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GM-CSF has been shown to be important for the survival and function of cells of dendritic cell/Langerhans cell (LC) lineage in vitro. Since these cells have been demonstrated to infiltrate human lung and some lung carcinomas, we hypothesized that the production of GM-CSF in the lung could be important in their recruitment and differentiation. Using both immunohistochemistry and in situ hybridization, we have shown that: (a) GM-CSF was produced by normal bronchiolar epithelium, the only site were CD1a+ LC are observed in the normal lung, whereas neither GM-CSF production nor LC were identified in normal alveolar epithelium. (b) In inflamed pulmonary tissue, hyperplastic alveolar cells produced GM-CSF, and CDla+ LC accumulated adjacent to these cells. (c) Some, but not all, lung carcinomas produced this cytokine, and a close correlation was found between the production of GM-CSF and the number of CDla+ LC infiltrating these tumors. Since GM-CSF was produced at all sites where CDla+ LC are known to accumulate, but not at other locations within the lung, these data suggest that the local production of GM-CSF by certain lung cells may play an important role in determining the distribution and differentiated state of dendritic cell/LC in the human lung. (J. Clin. Invest. 1993. 91:566-576.)

Administration of alpha/beta interferon (IFN-␣/␤) to mice infected with Mycobacterium tuberculosis has been shown to increase mycobacterial growth. Because IFN-␣/␤ has direct pleiotropic effects on the differentiation and functional activities of macrophages, we evaluated the effect of IFN-␣/␤ on mycobacterial growth in human monocytes/macrophages in vitro. Monocytes cultured at optimal cell density could control the growth of M. bovis BCG, as assessed both by measurement of luciferase activity expressed by a mycobacterial reporter strain and by counting of CFU. In contrast, unrestrained mycobacterial growth was observed when monocytes were treated with alpha interferon (IFN-␣) 3 days prior to or concomitant with infection. This striking loss of mycobacteriostatic activity was observed with IFN-␣ and IFN-␤ and was induced in both freshly isolated monocytes and culture-derived macrophages. Pretreatment of monocytes with IFN-␣ modified cellular morphology and reduced viability following culture, but neither was observed for culture-derived macrophages, indicating that the effects of IFN-␣ on mycobacteriostatic activity and cell differentiation and death could be dissociated. These results are compatible with the possibility that the secretion of IFN-␣/␤ could directly promote mycobacterial growth in patients harboring these organisms.Following infection with Mycobacterium tuberculosis, most immunocompetent adults are able to develop an immune and inflammatory response that controls mycobacterial proliferation, and although these individuals continue to harbor small numbers of viable mycobacteria, clinical signs of tuberculosis do not develop (36,37). In a minority of individuals, a variety of factors, including aging, stress, malnutrition, alcoholism, intercurrent infections, and cancer, can subsequently blunt this acquired resistance, resulting in the reactivation of dormant mycobacteria and the development of overt disease.The changes in the immune and inflammatory response that predispose individuals to the reactivation of mycobacterial infection are not completely defined (35). Considerable evidence indicates that antigen-specific T-cell-mediated immunity is required for the control of infection by pathogenic mycobacteria (14,35). The development of anergy and reductions in the numbers of CD4 T cells in patients with certain viral diseases (e.g., infection with human immunodeficiency virus [HIV] and measles) undoubtedly contribute to the susceptibility of these individuals to tuberculosis (15,20,26,29,37). It is also recognized that numerous cytokines produced by host immune and inflammatory cells can modify the effector functions of macrophages, and changes in production of these mediators could have deleterious effects on the control of mycobacterial infections (42). In this regard, Manca et al. (28) recently showed that infection of mice with a highly virulent clinical isolate of M. tuberculosis resulted in higher levels of mRNA for alpha interferon (IFN-␣) in the lungs of these animals than those observe...

Some patients infected with human immunodeficiency virus (HIV) who are experiencing antiretroviral treatment failure have persistent improvement in CD4+ T cell counts despite high plasma viremia. To explore the mechanisms responsible for this phenomenon, 2 parameters influencing the dynamics of CD4+ T cells were evaluated: death of mature CD4+ T cells and replenishment of the CD4+ T cell pool by the thymus. The improvement in CD4+ T cells observed in patients with treatment failure was not correlated with spontaneous, Fas ligand-induced, or activation-induced T cell death. In contrast, a significant correlation between the improvement in CD4+ T cell counts and thymic output, as assessed by measurement of T cell receptor excision circles, was observed. These observations suggest that increased thymic output contributes to the dissociation between CD4+ T cell counts and viremia in patients failing antiretroviral therapy and support a model in which drug-resistant HIV strains may have reduced replication rates and pathogenicity in the thymus.

The mechanisms through which immune and inflammatory responses stimulate the expression of antimycobacterial activity by human macrophages remain poorly defined. To study this question, we developed a method permitting the rapid quantification of viable mycobacteria, based on the detection of luciferase activity expressed by a Mycobacterium bovis Bacillus Calmette-Guerin (BCG) reporter strain, and used this approach to evaluate mycobacterial survival in human monocyte-derived macrophages following stimulation with cytokines and through crosslinking of costimulatory molecules expressed on the cell surface. Modest proliferation, followed by persistence of mycobacteria, was observed in unpretreated macrophages as assessed both by measurement of luciferase activity and by the evaluation of colony forming units. Of the 19 cytokines tested, only granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) were found to improve the mycobactericidal activity of monocyte-derived macrophages. In both cases, this effect was observed only when macrophages were pretreated with the cytokines prior to infection. In contrast, pretreatment of human macrophages with interferon-gamma, either alone or in combination with other mediators (including tumor necrosis factor-alpha and 1,25[OH]2-vitamin D3), did not improve mycobacterial killing. The stimulation of macrophages through several different costimulatory molecules known to participate in macrophage-lymphocyte interactions (CD4, CD40, CD45, CD86, CD95 [Fas/Apo-1]) also failed to improve mycobactericidal activity. This study shows that GM-CSF and IL-3, cytokines whose receptors are known to share a common subunit and to use common second messengers, may contribute to the stimulation of mycobactericidal activity in humans. The ability to rapidly screen the effects of different macrophage stimuli on mycobacterial survival through the detection of luciferase activity should help define additional signals required for optimal antimycobacterial responses.

The mechanisms through which granuloma formation helps control mycobacterial infection are poorly understood, but it is possible that the accumulation of macrophages at high density at sites of infection promotes the differentiation of macrophages into cells with improved mycobactericidal activity. To test this possibility, varying numbers of monocytes were cultured in 96-well plates for 3 days, infected with Mycobacterium bovis bacillus Calmette-Guérin, and mycobacterial number was assessed 7 days after infection based on the measurement of luciferase activity expressed by a mycobacterial reporter strain or by counting CFU. Mycobacterial growth was optimal in cultures containing 5 × 104 cells/well, but increasing the number of cells to 2 × 105 cells/well resulted in complete inhibition of mycobacterial growth. This effect could not be explained by differences in mycobacterial uptake, multiplicity of infection, acidification of the extracellular medium in high density cultures, enhanced NO production, or paracrine stimulation resulting from secretion of cytokines or other proteins. The morphology of cells cultured at high density was strikingly different from that of monocytes cultured at 5 × 104 cells/well, including the appearance of numerous giant cells. The bacteriostatic activity of monocyte-derived macrophages was also dependent on cell number, but fewer of these more mature cells were required to control mycobacterial growth. Thus, the ability of human macrophages to control mycobacterial infection in vitro is influenced by the density of cells present, findings that may help explain why the formation of granulomas in vivo appears to be a key event in the control of mycobacterial infections.

Studies in experimental animals have suggested that gamma/delta T-cells play an important role in the immune response against mycobacteria, but evidence for the participation of these cells in the course of human tuberculosis remains fragmentary. We have evaluated the number and state of activation of gamma/delta T-cells in the peripheral blood of patients with active tuberculosis using two-color cytofluorometry, and we have sought evidence that these cells might play a role in the impaired responses to recall antigens seen in some patients by comparing the proliferation of blood T-lymphocytes before and after removing gamma/delta T-cells by panning. Results were compared with those obtained for cells from normal subjects and from patients with sarcoidosis. The proportion and absolute number of circulating CD3+ gamma/delta T-cells were not significantly different comparing blood from patients with tuberculosis and that from control subjects [54.6 +/- 39.9 (n = 17) and 59.1 +/- 30.2 cells/microliters (n = 10), respectively, p > 0.2], and the proportion of cells expressing receptors using the V delta 1 variable region remained unchanged in patients with tuberculosis. Few gamma/delta T-cells from patients with tuberculosis expressed surface antigens associated with activation (IL-2R, < 1%; HLA-DR, 2.6 +/- 3.4%). Four of 15 patients with sarcoidosis had a proportion of gamma/delta T-cells that was outside the range observed in normal subjects, but the absolute number of CD3+ gamma/delta T-lymphocytes was not different comparing the two groups (p > 0.2).(ABSTRACT TRUNCATED AT 250 WORDS)

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