Stimulation of macrophages by lipopolysaccharide (LPS) leads to the rapid activation of MAP kinases (MAPK) and the subsequent induction of cytokine gene expression. We sought to determine whether LPS-inducible cytokine genes were differentially regulated in macrophages derived from different tissues. Our studies revealed that PD98059, an inhibitor of the extracellular-regulated kinase (ERK) pathway, blocked LPS-induced activation of tumor necrosis factor ␣ (TNF-␣) gene expression in a murine cell line derived from alveolar macrophages but not in a nonpulmonary macrophage cell line. These findings were confirmed using primary murine alveolar and peritoneal macrophages. This suggests that the TNF-␣ promoter contains MAPK-dependent and -independent regulatory elements that are used in a cell type-specific manner. We also found that differences in MAPK-regulated signaling were not mediated by NF-B, LITAF, Egr-1, CREB, or ATF2/ c-Jun. Together, these studies demonstrate that transcriptional activation of the TNF-␣ gene requires the ERK signaling cascade in selected macrophage populations. J. Leukoc. Biol. 67: 885-893; 2000.

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Gamma interferon (IFN-␥) is a cytokine which plays a critical role in resistance to Mycobacterium tuberculosis infection. While T lymphocytes and natural killer cells are a major source of IFN-␥, previous demonstrations that it can be produced by murine macrophages prompted us to examine the capacity of human alveolar macrophages to express IFN-␥. Here we report that in vitro infection of alveolar macrophages with M. tuberculosis induces both the release of IFN-␥ protein and a transient increase in IFN-␥ mRNA levels. The IFN-producing cells were shown to be macrophages by reverse transcription-in situ PCR. We also observed that M. tuberculosis stimulation resulted in IFN-␥-dependent expression of the chemokines IFN-␥-inducible protein 10 and monokine induced by IFN-␥, suggesting that macrophage-derived IFN-␥ can function in an autocrine and/or paracrine manner. The existence of a positive regulatory loop was suggested by the observation that exogenous IFN-␥ protein could induce IFN-␥ mRNA expression in uninfected alveolar macrophages. Interleukin-12 was also found to be a potent inducer of IFN-␥ production, and M. tuberculosis-induced IFN-␥ production appears to be mediated, at least in part, by IL-12. In contrast, M. tuberculosis-induced IFN-␥ production by alveolar macrophages could be blocked by exogenous interleukin-10. These studies are the first to demonstrate an autoregulatory role for IFN-␥ produced by alveolar macrophages infected in vitro with M. tuberculosis.

It has previously been established that muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine) is an effective immunostimulant whose primary target cell type is the macrophage. Muramyl dipeptide is known to be structurally identical to a portion of the monomer unit of peptidoglycan, a nearly ubiquitous component of bacterial cell walls. To establish whether muramyl dipeptide or glycopeptides structurally related to it are formed as a result of macrophage processing of peptidoglycan, Bacillus subtilis cell walls radiolabeled in the muramic acid, glucosamine, and alanine residues of the constituent peptidoglycan were incubated in the presence of the cultured macrophage-like cell line RAW264, and the glycopeptides which released into the medium were fractionated and analyzed. Although muramyl dipeptide was not found in the culture medium, at least three glycopeptides structurally related to it were found, namely GlcNAc-MurNAc-Ala-isoGln-Dap-Ala, GlcNAc-MurNAc-Ala-isoGln-Dap, and GlcNAc-MurNAc-Ala-isoGln.

Although type-specific IgG directed to the O-polysaccharide antigen of bacterial lipopolysaccharide (LPS) is protective in most models of LPS or bacterial challenge, no currently available IgG binds to LPS from all gram-negative bacteria. The ability of a peptide-IgG conjugate, CAP18(106-138)-IgG, to bind and neutralize LPS, to kill gram-negative bacteria, and to protect in a sensitized mouse model of LPS toxicity was studied. CAP18(106-138)-IgG bound LPS from multiple gram-negative bacteria in four different binding assays. In a fluid-phase RIA, half-maximal binding of 5 microg/mL 3H-labeled LPS occurred at 5-10 microg/mL CAP18(106-138)-IgG, similar to binding with monoclonal type-specific IgG. CAP18(106-138)-IgG neutralized LPS, as assessed by LPS-induced coagulation of limulus amebocyte lysate and production of tumor necrosis factor in vitro, was bactericidal for a wide range of gram-negative bacteria, and decreased LPS-induced lethality in sensitized mice. Antibacterial peptide-IgG conjugates merit further study as a novel adjunctive therapy for gram-negative sepsis.