Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known as an inducer of proliferation and functional activation of myeloid cells. This study was carried out to characterize the effects of GM-CSF on polymorphonuclear leukocytes (PMN) more extensively. Using Northern blot analysis, we show that PMN are able to accumulate mRNAs for different cytokines, including tumor necrosis factor-a (TNF-a); G-CSF, and M-CSF, all of which are involved in inflammation and hematopoiesis. Biological assays and immunoassays demonstrate that PMN translate these mRNAs, except TNF-a, into secretory proteins. However, the expression of these cytokines is dependent on stimulation by exogenous signals, preferentially provided by the T cell-derived lymphokine GM-CSF.

SUMMARYMonocytes (MO) and macrophages (MAC ) are important producers of cytokines involved in the pathophysiology of bacterial sepsis. Most studies concentrate on the effects of bacterial lipopolysaccharides (LPS) regarding the induction of cytokine gene expression and secretion in MO/MAC. Here we report that besides LPS, the synthetic lipoprotein analogue lipopeptide N-palmitoyl-S-(2,3-bis(palmitoyl )-(2RS)-propyl )-(R)-cysteinyl-alanyl-glycine (Pam3-Cys-Ala-Gly), another component of the outer membrane of Gram-negative bacteria, as well as heat-killed Staphyloccocus aureus (S. aureus/SAC ) are potent stimuli for cytokines in human MO. For all three investigated stimuli we found an individual pattern of cytokine induction: LPS was most potent in inducing interleukin-6 (IL-6) synthesis, whereas for tumour necrosis factor-a ( TNF-a) secretion SAC was the best stimulus. Comparable amounts of IL-8 were induced by either LPS or Pam3-Cys-AlaGly, with SAC being less effective even at higher concentrations. The addition of serum led to an increase in LPS-, SAC-and Pam3-Cys-Ala-Gly-stimulated TNF-a secretion, indicating that the presence of serum is critical not just for LPS stimulation. Furthermore, as is known for LPS, Pam3-Cys-Ala-Gly and SAC rendered MO refractory to a second bacterial stimulus. Pam3-CysAla-Gly and SAC induced tolerance for itself, but LPS could partially overcome this effect. As the CD14 molecule is discussed as a common receptor for different bacterial components, we investigated whether the TNF-a response of MO could be blocked by anti-CD14 antibodies. MY4, a CD14 antibody, selectively blocked the TNF-a secretion induced by LPS but not by Pam3-Cys-Ala-Gly or SAC. In summary, we conclude that besides LPS, lipopeptide Pam3-CysAla-Gly and SAC are potent stimuli for human MO, while the mechanisms of activation seem to be partially different from LPS.

Macrophages (MAC) and polymorphonuclear granulocytes (PNG) are professional phagocytes which perform essential functions in antibacterial defense. The intracellular bacterium Mycobacterium tuberculosis persists and replicates in resting macrophages. Although it is generally assumed that activated MAC are central to protection against M. tuberculosis, PNG may also contribute to defense. We wondered whether PNG produce proinflammatory chemokines after stimulation by M. tuberculosis or its major cell wall component, lipoarabinomannan (LAM). In this study, we showed that M. tuberculosis-and LAM-activated human PNG secrete the leukocyte attractant interleukin-8 (IL-8) and the PNG-specific chemokine GRO-␣ in a dosedependent manner. Treatment of PNG with the leukotriene-B4 inhibitor MK-886 prior to stimulation with M. tuberculosis or LAM partially blocked IL-8 and GRO-␣ induction, suggesting involvement of the 5-lipoxygenase pathway in the secretion of these chemokines. We conclude that PNG contribute to early resistance to M. tuberculosis via chemokine secretion.

Infection with Mycobacterium tuberculosis or phagocytosis of large latex beads induced interleukin-12 (IL-12) production in macrophages. In contrast, tumor necrosis factor (TNF) was produced only in response to M. tuberculosis infection, not after phagocytosis of latex beads. Comparable results were obtained with cells from immunocompetent C57BL/6 and gamma interferon receptor-deficient mutant mice. Thus, phagocytosis by mechanisms not specific for M. tuberculosis was a sufficient trigger for IL-12 secretion, emphasizing the central role of this cytokine in the initiation of anti-infective immunity.

Colony-stimulating factors are required for survival proliferation, differentiation and functional activation of granulocytes, macrophages and their precursor cells. In the present report, however, we demonstrate antiproliferative activity of recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) on monoblast cell line U-937 and provide evidence for the involvement of tumor necrosis factor alpha TNF-alpha and interleukin 1 beta (IL 1 beta) in its growth inhibitory action. GM-CSF (but not granulocyte CSF, G-CSF or macrophage CSF, M-CSF) suppressed DNA synthesis and self renewal of U-937 cells. Similarly, medium conditioned by U-937 cells in response to GM-CSF (GM-CSF U-937-CM) was able to reduce clonogenicity and [3H]thymidine uptake by U-937 cells. Since neutralization of GM-CSF present in GM-CSF U-937-CM by monoclonal antibody to GM-CSF did not abrogate the autoinhibitory activity present in GM-CSF U-937-CM, we considered the possibility that other soluble molecules are released by U-937 cells upon GM-CSF stimulation. Neutralization by antibodies to IL 1 beta and TNF-alpha suggested that both monokines could be the antiproliferative principle operating in GM-CSF U-937-CM. Moreover, employing IL 1 beta-specific enzyme-linked immunosorbent assay, TNF-alpha specific radioimmunoassay, Northern analysis using a cloned TNF-alpha-specific cDNA and an oligonucleotide probe for IL 1 beta, we demonstrate GM-CSF-inducible IL 1 beta and TNF-alpha gene expression by U-937 cells at the mRNA and protein level. Although M-CSF expression was induced under similar conditions, M-CSF failed to inhibit growth of U-937 cells.