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Migration of neutrophils through endothelial cells (EC) and induction of cytokine secretion are two well-documented events during the inflammatory reaction. The inflammatory, chemotactic cytokine interleukin-8 (IL-8) is secreted by EC in response to IL-1 stimulation. In this study, we show that platelets activated with either adenosine-5'-diphosphate or epinephrine induce IL-8 secretion by EC. This stimulatory activity was found to be associated with sedimented platelets after activation. Blockade of IL-1 receptors on EC with IL-1 receptor antagonist (IL-1Ra) decreased the stimulatory effect of whole activated platelet preparations by 59% (P < .05). Similarly, IL-1Ra pretreatment of EC reduced the stimulatory effect of sedimented activated platelets by 60% (P < .01). In addition, we treated human blood donors with 750 mg of oral aspirin, and evaluated the stimulatory effect of epinephrine-activated platelets on IL-8 secretion by EC. IL-8 synthesis after aspirin ingestion was inhibited by 90% (P < .01) as compared with the preaspirin stimulation. These observations show that activated platelets induce IL-8 secretion via membrane-associated IL-1 activity, and provide a novel relationship between coagulation and inflammation that could be relevant to several diseases.

Plasma samples from cancer patients undergoing immunotherapy with high-dose recombinant interleukin-2 (IL-2) were obtained over a 5-day course of treatment and assayed by radioimmunoassay or enzyme-linked immunosorbent assay for the complement degradation products, C3a, iC3b, Ba, Bb, C4d, and SC5b-9. In the majority of patients, pretreatment C3a, Ba, Bb, and SC5b-9 plasma levels were comparable with those measured in normal donor plasma. However, by the end of the 5-day treatment course, C3a levels had increased 15.6-fold. In several patients, peak concentrations of C3a were as high as those reported in patients with sepsis or burn injury. Plasma levels of alternative pathway components Ba and Bb also increased, 8.0- and 5.0-fold, respectively, during IL-2 treatment. Likewise, levels of one of the terminal complexes, SC5b-9, increased 5.0-fold and the plasma C4d and iC3b concentrations increased 4.8- and 2.9-fold, respectively, by the fifth day of treatment. To determine whether activated lymphocytes participate in IL-2-induced complement activation, peripheral blood mononuclear cells (PBMC) obtained from IL-2 recipients before and 5 days after beginning therapy were reacted with monoclonal antibodies (MoAbs) against C3c and the terminal complement complex SC5b-9. Dual-color cytofluorographic analysis showed that within the CD3(+) population, the percentage of cells binding the anti-C3c and anti-SC5b-9 MoAbs increased 6.2-fold and 5.1-fold, respectively, by day 5. The anti-C3c MoAb also bound to CD3(+) cells stimulated in vitro with IL-2 and then exposed to serum. Moreover, fluid-phase iC3b was generated from purified C3 by PBMC activated in vitro with IL-2, but not by unstimulated cells. Serum levels of C-reactive protein (CRP) are markedly elevated in patients undergoing IL-2 immunotherapy. This hepatic acute phase reactant has been shown to activate the classical pathway when bound to cell surfaces. Because levels of the classical component C4d increase markedly during IL-2 treatment, we sought to determine if CRP became bound to PBMC during IL-2 treatment and found that during therapy, the percentage of CD3(+) cells reactive with an anti-CRP MoAb increased from less than 2% to greater than 18%. When PBMC were activated with IL-2 in vitro and then exposed to exogenous CRP, greater than 20% of the CD3(+) cells reacted with the anti-CRP MoAb.(ABSTRACT TRUNCATED AT 400 WORDS)

We investigated the effects of recombinant C5a (rC5a) on gene expression and synthesis of interleukin-1 beta (IL-1 beta) and tumor necrosis factor (TNF) in fresh human peripheral blood mononuclear cells (PBMC). Total (cell-associated and secreted) cytokine synthesis was measured. In the strict absence of endotoxin (lipopolysaccharide [LPS]), rC5a resulted in a small but statistically insignificant increase in immunoreactive IL-1 beta and TNF, as well as in IL-1 and IL-6 bioactivity. On the other hand, rC5a induced marked transcriptional activation of IL-1 beta and TNF in a dose-dependent fashion with an optimal concentration of 50 ng/mL. The rC5a-induced cytokine messenger RNA (mRNA) was not spontaneously translated into protein. At 50 ng/mL, rC5a induced the same levels of mRNA for IL-1 beta and TNF as 1 ng/mL of LPS, whereas LPS induced 12 times more IL-1 beta protein and 70 times more TNF protein than rC5a alone. The C5a-induced mRNA half-life was the same as that induced by LPS. Formyl-Meth-Leu-Phe (fMLP) did not induce cytokine transcription. Pretreatment with rC5a enhanced cytokine synthesis induced by other stimuli. After 2 hours of preincubation with rC5a, PBMC synthesized 3 to 10 times more IL-1 beta and TNF on subsequent stimulation by LPS or IL-1 itself. We conclude that rC5a provides primarily a transcriptional but not translational signal for IL-1 beta and TNF; the half-life of the untranslated mRNA is the same as that of translated message; rC5a-induced transcription upregulates PBMC for enhanced synthesis of these cytokines; and a translational signal can be provided by LPS or IL-1 itself.

Morphologic evaluation of synthetic grafts (both seeded and unseeded) harvested within 2 weeks of implantation has revealed heavy infiltration with polymorphonuclear leukocytes (PMNs). Since complement-activated PMNs are known to damage endothelial cells, we hypothesized that complement activation might prove a barrier to optimal endothelial cell seeding of prosthetic grafts. To determine whether synthetic vascular prostheses activate complement and whether the type of graft material influences the degree of activation, we assayed the plasma of 10 healthy donors for complement activity following incubation with short segments of knitted Dacron and polytetrafluoroethylene (PTFE) graft material. C5a generation was measured by radioimmunoassay and granulocyte aggregometry. Standard hemolytic assays were used to determine depletion of functional classical pathway (CH50 and C4) alternative pathway (APH50) activity. Results indicated substantial complement activation by Dacron and virtually none by PTFE. Activation by Dacron appeared to occur via both complement pathways. Such complement "reactivity" may have important implications for the performance of prosthetic materials as small-caliber vascular grafts, whether seeded with endothelial cells or not.

A microhemolytic assay for measurement of murine alternative complement pathway activity is described. The assay uses 51Cr release from neuraminidase-treated rabbit erythrocytes incubated with Mg2+ EGTA-chelated murine serum. Neuraminidase pretreatment of rabbit erythrocytes increases the sensitivity of the assay 8--10-fold, enable the use of small volumes of individual mouse sera. The assay affords a simple, sensitive and reproducible method for measuring murine alternative complement pathway activity. Significant differences were found between strains alternative complement pathway activity of serum from various inbred murine lines was measured.