites (lobar bronchus and nasal turbinates). To determine whether airway epithelial cells bear functional class II molecules that allow for their cognate interaction with T lymphocytes, cells isolated from these sites were used in mixed lymphocyte cultures (MLR), as an in vitro model of accessory cell function. Freshly isolated cells (11 bronchi/3 turbinates) stimulated allogeneic T lymphocytes (stimulation index [S.I.] = 9.3 [mean]; P less than 0.001 compared to T cells alone). In order to assess the potential role of contaminating conventional accessory cells, bronchial epithelial cell isolates were first preincubated in a serum-free, growth factor-supplemented medium that functionally eliminates potential non-epithelial stimulators prior to MLR culture. Conventional accessory cell-depleted epithelial cells were still capable of stimulating allogenic T lymphocytes in 18 of 23 MLR cultures (S.I. = 5.5 [mean]; P less than 0.0005 compared to T cells alone). The addition of an anti-class II monoclonal antibody (VG2.2) at the onset of culture completely inhibited the MLR response (n = 10). No shift in the CD4+/CD8+ ratio was detected between lymphocytes harvested from airway epithelial cell MLR (1.42 +/- 1.29) and the ratio from T lymphocytes cultured alone (1.3 +/- 0.75), suggesting that both CD4+ and CD8+ T lymphocytes were proliferating in response to stimulation from alloepitopes recognized on airway epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
We investigated accessory cell function, antigen (Ag) trafficking, and uptake of immune complexes in isolated nasal epithelial cells (NEC) and airway epithelial cells (AEC), as well as in the two respiratory epithelial cell lines A549 and BEAS-2B. The NEC and AEC were capable of supporting Ag-specific as well as phytohemagglutinin-induced and anti-CD3 antibody-induced T-cell proliferation. We colocalized fluorescein isothiocyanate (FITC)-labeled Ags with human leukocyte antigen (HLA)-DR in A549 and BEAS-2B, utilizing laser confocal microscopy. Respiratory epithelial cells stimulated and unstimulated with interferon (IFN)-gamma were pulsed with FITC-labeled Ags for varying periods and evaluated for their ability to internalize Ag. In the unstimulated cells, intracellular punctate staining was evident at 60 min and persisted up to 120 min. In the IFN-gamma-stimulated cells (100 U/ml for 48 h), uptake occurred at 30 min, was maximal at 60 min, and diminished at 120 min. We conducted kinetic studies in the A549 and BEAS-2B cells, utilizing electron microscopy with colloidal gold-conjugated Ags (Au-OVA). At 15 min, Au-OVA was evident in the early compartments resembling the compartment of uncoupling of receptor and ligand. At 30 min, multivesicular bodies were labeled with Au-OVA, and by 60 min Au-OVA was present in the primary and secondary lysosomes. The FITC-labeled Ags colocalized with an early endosomal marker (anti-cathepsin D), a late endosomal marker (M6PR), a lysosomal marker (CD63), and with 3-(2, 4-dinitroanilino)-3'-aminomethyldipropylamine, a marker of acidic vesicles. The BEAS-2B and A549 cells, and NEC and AEC, expressed surface Fcgamma receptor and internalized IgG immune complexes. The NEC and AEC also expressed the costimulatory molecules CD80 and CD86 as determined with flow cytometry, the reverse transcription-polymerase chain reaction for RNA, and immunohistochemistry, and T-cell proliferation could be blocked by treating NEC and AEC with anti-CD80 and anti-CD86 antibodies. Our findings suggest that respiratory epithelial cells may have a role in local Ag presentation.
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