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Leukotriene B4 (LTB4) release initiated by interaction of immune complexes (ICs) with Fc gamma RII and Fc gamma RIII receptors on human neutrophils was studied using well-defined complexes. Immune complexes consisting of polyclonal rabbit antibody to human albumin were prepared at equivalence (insoluble complex) and at five times antigen excess (soluble complex). Incubation of human neutrophils with soluble and insoluble ICs led to the synthesis of LTB4 from endogenous arachidonic acid (AA). LTB4 release induced by ICs was markedly inhibited by monoclonal antibodies against either Fc gamma RII or Fc gamma RIII receptor. Treatment of neutrophils with pertussis toxin significantly inhibited the release of LTB4 induced by soluble ICs. However pertussis toxin treatment minimally inhibited the LTB4 release induced by insoluble ICs. Crosslinking of either Fc gamma RII and Fc gamma RIII receptors on neutrophil surfaces induced LTB4 release. This is the first experimental observation showing that both Fc gamma RII and Fc gamma RIII directly induce neutrophil LTB4 metabolism in the absence of exogenous AA. These studies also suggest the involvement of novel pertussis toxin insensitive signal transduction pathways in insoluble ICs stimulation of neutrophils.

Interactions between T lymphocytes, neutrophils, and epidermal cells are believed to play a central role in the pathophysiology of psoriasis and other inflammatory cutaneous disorders. Although there is strong evidence that lymphocyte-function-associated antigen-1 (LFA-1) positive T cells are retained in the epidermis via intercellular adhesion molecule-1 (ICAM-1) expression induced on keratinocytes, the molecular basis for the directed migration of T cells or neutrophils towards the epidermis is not known. To investigate whether epidermal keratinocyte-derived products may be important in the migration of T cells and neutrophils into the epidermis, human keratinocytes were cultured in the presence of various cytokines and chemotactic activity of the supernatants were assessed. TNF-alpha stimulation produced directed migrational responses for both neutrophils and T-lymphocytes (both CD4 and CD8), but not B lymphocytes; 69% of T-cell movement and 80% of neutrophil migration induced by the TNF-alpha treated keratinocyte cell supernatants could be inhibited by anti-interleukin-8 (IL-8) serum. Using the same antibody, IL-8 was immunoprecipitated from the supernatants of TNF-stimulated 35S-labelled keratinocytes, and a single 7-kd band product detected by SDS-PAGE. In keeping with these biological activities and protein data, Northern blot analysis of total cellular RNA extracted from keratinocyte monolayers hybridized with a 32P-labelled 1-kb cDNA to IL-8 mRNA, revealed induction of the IL-8 gene in the presence of TNF-alpha and IL-1 beta, but not IFN-gamma. The protein kinase C agonist, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known stimulator of psoriasiform cutaneous inflammation when applied directly to murine epidermis, strongly induced keratinocyte elaboration of IL-8 mRNA. These studies demonstrate that activated human keratinocytes are capable of producing biologically active IL-8, and provide evidence that keratinocytes can play a key role in mediating the influx of T cells and neutrophils into the epidermis.

The ability of human peripheral blood monocytes to produce tumor necrosis factor (TNF) and interleukin-1 (IL-1) in an in vitro model of immune complex-mediated glomerulonephritis was investigated. When isolated monocytes were incubated with human glomerular basement membrane (GBM) containing anti-GBM immune complexes, both TNF and IL-1 were produced and secreted into the medium. The time course of secretion differed, with IL-1 production being maximal after approximately 8 hours, whereas TNF levels continued to rise for 30 hours. The activities of the monocyte-derived TNF and IL-1 were inhibitable by specific antibodies. No effect was seen when monocytes were incubated separately with either GBM alone or anti-GBM IgG. The levels of TNF and IL-1 released were comparable with those induced by high concentrations of LPS, indicating that production was close to the maximal levels reported for these cells. High levels of TNF and IL-1 also were produced in response to soluble immune complexes. The results show that monocytes can produce significant levels of TNF and IL-1 in response to both surface-bound and soluble immune complexes and provide support for the participation of these monokines in glomerulonephritis.

It has been well documented that both natural and synthetic chemotactic peptides can induce lysosomal enzyme release from neutrophils treated with cytochalasin B. These same peptides are also potent inducers of unidirectional movement, as demonstrated by the chemotactic response in Boyden chambers. In this study, the ability of another family of leukocytes, eosinophils, to release lysosomal enzymes and exhibit a chemotactic response to both natural and synthetic chemotactic peptides was examined. A striking fundamental difference between neutrophil and eosinophil chemotaxis and enzyme release was shown using C5a, formyl met-leu-phe (FMLP), and ala-gly-ser-glu (AGSG) peptides. The 50% effective doses (ED50) for chemotactic responses to C5a, FMLP, or AGSG by neutrophils and eosinophils were 0.05 microgram/ml and 1.0 microgram/ml, 10(-12) M and 10(-10) M, and 10(-7) M and 10(-7) M, respectively. At the same concentrations, these peptides (C5a, f met-leu-phe, and ala-gly-ser-glu) induced the following release of glucosaminidase from neutrophils and eosinophils, respectively: 42% and 2%, 42% and 2%, and 29% and 2%. In striking contrast, immune complexes and opsonized zymosan particles induced the release of 39% and 42% of the total glucosaminidase from neutrophils, while eosinophils released 32% and 43% of the total glucosaminidase from immune complexes and opsonized zymosan particles, respectively. These data indicate fundamental differences between neutrophils and eosinophils in unidirectional movement induced by chemotactic factors and enzyme release mechanism(s).

Numerous synthetic N-formylated peptides, believed to be the analogs of the naturally occurring initiating signal peptides produced by bacteria, are potent chemotactic agents for phagocytic cells in several species. The authors have characterized the receptor with moderately high affinity for the chemotactic peptide f-Met-Leu-[3H]Phe on the rat peritoneal neutrophils. When neutrophils are incubated with f-Met-Leu-[3H]Phe at 24 C, the binding is saturable and reversible. The receptor on the inflammatory rat neutrophils has an equilibrium dissociation constant (KD) of 3.4 x 10(-8) M at 24 C, and there are approximately 65,000 sites per cell. In addition, the potency of several of these chemotactic peptides in inducing lysosomal enzyme secretion and superoxide production correlated well with their ability to compete with f-Met-Leu-[3H]Phe for receptor binding. Structure activity studies further demonstrate that the fine specificity of the formyl peptide receptor has been conserved across species lines.

Prostaglandins (PGs) of the E series and PGI2 have been shown to inhibit acute inflammatory reactions in vivo and polymorphonuclear leukocyte (PMN), chemotaxis, lysosomal enzyme release, and superoxide anion (O-2) production in vitro. This inhibition of neutrophil stimulation by PGEs and PGI2 has been correlated with their ability to increase intracellular cyclic adenosine monophosphate (cAMP) levels. However, the mechanism(s) by which PGEs and PGI2 alter the complex biochemical and biophysical events associated with stimulus-response coupling in the neutrophil are not clear. It is reported here that both PGEs and PGI2 in micromolar concentrations inhibit formyl-methionyl-leucyl-phenylalanine (FMLP)- and zymosan-induced lysosomal enzyme secretion and superoxide anion production in a dose-dependent manner. No preincubation time of PMNs with the prostaglandins is required for inhibition. Addition of PGEs 10 seconds or later after FMLP stimulation does not alter the biologic response of the neutrophils to the stimulus, suggesting that the prostaglandin inhibition effects early events associated with stimulus-response coupling in the neutrophil. Prostaglandin inhibition of lysosomal enzyme release by the calcium ionophore A23187 was overcome by increasing the extracellular ionophore and/or calcium concentration, suggesting that PGs may modulate intracellular free calcium levels in a manner similar to that observed with platelets. Inhibition of phorbol myristate acetate (PMA)-induced neutrophil lysosomal enzyme secretion by PGEs and PGI2 was overcome by increasing concentrations of PMA. However, neither PGEs nor PGI2 altered O-2 production by PMA-treated neutrophils. These data indicate a dissociation between PMA-stimulated O-2 production and lysosomal enzyme release. These findings are consistent with the hypothesis that inhibition of neutrophil stimulation by PGEs and PGI2 is a result of increased intracellular cyclic AMP levels and modulation of calcium-dependent events. In addition, the data indicate that there are at least two mechanisms by which PMNs can be stimulated to produce O-2, one inhibited by PGEs and PGI2 and a second independent of prostaglandin modulation.