Protein complexes of the 28-kDa proteasome activator (PA28) family activate the proteasome and may alter proteasome cleavage specificity. Initial investigations have demonstrated a role for the IFN-γ-inducible PA28α/β complex in Ag processing. Although the noninducible and predominantly nuclear PA28γ complex has been implicated in affecting proteasome-dependent signaling pathways, such as control of the mitotic cell cycle, there is no previous evidence demonstrating a role for this structure in Ag processing. We therefore generated PA28γ-deficient mice and investigated their immune function. PA28γ−/− mice display a slight reduction in CD8+ T cell numbers and do not effectively clear a pulmonary fungal infection. However, T cell responses in two viral infection models appear normal in both magnitude and the hierarchy of antigenic epitopes recognized. We conclude that PA28γ−/− mice, like PA28α−/−/β−/− mice, are deficient in the processing of only specific Ags.

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Pulmonary inflammation, abnormalities in alveolar type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS). We used the naturally occurring “pearl” HPS2 mouse model to investigate the mechanisms of lung inflammation observed in HPS. Although baseline bronchoalveolar lavage (BAL) cell counts and differentials were similar in pearl and strain-matched wild-type (WT) mice, elevated levels of proinflammatory (MIP1γ) and counterregulatory (IL-12p40, soluble TNFr1/2) factors, but not TNF-α, were detected in BAL from pearl mice. After intranasal LPS challenge, BAL levels of TNF-α, MIP1α, KC, and MCP-1 were 2- to 3-fold greater in pearl than WT mice. At baseline, cultured pearl alveolar macrophages (AMs) had markedly increased production of inflammatory cytokines. Furthermore, pearl AMs had exaggerated TNF-α responses to TLR4, TLR2, and TLR3 ligands, as well as increased IFN-γ/LPS-induced NO production. After 24 h in culture, pearl AM LPS responses reverted to WT levels, and pearl AMs were appropriately refractory to continuous LPS exposure. In contrast, cultured pearl peritoneal macrophages and peripheral blood monocytes did not produce TNF-α at baseline and had LPS responses which were no different from WT controls. Exposure of WT AMs to heat- and protease-labile components of pearl BAL, but not WT BAL, resulted in robust TNF-α secretion. Similar abnormalities were identified in AMs and BAL from another HPS model, pale ear HPS1 mice. We conclude that the lungs of HPS mice exhibit hyperresponsiveness to LPS and constitutive and organ-specific macrophage activation.

Numerous in vitro studies have demonstrated that Histoplasma capsulatum is engulfed by the diverse populations of phagocytic cells including monocytes/macrophages (Mphi), immature dendritic cells (DC), and neutrophils. The in vivo distribution of H. capsulatum has yet to be examined following an intrapulmonary challenge. To accomplish this goal, we engineered GFP into two genetically dissimilar strains of H. capsulatum, G217B and 186R. C57BL/6 mice were infected with each of these strains, and we analyzed the distribution of this fungus in the three major phagocytic populations on successive days. Yeast cells were found in all three populations of cells from Days 1 through 7. Proportionally, DC dominated at Day 1, whereas the majority of yeast cells was detected in neutrophils thereafter. Yeast cells were present in inflammatory and resident Mphi on Day 3, but on Day 7, they were chiefly in inflammatory Mphi. Yeast cells were predominantly in a CD11c(+intermediate/high), F4/80(-), CD11b(+), Ly-6C(+), CD205(+) DC population. Neutralization of TNF-alpha or IFN-gamma produced a significant redistribution of yeast cells. These results reveal the complex nature of intracellular residence of this fungus. Moreover, the findings demonstrate that there is a skewing in the subpopulations of cells that are infected, especially DC.

Vaccination with recombinant (r) hsp60 protects mice against Histoplasma capsulatum. To map the domain of rhsp60 that confers protection, four gene segments were cloned into pET19b and expressed in Escherichia coli. rhsp60 fragments were tested for their capacity to induce proliferation by sensitized splenocytes and to protect BALB/c and C57BL/6 mice. All polypeptides stimulated cells from BALB/c mice immunized with yeasts or with rhsp60. Fragment 3 caused the most vigorous response by cells from mice immunized yeasts, and fragment 1 caused the most vigorous response by cells from mice immunized with rhsp60. Splenocytes from yeast-immunized C57BL/6 mice did not recognize any polypeptide. In contrast, cells from C57BL/6 mice inoculated with rhsp60 responded to all fragments but most intensely to fragment 2. In both strains, fragment 3 conferred protection against sublethal and lethal challenges. Thus, a common protective domain of hsp60 has been identified.