Intense neutrophilic lung infiltration characterizes severe Pneumocystis carinii pneumonia, with considerable morbidity and mortality attributed to host responses. The P. carinii cell wall constituent β-1,3-glucan has been shown to induce lower respiratory tract inflammatory responses. Our laboratory has also recently documented elaboration of inflammatory cytokines by alveolar epithelial cells in response to P. carinii β-glucan. This investigation explores the signaling events promoting inflammatory cytokine expression from alveolar epithelial cells in P. carinii pneumonia.
Rat type II alveolar epithelial cells were isolated and cultured prior to challenge with P. carinii β-glucan-rich cell wall isolate (PCBG). Elaboration of inflammatory cytokines was determined by ELISA in the presence or absence of inhibitors of NF-κB, protein kinase C (PKC), MEK1 and p38. Activation of NF-κB was shown by nuclear translocation of the p50/p65 heterodimer, as demonstrated by immunofluorescence microscopy and electophoretic mobility shift assay (EMSA). Inhibition of NF-κB activation was shown by Northern blot analysis for PCBG-induced inflammatory cytokine mRNA in the presence or absence of pyrridoline dithiocarbamate (PDTC). Activation of PKC was revealed by Western blot analysis for phosphorylated PKC before and after PCBG challenge.
Consistent with our previous data, macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-a (TNF-α) were elaborated in response to PCBG. This effect was abrogated by inhibitors of NF-κB or PKC, though no change was noted with inhibition of MEK1 or p38. Prompt nuclear translocation of the NF-κB p50/p65 heterodimer was observed following PCBG challenge by both immunofluorescence and EMSA. PDTC efficiently blocked PCBG-induced MIP-2 and TNF-α mRNA production. Significantly increased levels of phosphorylated PKC were observed following PCBG exposure.CONCLUSIONS: PCBG-induced MIP-2 and TNF-α expression from alveolar epithelial cells requires activation of both PKC and NF-κB. Activation is likely sequential, with PKC phosphorylation resulting in nuclear translocation of NF-κB.
Pneumocystis pneumonia results in substantial morbidity and mortality, despite effective antimicrobial agents. This investigation suggests two novel therapeutic targets which may moderate the exaggerated host responses to infection.
S.E. Evans, None.