Members of the Mycobacterium avium-intracellulare complex (MAC) can exist in a transparent or opaque colonial morphology when cultured on synthetic medium. An opaque variant was developed from a transparent strain of a clinical MAC isolate. Comparison of the two variants showed a greater ability of the transparent colonial variant to infect normal human monocytes as measured by growth in monocyte-bacteria cocultures. Further analyses indicated diminished ability of the transparent variant to induce extracellular secretion of interleukin (IL)-1 and IL-6, as well as membrane-associated IL-1 when compared with the opaque isotype. At the molecular level, induction of specific IL-1 alpha, IL-1 beta, and IL-6 mRNAs was consistent with the protein results. These results suggest that the virulent transparent MAC, as opposed to the avirulent opaque type, may escape host defenses by failing to induce IL-1 and IL-6, key factors in the initiation of a normal immune response.

Lipopolysaccharides (LPSs) from Escherichia coli, Serratia marcescens, and SabnoneUa typhimurium, at doses from 1 to 100 ng/ml, strongly enhanced growth inhibition of Candida albicans by human polymorphonuclear leukocytes (PMN) in vitro. Flow cytometry analysis demonstrated that LPS markedly augmented phagocytosis of Candida cells by increasing the number of yeasts ingested per neutrophil as well as the number of neutrophils capable of ingesting fungal cells. LPS activation caused augmented release of lactoferrin, an iron-binding protein which itself could inhibit the growth of C. albicans in vitro. Antibodies against lactoferrin effectively and specifically reduced the anti-C. albicans activity of both LPS-stimulated and unstimulated PMN.Northern (RNA blot) analysis showed enhanced production of mRNAs for interleukin-1 13, tumor necrosis factor alpha, and interleukin-6 and in neutrophils within 1 h of stimulation with LPS. The cytokines were also detected in the supernatant of the activated PMN, and their synthesis was prevented by pretreatment of LPS-stimulated PMN with protein synthesis inhibitors, such as emetine and cycloheximide. These inhibitors, however, did not block either lactoferrin release or the anti-Candida activity of LPS-stimulated PMN. These results demonstrate the ability of various bacterial LPSs to augment neutrophil function against C. albicans and suggest that the release of a candidastatic, iron-binding protein, lactoferrin, may contribute to the antifungal effect of PMN. Moreover, the ability to produce cytokines upon stimulation by ubiquitous microbial products such as the endotoxins points to an extraphagocytic, immunomodulatory role of PMN during infection.

Mycobacterium avium-intracellulare (MAI) is an opportunistic pathogen commonly found in acquired immunodeficiency syndrome patients, whose immune systems are severely compromised. However, normal responses to this bacterium are apparently sufficient to prevent disseminated infection because disease is rarely found unless an immunocompromised state is present. Because interleukin-6 (IL-6) is an inflammatory cytokine with a multitude of activities, we investigated the potential of MAI to induce IL-6 from normal human leukocytes. Peripheral blood mononuclear cells were fractionated into monocytes (Mo), large granular lymphocytes (LGL), and T cells and stimulated with bacteria. Culture supernatants were collected and assayed for IL-6 activity by bioassay. Mo and LGL, but not T cells, were found to release IL-6 within 12 hours of stimulation, with optimal production occurring by 2 days of culture. Production of IL-6 from human leukocyte subsets was confirmed by Northern blot analysis and by neutralization of biologic function of the culture supernatants with specific antisera. Taken together, these results indicate that production of IL-6 is a key response of Mo and LGL to MAI. The role of IL-6 in MAI infection, therefore, needs to be further investigated.

Treatment of monocytes with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was shown to enhance their antimycobacterial activity in an in vitro assay. Furthermore, Mycobacterium avium-M. intracellulare was found to induce the production of this hemopoietic growth factor. Human peripheral blood mononuclear cells were fractionated by plastic adherence and Percoll density centrifugation, and each population of cells was stimulated with mycobacteria. GM-CSF was produced by both monocytes and large granular lymphocytes (LGL) but not T lymphocytes. The phenotype of the GM-CSF-producing LGL was found to be CD2+, CD16+, and HLA-DR+ but negative for T-cell and monocyte markers. Kinetic studies demonstrated that GM-CSF appeared in the supernatant fluids within 2 days of culture of either monocytes or LGL and continued to be produced up to 7 days of incubation. Northern (RNA) blot analysis of RNA from both cell types demonstrated the expression of GM-CSF message within 24 h of stimulation. From these studies, LGL and monocytes are capable of responding to M. avium-M. intracellulare by producing factors that augment normal immune functions, including the antibacterial capability of monocytes.