Blood mononuclear cell production of TNF-alpha and IL-8: engagement of different signal transduction pathways including the p42 MAP kinase pathway.

Recent studies of human peripheral blood mononuclear cells (PBMC) stimulated with IgG subclasses have suggested that tumor necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8) production proceed along different signal transduction pathways. To investigate this possibility, inhibitors of signal transduction pathways were employed. Human PBMC were pretreated with various inhibitors before being added to IgG2-coated wells and 4-h supernatant fluids evaluated for cytokine content. The effects of various inhibitors on MAP kinase activation were determined. Inhibitors of protein tyrosine kinases, phosphatases, and phospholipase C decreased TNF-alpha and IL-8 production, suggesting that all three enzyme pathways are involved in cytokine generation. Inhibitors of G-proteins had differing effects: pertussis toxin inhibited IL-8 but not TNF-alpha production, whereas cholera toxin inhibited TNF-alpha but not IL-8 production. Pretreatment of PBMC with pertussis toxin resulted in reduced IgG2-induced calcium mobilization, whereas cholera toxin had no effect, correlating with the effects of pertussis toxin on IL-8 expression. Inhibitors of protein kinase C (PKC) completely blocked TNF-alpha generation but had no effect on IL-8 production. Gö6976, which inhibits certain isoforms of PKC, inhibited production of both IL-8 and TNF-alpha. Isoforms of PKC may have opposing effects on cytokine production. PD 98059, a compound that specifically inhibits the activation of mitogen-activated protein kinase kinase (MEK1), inhibited TNF-alpha production, but had insignificant effects on IL-8 production. Pretreatment of PBMC with either PD 98059 or genistein reduced the extent of phosphorylation of p42 MAP kinase in cells activated on contact with IgG2. These findings suggest distinct signal transduction pathways for cytokine production in PBMC stimulated with IgG2.

Cytokine responses of human blood monocytes stimulated with Igs.

Using an in vitro system for stimulating human peripheral blood mononuclear cells (PBMC) with immobilized Ig, patterns of cytokine production as a function of different Ig classes and subclasses were elucidated. Wells were coated with IgA, IgG1, IgG2, IgG3 or IgG4. Equivalent protein content on surfaces of wells was demonstrated by a human kappa chain ELISA. Isolated human PBMC were added to Ig-coated wells and incubated for 24 hrs before supernatants were assayed for cytokines. The IgG subclasses showed differences in cytokine production stimulated from PBMC, with the relative stimulation for TNF alpha being IgG2 > or = IgG3 > or = IgG1 > IgG4 and for IL-6 production, IgG2 > or = IgG3 > IgG1 = IgG4. In contrast, the relative stimulation for IL-8 was IgG1 = IgG2 = IgG3 = IgG4. IgA caused less production of TNF alpha when compared to IgG2, but similar levels of IL-8. Such differences may have important implications in the pathogenesis of immune complex mediated diseases.

Protective effects of IL-4, IL-10, IL-12, and IL-13 in IgG immune complex-induced lung injury: role of endogenous IL-12.

Using the IgG immune complex (BSA-anti-BSA) model of acute lung injury in rats, we have compared four intratracheally administered cytokines for their protective effects on parameters of injury (albumin leak and hemorrhage) and on neutrophil accumulation (lung content of myeloperoxidase). The descending rank order of protective effects was: IL-10 > or = IL-13 > IL-4 > > IL-12. In animals receiving an intratracheal instillation of 1.0 microg murine rIL-4, IL-10, IL-12 or IL-13, the levels of TNF-alpha in bronchoalveolar (BAL) fluids after intrapulmonary deposition of IgG immune complexes were reduced by 98, 98, 34, and 97%, respectively, implying a corresponding reduction in up-regulation of lung vascular intercellular adhesion molecule-1. The unexpected findings with IL-12 were further evaluated. In spite of reduced BAL levels of TNF-alpha in IL-12-treated animals, BAL levels of IFN-gamma were elevated sixfold, indicative of the expected biologic response to IL-12. Alveolar macrophages obtained from the same animals showed a 68% reduction in formation in vitro of NO2-/NO3-. When rats undergoing intrapulmonary deposition of IgG immune complexes were treated either i.v. or intratracheally with blocking Ab to murine IL-12, there were significant increases in lung permeability and myeloperoxidase values, suggesting that in this model intrinsic IL-12 functions in a regulatory manner. In homogenates of injured lungs, this Ab detected heterodimeric complex, consistent with rat IL-12. These data confirm the ability of certain cytokines to suppress in vivo lung inflammatory responses and underscore the unexpected anti-inflammatory activities of IL-12.

Regulatory effects of interleukin-6 in immunoglobulin G immune-complex-induced lung injury.

Interleukin-6 (IL-6) is a cytokine produced in response to a variety of inflammatory stimuli. Although IL-6 is often observed in increased amounts in acute respiratory distress syndrome, its role in the development of lung injury is unclear. The role of IL-6 was studied in the rat model of lung injury induced by the intra-alveolar deposition of IgG immune complexes. IL-6 induction, as determined by Northern blot analysis and bioactivity, was found as a function of time during the course of development of injury. Recombinant IL-6 instilled intratracheally at commencement of injury led to substantial reductions in lung vascular permeability, neutrophil accumulation, and levels of tumor necrosis factor (TNF)-alpha and macrophage inflammatory protein (MIP)-2 in bronchoalveolar lavage fluids. Conversely, blocking of intrinsic IL-6 by a neutralizing antibody resulted in increases in lung vascular permeability, neutrophil content, and TNF-alpha levels in bronchoalveolar lavage fluids. Rat alveolar macrophages stimulated in vitro with lipopolysaccharide in the presence of IL-6 showed a significant reduction in TNF-alpha expression. Together, these findings suggest that IL-6 acts as an intrinsic regulator of lung inflammatory injury after deposition of IgG immune complexes and that the protective effects of exogenously administered IL-6 may be in part linked to suppressed TNF-alpha production.