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Background: Patients with acute respiratory distress syndrome who retain maximal alveolar fluid clearance (AFC) have better clinical outcomes. The release of endogenous catecholamines associated with shock or the administration of β2-adrenergic receptor (β2AR) agonists enhances AFC via a 3'-5'-cyclic adenosine monophosphate-dependent mechanism. The authors have previously reported that transforming growth factor-β1 (TGF-β1) and interleukin-8 (IL-8), two major mediators of alveolar inflammation associated with the early phase of acute respiratory distress syndrome, inhibit AFC upregulation by β2AR agonists via a phosphoinositol-3-kinase (PI3K)-dependent mechanism. However, whether TGF-β1 and IL-8 cause an additive or synergistic inhibition of AFC is unclear. Thus, the central hypothesis of the study was to determine whether they synergistically inhibit the β2AR-stimulated AFC by activating two different isoforms of PI3K., Methods: The effects of TGF-β1 or IL-8 on β2AR agonist-stimulated net alveolar fluid transport were studied using short-circuit current studies. Molecular pathways of inhibition were confirmed by pharmacologic inhibitors and Western blotting of p-Akt, G-protein-coupled receptor kinase 2, protein kinase C-ζ, and phospho-β2AR. Finally, our observations were confirmed by an in vivo model of AFC., Results: Combined exposure to TGF-β1 and IL-8/cytokine-induced neutrophil chemoattractant-1 caused synergistic inhibition of β2AR agonist-stimulated vectorial Cl across alveolar epithelial type II cells (n = 12 in each group). This effect was explained by activation of different isoforms of PI3K by TGF-β1 and IL-8/cytokine-induced neutrophil chemoattractant-1 (n = 12 in each group). Furthermore, the inhibitory effect of TGF-β1 on 3'-5'-cyclic adenosine monophosphate-stimulated alveolar epithelial fluid transport required the presence of IL-8/cytokine-induced neutrophil chemoattractant-1 (n = 12 in each group). Inhibition of cytokine-induced neutrophil chemoattractant-1 prevented TGF-β1-mediated heterologous β2AR downregulation and restored physiologic β2AR agonist-stimulated AFC in rats (n = 6 in each group)., Conclusions: TGF-β1 and IL-8 have a synergistic inhibitory effect on β2AR-mediated stimulation of pulmonary edema removal by the alveolar epithelium. This result may, in part, explain why a large proportion of the patients with acute respiratory distress syndrome have impaired AFC.

Objective: Macrophages play a central role in the immune response against infectious organisms. Once activated, macrophages secrete proinflammatory cytokines and chemokines. Interleukin (IL)-8 and related CXC chemokines play a role in the recruitment and activation of phagocytes acting through CXCR1 and CXCR2 receptors. The nuclear receptor peroxisome proliferator-activated receptor (PPAR) gamma exerts antiinflammatory properties in macrophages, by inhibiting cytokine and CC chemokine production. In this study, we investigated whether PPAR-gamma also plays a role in the regulation of the CXC chemokine pathway., Methods and Results: Synthetic PPAR-gamma ligands increase CXCR2 but not CXCR1 gene expression in a PPAR-gamma-dependent manner in primary human macrophages in vitro and in atherosclerotic plaques in vivo. The increase of CXCR2 mRNA was paralleled by an increase in membrane protein expression. EMSA, ChIP, and transient transfection assays indicate that PPAR-gamma activates the CXCR2 promoter by binding to a PPAR response element (PPRE). Finally, human macrophages acquire responsiveness to the CXCR2 ligands (IL-8 and Grobeta), as measured by superoxide anion production, after induction of CXCR2 expression by PPAR-gamma ligands., Conclusions: Our results provide a novel mechanism via which PPAR-gamma can enhance the immune response in human macrophages.

Study Design: Human nucleus pulposus cells from intervertebral disc specimens were cultured to study the effects of tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 on the focal adhesion kinase (FAK) expression by these cells. The effect of co-stimulation with dexamethasone on the FAK expression by nucleus pulposus cells was also studied., Objectives: To evaluate the possible role of activated FAK expressed by the human nucleus pulposus cells and its correlation with inflammatory cytokines (TNF-alpha, IL-8) and dexamethasone., Summary of Background Data: There have been no reported studies showing the correlation between the activated FAK expression by human nucleus pulposus cells with inflammatory cytokines and dexamethasone., Methods: The FAK expression in cultured human nucleus pulposus cells was studied, and Western blot and immunofluorescence analysis were performed to assess its relation to TNF-alpha, IL-8, and dexamethasone., Results: Treatments of TNF-alpha and IL-8 up-regulated the activated FAK expression. Dexamethasone attenuated the cytokine-induced FAK expression. The effects of inflammatory cytokines on the FAK expression were found to be concentration dependent, with greater correlation shown by IL-8 than TNF-alpha., Conclusion: TNF-alpha and IL-8 stimulation up-regulated the FAK expression of human nucleus pulposus cells, and the coadministration of dexamethasone attenuated it.

Background: Post-traumatic MOF results from local tissue injury because of migration and activation of dysfunctional polymorphonuclear leukocytes (PMN). Although fracture surgery exacerbates the postinjury inflammatory response, it is usually beneficial. This study compared changes in PMN receptor expression and migratory activity, in whole blood and following PMN isolation., Methods: IL-8 mediated PMN migration and expression of CXCR-1, CD11b, and CD18 was studied in isolated and whole blood PMN in normal controls. Migration was studied at admission and day 5 after surgery in trauma patients undergoing fracture surgery., Results: PMN isolation results in increased expression of surface receptors and enhanced migration in normal controls. In trauma patient samples, isolated PMN migration is enhanced after injury, but suppressed when migration from whole blood is studied, both after injury and fracture surgery., Conclusion: PMN isolation results in priming for migration, which has a relatively greater impact upon PMN in trauma patients. The observation that PMN activity may decline but priming potential remains enhanced is novel. Further refinements of whole blood and isolated PMN techniques are clearly warranted. This may help to resolve the mismatch in clinical and scientific experience in those patients with major fractures requiring surgical stabilization.

Background: Pig-to-human xenotransplantation is hampered by strong humoral and cellular immune responses, including acute vascular rejection (AVR). Infiltration of vascular xenografts by recipient polymorphonuclear neutrophils (PMN) is an early feature of AVR. Since little is known about the initiation of PMN recruitment, the present study investigated whether activated porcine endothelial cells (EC) release factors that induce human PMN recruitment., Methods: Primary and immortalized porcine aortic EC cultures were stimulated with phorbol-myristate acetate/ionomycin, lipopolysaccharide, tumor-necrosis factor-alpha, or interferon-gamma. The interleukin (IL)-8 concentration of porcine EC supernatants was tested by ELISA. Human and porcine PMN were isolated from peripheral blood by Ficoll sedimentation and centrifugation, characterized by morphology and flow cytometry, and analyzed for chemotaxis using Boyden chambers or Transwells. PMN chemokine receptor desensitization was determined by intracellular calcium-flux measurements., Results: Porcine EC supernatants contained significant amounts of porcine IL-8 and triggered chemotaxis in both human and porcine PMN. Chemotaxis of porcine, but not human, PMN was inhibited by anti-porcine IL-8 antibodies and recombinant porcine IL-8 induced strong chemotaxis only in porcine PMN. Porcine EC supernatants desensitized human PMN CXC-chemokine receptor (CXCR) 2, but not CXCR1, a receptor for human IL-8. Human PMN chemotaxis induced by porcine EC supernatants was significantly inhibited by blocking CXCR2 and platelet-activating factor (PAF)., Conclusions: Both chemokines acting via CXCR2 and PAF are released by porcine EC inducing efficient chemotaxis of human PMN. These mechanisms responsible for the recruitment of human PMN to porcine endothelium during cell-mediated rejection of xenografts represent potential targets for preventive strategies.

Leukocyte adhesion to endothelial cells plays a pivotal role in the early stage of endotoxin shock. The attenuation of the leukocyte response to endotoxin may contribute to the prevention of further organ dysfunction. Recent evidence implies that N-acetyl-cysteine (NAC) attenuates endotoxin-induced pathophysiological changes. We investigated the effect of NAC on the expression of CD11b and CD62L in endotoxin-stimulated human whole blood. NAC (>10 mM) significantly inhibited the lipopolysaccharide (LPS)-induced upregulation of CD11b in a concentration-dependent manner. However, NAC did not affect the LPS-induced downregulation of CD62L. We also analyzed the effect of NAC on interleukin-8 (IL-8)-induced expression of CD11b in human whole blood. IL-8 (10 ng/mL) significantly upregulated the expression of CD11b, and the IL-8-induced upregulation was significantly attenuated by NAC (>10 mM) in a dose-dependent manner. We conclude that NAC attenuates the increased expression of CD11b in either LPS or IL-8-stimulated human whole blood.

Adipose tissue injection as a free graft for the correction of soft-tissue defects is a widespread procedure in plastic surgery. The main problem in achieving long-term soft-tissue augmentation is partial absorption of the injected fat and hence the need for overcorrection and re-injection. The purpose of this study was to improve the viability of the injected fat by the use of interleukin-8. The rationale for the use of interleukin-8 was its abilities to accelerate angiogenesis and attract inflammatory cells and fibroblasts, providing the injected adipocytes more feeding vessels and a well-established graft bed to enhance their viability. Human adipose tissue, obtained by suction-assisted lipectomy, was re-injected into the subcutis in the scalp of nude mice. Interleukin-8 (0.25 ng) was injected subcutaneously to the scalp as a preparation of the recipient site 24 hours before the fat injection and was added to the fat graft itself (25 ng per 1 cc of injected fat). In the control group, pure fat without interleukin-8 was injected and no interleukin-8 was added for the preparation of the recipient site. One cubic centimeter of fat was injected in each animal in both the study and control groups. There were 10 animals in each group. The animals were euthanized 15 weeks after the procedure. Graft weight and volume were measured and histologic evaluation was performed. In addition, triglyceride content and adipose cell sizes were measured as parameters for fat cells viability. Histologic analysis demonstrated significantly less cyst formation in the group treated with interleukin-8. No significant differences were found between the groups with regard to graft weight and volume or the other histologic parameters investigated. No significant differences were demonstrated in adipose cell sizes and their triglyceride content. In conclusion, less cyst formation, indicating improved quality of the injected fat, can be obtained by the addition of interleukin-8. Further studies of various dosages of interleukin-8 and their long-term effect are required before these encouraging results could be applied clinically.

Platelet factor-4 (PF-4)/CXCL4 was the first chemokine described to inhibit neovascularization. Here, the product of the nonallelic variant gene of CXCL4, PF-4var1/PF-4alt, designated CXCL4L1, was isolated for the first time from thrombin-stimulated human platelets and purified to homogeneity. Although secreted CXCL4 and CXCL4L1 differ in only three amino acids, CXCL4L1 was more potent in inhibiting chemotaxis of human microvascular endothelial cells toward interleukin-8 (IL-8)/CXCL8 or basic fibroblast growth factor (bFGF). In vivo, CXCL4L1 was also more effective than CXCL4 in inhibiting bFGF-induced angiogenesis in rat corneas. Thus, activated platelets release CXCL4L1, a potent regulator of endothelial cell biology, which affects angiogenesis and vascular diseases.

Objective: We have previously reported increased monocyte adhesion to human aortic endothelial cells (HAECs) cultured in 25 mmol/L glucose (HG) compared with normal glucose (NG) (5.5 mmol/L). In this study, we explored mechanisms that contribute to increased monocyte adhesion by elevated glucose., Methods and Results: We found that HAECs cultured in HG have increased production of the chemokine interleukin-6 (IL-6). We examined whether IL-6 directly modulated monocyte adhesion to EC. Inhibition of IL-6 using a neutralizing antibody significantly reduced glucose-mediated monocyte adhesion by 50%, and addition of IL-6 directly to human EC stimulated monocyte adhesion. PPARalpha has been reported to negatively regulate expression of IL-6 in vascular cells, so we examined PPARalpha-associated signaling in EC. A known PPARalpha agonist, Wy14,643, prevented glucose-mediated IL-6 production by EC and reduced glucose-mediated monocyte adhesion by 40%. HG-cultured HAEC had a 50% reduction in expression of PPARalpha compared with control EC. Primary aortic EC isolated from PPARalpha knockout (KO) mice showed increased monocyte adhesion compared with EC isolated from control mice. PPARalpha KO EC also had increased production of IL-6. Finally, we measured IL-6 levels in diabetic db/db mice and found significant 6-fold elevations in IL-6 levels in db/db EC., Conclusions: These data indicate that IL-6 production is increased in diabetes and contributes to early vascular inflammatory changes. PPARalpha protects EC from glucose-mediated monocyte adhesion, in part through regulation of IL-6 production.

Objectives: To investigate the mechanisms of metastasis formation in human pancreatic carcinoma, we examined the angiogenic capabilities of human pancreatic cancer cell lines with different metastatic potentials and the roles of inflammatory cytokines., Methods: Interleukin (IL)-8 secretion by human pancreatic cancer cells stimulated with IL-1alpha or IL-1 receptor antagonist (IL-1ra) was measured by enzyme-linked immunosorbent assay (ELISA). We then examined how cancer cells with different metastatic potentials influenced the proliferation and tube formation of human umbilical vein endothelial cells (HUVECs) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction method (MTT assay) and an angiogenesis assay, respectively. We also examined the role of inflammatory cytokines in relation to tumor metastatic potential and angiogenesis., Results: IL-8 secretion levels by pancreatic cancer cells were regulated by IL-1alpha and correlated with metastatic potential. Both HUVEC proliferation and tube formation were strongly enhanced by coculture with metastatic pancreatic cancer cells and were enhanced to a similar extent by culture in the presence of IL-1alpha and IL-8. In contrast, blockade of IL-1alpha or IL-8 inhibited HUVEC proliferation and angiogenesis., Conclusions: The inflammatory cytokines IL-1alpha and IL-8 may have an important role in metastasis via vascular endothelial cell proliferation and angiogenesis.

It has been previously described that daily intraplantar (i.pl.) injections of prostaglandin E2 (PGE2) and dopamine in rats for 14 days cause the development of a persistent mechanical nociceptor hypersensitivity state lasting more than 30 days. Considering that during inflammation, the release of these hyperalgesic agents are mediated by cytokines, we investigated in the present study whether interleukin-1beta (IL-1beta), IL-8 and tumour necrosis factor-alpha (TNF-alpha) are able to induce persistent mechanical nociceptor hypersensitivity. Daily i.pl. administration of TNF-alpha, IL-1beta or IL-8 for 18 days led to persistent mechanical nociceptor hypersensitivity, which lasted at least 30 days after the cessation of treatment. The co-treatment of the animals with IL-1beta plus indomethacin, but not with atenolol, prevented the induction of persistent mechanical nociceptor hypersensitivity. The co-treatment of the animals with IL-8 plus atenolol, but not with indomethacin, prevented the induction of persistent mechanical nociceptor hypersensitivity. The daily co-treatment of TNF-alpha with either indomethacin or atenolol partially inhibited (+/-50%) the induction of persistent mechanical nociceptor hypersensitivity. However, the combined treatment with indomethacin plus atenolol abolished the induction of the persistent mechanical nociceptive hypersensitivity by TNF-alpha.A single injection of cytokines in the contralateral paws of the animals with persistent hypersensitivity caused only an acute nociceptive response. This observation, together with the demonstration of undetectable levels of immunoglobulins against TNF-alpha, IL-1beta or IL-8 in the sera of animals after the development of the persistent hypersensitivity induced by those cytokines, indicate that this event is not due to an ongoing immunological response against the cytokines. In conclusion, our results support the suggestion that IL-1beta- and IL-8-induced persistent mechanical nociceptor hypersensitivity results from the endogenous release of eicosanoids and sympathetic amines, respectively. However, TNF-alpha-induced mechanical nociceptor hypersensitivity results from the concomitant endogenous release of eicosanoids and sympathomimetic mediators.

Dysregulated polymorphonuclear leukocyte (PMN) apoptosis and PMN-mediated organ damage have been associated with several medical conditions such as systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), and ischemia/reperfusion injury. IL-1beta and IL-8 are two cytokines that are elevated under similar conditions. Therefore, we hypothesized that PMN exposed to these cytokines would secrete factors that could affect PMN apoptosis in a cell contact-independent manner. We have previously shown that media conditioned by IL-1beta-stimulated PMN (CM-IL1beta) for 2 h suppressed spontaneous PMN apoptosis. Data presented here demonstrate that media conditioned by IL-8-stimulated PMN (CM-IL8) also have the ability to suppress spontaneous, as well as FasL- and TNF-alpha-induced apoptosis. In contrast, CM-IL1beta was able to suppress FasL-induced, but not TNF-alpha-induced, apoptosis. To elucidate the mechanisms these media use to elicit their effects, we examined the expression and function of several apoptosis-related proteins. Experimental results demonstrate that both CM-IL1beta and CM-IL8 have the ability to delay caspase activation, but have no effect on the expression of their upstream activator, Fas, or its ligand, FasL. Examination of several Bcl-2 family members revealed a selective regulation by each media: CM-IL1beta up-regulated Bcl-X(L), while CM-IL8 down-regulated Bak expression. Additionally, CM-IL1beta, but not CM-IL8, promoted the activation of NF-kappaB, which has anti-apoptotic activity. Together, we can conclude that IL-1beta- and IL-8-stimulated PMN have the ability to suppress PMN apoptosis in a paracrine manner, and that the extent and mechanism of suppression is specific for each.

Systemic candidasis is a life-threatening complication of antibiotic and immunosuppressive therapies and can alter host defense mechanisms through pathways that are poorly understood. Promotion of polymorphonuclear leukocyte (PMN) chemotaxis by beta-glucan towards fMLP or IL-8 gradients demonstrates a fundamental effect on host defenses by pathogenic fungi. The aim of the present study was to determine whether recognition of beta-glucan is sufficient to alter PMN motility in the absence of agonists of G-coupled protein chemotactic receptors. Present findings demonstrate a profound increase in PMN motility by beta-glucan supplementation of a fibronectin substratum in an underagarose migration assay. Motility on beta-glucan included a 3-fold increase in distance of migration, as well as a 5-fold increase in the number of PMNs recruited into the motile phase as compared to motility on fibronectin alone. This promotion of motility is determined by the beta2 integrin complement receptor 3 (CR3) (CD11b/CD18) rather than the beta1 integrin very late antigen 3 (VLA-3), which mediates chemotaxis on beta-glucan-supplemented matrix towards fMLP. PMN motility on beta-glucan-supplemented fibronectin was selectively decreased by inhibitors of pp60 src and ras, whereas motility was promoted by inhibition of p38-MAPK. No effect of these inhibitors was seen on PMNs migrating on fibronectin alone. Migration on beta-glucan-supplemented fibronectin, but not on fibronectin alone, was negatively regulated by protein kinase C (PKC) or cAMP activation. These findings indicate that beta-glucan is sufficient to alter the migratory capacity of PMN in the absence of costimulation by fMLP. Enhanced PMN migration on beta-glucan is mediated through specific integrins and second messenger pathways that are distinct from those utilized by PMNs migrating in the absence of beta-glucan.

Background: Trauma modulates polymorphonuclear neutrophil (PMN) function, predisposing to organ failure and infection. Many chemoattractants released by injury activate PMNs via G-protein-coupled (GPC) receptors, which elevate PMN cytosolic calcium ([Ca2+]i). Nonetheless, PMN GPC receptor function after injury is unstudied., Methods: PMNs from 11 major trauma patients (Injury Severity Score = 31 +/- 3, eight men and three women, age = 38 +/- 3) were obtained on days 1, 3, and 7 after injury. Nine developed organ failure and one died. PMNs were exposed to interleukin-8 (IL-8), growth regulated oncogene-alpha (GRO-alpha), and platelet-activating factor (PAF) to stimulate the CXCR1, CXCR2, and PAF receptors. [Ca2+]i flux measurements were used to quantify receptor responses. Receptor responses to individual as well as serial GPC agonists were studied over the week after injury and compared with the responses of PMNs from healthy volunteers (n = 10-23). Results were evaluated by one-way analysis of variance, and paired and unpaired t tests., Results: Responses to GRO-alpha and PAF were significantly depressed early after injury (p < 0.01). Responses to all agonists tested tended to be lowest on day 1, to peak on day 3, and to decrease again by day 7, but variations in response to GRO-alpha were the most marked (p < 0.03, analysis of variance). Whereas GRO-alpha primed IL-8 and IL-8 primed PAF in normal PMNs, GRO-alpha paradoxically suppressed IL-8 responses and IL-8 suppressed PAF responses in trauma PMNs. PAF priming of IL-8 responses was unaffected by injury., Conclusion: Receptor responses to individual GPC agonists are suppressed early after trauma, but increase by day 3. Normal chemokine priming of PMN calcium mobilization is reversed by injury; priming by PAF is intact. PMN GPC responses depend on the sequence in which agonists are encountered. Injury appears to alter these interactions, thus priming some aspects of PMN function while simultaneously suppressing others.

Delayed neutrophil apoptosis is a feature of persistent acute inflammation. Neutrophil-mediated damage has been shown to be associated with the development of inflammatory bowel disease (IBD). Persistence of these cells both at the colonic site and circulation may further contribute to IBD. The aims of this study were to determine whether neutrophils isolated from IBD patients delay apoptosis and to investigate possible mechanisms involved in this delay. We studied 20 patients with IBD, 13 with Crohn's disease, and 7 with ulcerative colitis, all of whom were undergoing intestinal resection for symptomatic disease. Seventeen patients undergoing elective resection of colon cancer acted as operative controls. Systemic, mesenteric arterial, and mesenteric venous blood was harvested. Neutrophils isolated from patients with IBD showed decreased spontaneous apoptosis compared to cancer patients. Mesenteric venous serum of IBD patients contributed to this delay, which contained higher concentrations of interleukin-8 (IL-8). Pro-caspase 3 expression was also reduced in IBD neutrophils, which may contribute to decreased spontaneous and Fas antibody-induced apoptosis. Neutrophil apoptosis may be altered in Crohn's disease and ulcerative colitis through release of anti-apoptotic cytokines and altered caspase expression. The alterations in cell death mechanisms may lead to persistence of the inflammatory response associated with IBD.

Purpose: Chemotaxins from inflammatory sites prime or activate neutrophils (PMN) by using cytosolic calcium ([Ca2+]i) fluxes as second messengers. [Ca2+]i can be mobilized rapidly by receptor-mediated entry or store-release, or more slowly by store-operated calcium influx (SOCI). We studied [Ca2+]i mobilization by chemotaxins and how trauma impacts the calcium entry mechanisms used by chemotaxins., Methods: [Ca2+]i flux was studied by spectrofluorometry. The contributions of early and late [Ca2+]i currents to net calcium flux were compared after stimulation by more potent (fMLP, C5a, PAF) or less potent (IL-8, GRO-alpha, and LTB4) agonists. Store operated [Ca2+]i mobilization was reflected by the ratio of area under the [Ca2+]i efflux curve to peak [Ca2+]i (efflux curve). PMN from trauma patients (ISS > 25) and pair-matched volunteer (n = 7 pairs) were then primed and stimulated with thapsigargin to compare cell calcium stores and SOCI., Results: Late [Ca2+]i mobilization made more important contributions to fMLP, PAF, and C5a signals than to IL-8, GRO-alpha, or LTB4 (p < 0.01 all comparisons). Calcium stores and store release were only marginally lower after injury (p = not significant), but trauma PMN showed far higher [Ca2+]i influx after thapsigargin (p = 0.007), and greater net SOCI (p = 0.034)., Conclusions: SOCI may play an important role in PMN activation, and trauma increases PMN SOCI. Prolonged elevations of [Ca2+]i due to enhanced SOCI may alter stimulus-response coupling to chemotaxins and contribute to PMN dysfunction after injury.

Interleukin 8 (IL-8) and growth-related oncogene alpha (Gro-alpha) delay neutrophil apoptosis, which is thought to be important for the resolution of inflammation. We hypothesized that (IL-8) and Gro-alpha interfere with extracellular death receptor signaling or intracellular caspase activation to suppress neutrophil apoptosis. In addition, we sought to determine if prolonged neutrophil half-life was associated with preservation of function. Polymorphonuclear leukocytes (PMN) were cultured with IL-8 or Gro-alpha (0-100 ng/mL) in normoxia or hypoxia, and the extent of apoptosis was assessed by histology and TdT-mediated dUTP nick end labeling (TUNEL). Subsequently, to determine the role of apoptotic-associated receptors, PMN were cultured with IL-8 and neutralizing monoclonal antibody to Fas (CD95), TNFR55, and TNFR75. To establish the effect of IL-8 or Gro-alpha on pro-apoptotic caspase activity, the cleavage of specific colorimetric substrates was assessed. Functional changes in PMN included the capacity to produce superoxide anion and phagocytosis of Escherichia coli. At the 100 ng/mL dose, the addition of IL-8 and Gro-alpha maximally suppressed PMN apoptosis from 54% (untreated) to 5% and 6%, respectively. The addition of neutralizing antibodies to Fas, TNFR55, or R75 caused no change in IL-8 suppression of apoptosis. Caspase 3 activity was markedly suppressed at 24 h by the inclusion of either IL-8 and Gro-alpha. IL-8 and Gro-alpha-stimulated PMN released more superoxide anion and had an increased phagocytic index vs. control PMN. IL-8 and Gro-alpha suppress neutrophil apoptosis to a similar level that is not influenced by oxygen tension at high doses. The effect of IL-8 and Gro-alpha does not depend on activation of the Fas, TNFR55, or R75 receptor pathways but involves suppression of caspase 3 activity. IL-8 or Gro-alpha extends the functional half-life of neutrophils and may explain their role in disease states such as acute respiratory distress syndrome.

Objective: To study the effects of a spin trap agent and a CD18 antibody administered after stroke induction on intracerebral hemorrhaging. The drugs can prevent leukocyte adhesion., Methods: A rabbit embolic stroke model that produces intracerebral hemorrhage was used., Results: A time course study showed that hemorrhaging was grossly apparent in approximately 50% of the subjects at 5 hours and in 75% at 24 hours after embolization. MDL 101,002, a spin trap agent, administered IV 5 minutes after embolization, significantly decreased the volume of hemorrhage. It also improved the behavior score relative to vehicle-treated rabbits. The CD18 antibody tended to decrease hemorrhage volume., Conclusion: The beneficial effect of MDL 101,002 may be caused by inhibition of free radical injury to brain tissue, thereby protecting brain microvessel integrity. Acute therapy for intracerebral hemorrhage may be feasible.

Neutrophil (PMN) priming and subsequent responses to the IL-8 presented on pulmonary endothelial surfaces may be crucial determinants of the development of adult respiratory distress syndrome after injury. Elevated plasma ELR+ C-X-C chemokine (CXC) levels might contribute to PMN priming after trauma, but the role of CXCs in priming circulating PMNs is unstudied. We evaluated the interactions of IL-8 and GRO-alpha in priming human PMN calcium fluxes [Ca2+]i within circulatory environments. At physiologic concentrations, GRO-alpha primes PMN for IL-8 mediated [Ca2+]i mobilization, whereas IL-8 abolishes GRO-alpha responses. Repeated GRO-alpha exposures further enhance IL-8 responses. PMN priming for IL-8 responses in normal plasma was CXCR2 dependent. CXCR2 was more responsive than CXCR1 to low levels of IL-8, together suggesting that CXCR2 is the important CXC receptor at circulating (i.e., low) agonist concentrations. CXCR1 stimulation down-regulated CXCR2 surface expression, whereas CXCR2 stimulation upregulated CXCR1 expression. GRO-alpha/ CXCR2 signaling enhanced post-receptor IL-8 initiated PMN [Ca2+]i influx as well as efflux. Sufficient stimulation of the CXCR1 terminated this cooperative relationship by downregulating surface expression of CXCR2. This study is the first to report that at physiologic concentrations, C-X-C chemokines can act on circulating human PMNs as an integrated system where CXCR2 agonists, rather than cross-desensitizing CXCR1, act to enhance signaling of IL-8 at CXCR1 both by receptor and post-receptor mechanisms. Such CXCR2 mediated priming of CXCR1/ IL-8 interaction may enhance PMN attack on the lung after injury.

The alpha chemokine family is central to the participation of neutrophils in the acute inflammatory response. These substances interact with neutrophils through two cell surface receptors, CXCR-1 and CXCR-2 (formally known as IL-8R-1 and IL-8R-2). We investigated the possible regulatory effects of tumor necrosis factor alpha (TNFalpha) pretreatment on CXCR-1 and CXCR-2. To this end, we examined these receptors with flow cytometry, radioligand binding, Northern blot analyzes, calcium mobilization, and chemotaxis experiments on human neutrophils. In flow cytometry experiments, TNFalpha pretreatment substantially decreased cell surface CXCR-2 receptor levels but showed partial recovery at 120 min. On the other hand, CXCR-1 receptor levels had a sharp decline at 15 min and maintained that level to 120 min. Northern blot analyzes showed that mRNA levels of both IL-8 receptors were essentially unchanged after 45 min of TNFalpha pretreatment, but declined markedly following 2 h of pretreatment. Chemotaxis experiments on cells treated with TNFalpha for 5-120 min showed a substantial down-regulation of chemotaxis to IL-8 and GROalpha. This was noted to be much greater than the decline in cell surface receptors. Calcium mobilization experiments revealed minimal inhibition of the IL-8-induced increase in calcium after pretreatment with TNFalpha, but the response to NAP-2 was substantially inhibited. The data demonstrate differential regulation of the IL-8 receptor.

Clinical neutrophil (PMN) priming is the net result of multiple stimuli, with intracellular calcium ([Ca2+]i) being a key second messenger for PMN agonists such as the chemokines. Thus, [Ca2+]i measurement may be a robust tool for the assessment of global PMN activation. [Ca2+]i is difficult to measure in complex biologic environments, however, so data in this area are limited. We therefore developed an in vitro system to measure the effects of chemokines on PMN [Ca2+]i. PMN were isolated from volunteer blood. PMN [Ca2+]i responses to interleukin (IL)-8 and Growth-Related Oncogene (GRO)-alpha were studied by fura-2-acetoxymethyl ester fluorescence with or without reincubation in autologous plasma just prior to study. The effects of IL-8 and GRO-alpha on PMN [Ca2+]i at ascending doses, with or without plasma reincubation, given sequentially and in the presence or absence of extracellular calcium, were studied. PMN basal [Ca2+]i was increased by plasma, as were low-dose priming and higher-dose spike responses to IL-8. GRO-alpha caused a more pronounced priming of PMN [Ca2+]i than IL-8 at low doses, although significantly lower peak responses were observed with GRO-alpha than IL-8 at higher doses. Plasma suppressed both priming and spike responses to GRO-alpha. When given serially at clinically relevant agonist doses, GRO-alpha was permissive of IL-8 signaling, whereas IL-8 blocked GRO-alpha signaling. IL-8 generates high [Ca2+]i spikes using intracellular calcium stores only. GRO-alpha produces lower [Ca2+]i spikes despite using both intra- and extracellular stores. Plasma preincubation has profound effects on PMN [Ca2+]i responses to chemokines. These can be measured accurately, as described. In clinically relevant environments, IL-8 and GRO-alpha interact in a regulatory fashion. GRO-alpha may act as a priming agent, with IL-8 activating PMN functions requiring high [Ca2+]i. This cross-cooperation is probably terminated by IL-8 regulation of GRO-alpha activity at the C-X-C chemokine receptor 2.

The CXC chemokines have well-documented neutrophil chemotactic, angiogenic, and mitogenic properties. The current investigations evaluate the effects of interleukin-8 (IL-8), epithelial neutrophil activating protein (ENA-78), and macrophage inflammatory peptide-2 (MIP-2) on hepatocyte proliferation in vitro and liver regeneration in vivo. Primary rat hepatocytes were isolated by collagenase digestion and exposed to incremental doses of IL-8, ENA-78, or MIP-2, and cellular proliferation was measured via tritiated thymidine incorporation. These experiments demonstrated significant increases in hepatocyte proliferation in response to IL-8, ENA-78, and MIP-2. Next, rats were sacrificed in a time-dependent manner following 70% hepatectomy or sham laparotomy, and hepatic tissue levels of MIP-2 and ENA-78 were measured using an ELISA. ENA-78 and MIP-2 were significantly elevated following 70% hepatectomy as compared with sham-operated control animals. Rats undergoing 70% hepatectomy were then treated with neutralizing anti-ENA-78 serum, anti-MIP-2 serum, or preimmune control serum, and liver regeneration was evaluated. These experiments demonstrated that neutralization of ENA-78 or MIP-2 slowed the rate of liver regeneration. These data suggest that the CXC chemokines may be important agents for the induction of hepatocyte proliferation and may be important molecules in vivo in the setting of liver injury, repair, and regeneration.

Objective: To examine the changes in neutrophil deformability, aggregation, and adherence in response to stimulation with proinflammatory cytokines and bacterial toxins., Design: Prospective, randomized trial., Setting: Research laboratory., Subjects: Neutrophils isolated from healthy volunteers., Interventions: Neutrophils were exposed to tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-8, their combination, endotoxin (LPS), lipoteichoic acid (LTA), and staphyloccocal enterotoxin B (SEB). Neutrophil deformability was measured as percent neutrophils filtered through 5-microm diameter filters. Aggregation was measured using a platelet aggregometer. Adherence was determined by examining the binding of neutrophils to albumin-coated latex beads., Measurements and Main Results: Exposure to TNF-alpha and IL-1beta led to significant decreases in neutrophil filterability, which was attenuated by cytochalasin D pretreatment. LPS and LTA also decreased deformability, suggesting that these toxins directly stimulated neutrophils independent of cytokines. IL-8 and SEB did not significantly affect neutrophil deformability. TNF-alpha and LPS were associated with significant neutrophil aggregation, which was inhibited by pretreatment with anti-CD18 antibodies. Neutrophil aggregation was not affected by IL-1beta, LTA, or SEB. TNF-alpha, IL-8, and LPS increased neutrophil adherence, which also was attenuated by pretreatment with anti-CD18 antibodies. IL-1beta, LTA, and SEB did not significantly affect neutrophil adherence., Conclusions: Cytokines and bacterial toxins differ in their effects on neutrophil deformability, aggregation, and adherence. Of the cytokines examined, TNF-alpha appears to have the greatest direct effects on neutrophil rheology. Similarly, endotoxin appears to have greater direct effects on neutrophil rheology than the Gram-positive bacterial toxins, LTA, and staphylococcal enterotoxins.

Background and Purpose: Inducible cyclooxygenase (COX-2) has been found to play a pathological role in cerebral insult. We investigated the expression of COX-2 in the basilar artery after experimental subarachnoid hemorrhage (SAH)., Methods: In a canine "two-hemorrhage" model of SAH, the basilar arteries were obtained on day 2 after a cisternal injection of autologous blood or on days 4, 6, 7, or 9 after the second injection. Basilar arteries also were obtained 12 hours after intracisternal injection a cytokine: interleukin (IL)-1 beta (0.03 microgram), IL-6 (3 micrograms), or IL-8 (10 micrograms). Western blotting with a polyclonal anti-COX-2 antibody was performed in these arteries., Results: COX-2 protein was not demonstrated in the basilar artery in control animals without SAH. However, it was expressed in the basilar artery on days 2, 4, 6, and 7 after blood injection but not on day 9. Intracisternal injection of IL-1 beta, IL-6, or IL-8 also induced COX-2 in the basilar artery., Conclusions: COX-2 expression was detected in basilar arterial tissue in both acute and chronic stages after SAH. Elevation of inflammatory cytokines after SAH may be involved in the induction of COX-2, which may produce sufficient quantities of eicosanoids to affect hemodynamics after SAH.

Interleukin (IL)-6 and IL-8 are important regulators of inflammatory responses in myocardial infarction. Induction of monocyte procoagulant activity (PCA) by these cytokines could present a mechanism that links inflammatory responses to thrombotic events. We therefore investigated the effect of IL-6 and IL-8 on monocyte tissue factor (TF) expression. Recombinant human IL-6 and IL-8 caused a time- and dose-dependent increase in PCA (recalcification time) of monocytic U937 cells and of mononuclear leukocytes. Using blocking anti-TF monoclonal antibodies and factor VII-deficient control plasma, this PCA was shown to be TF dependent. Compared with unstimulated cells, mononuclear cell PCA increased by 4.5-fold to 17 +/- 2 mU/5x10(5) cells after exposure to 100 ng/L IL-6 for 4 hours and by 6.6-fold to 27 +/- 4 mU/5x10(5) cells after exposure to IL-8 under the same conditions. Northern blot analysis showed an increase in TF mRNA after stimulation with IL-6 or IL-8 for 2 hours, and after 4 hours an increase in cellular TF protein content was found by immunoassay. Flow cytometry demonstrated that IL-6 and IL-8 induced an increase in TF surface expression on monocytes. Thus, IL-6 and IL-8 induce monocyte PCA by increasing mRNA, protein content, and surface expression of TF.

Although circulating levels of interleukin 8 (IL-8), a potent pro-inflammatory chemokine, and many other inflammatory mediators increase in response to cardiopulmonary bypass, only a small proportion of patients develop a clinically significant systemic inflammatory response. The natural mechanisms that control the inflammatory response are poorly understood. To investigate the role of IL-8 in a human inflammatory model, 15 adult patients undergoing cardiopulmonary bypass for elective coronary artery bypass grafting were studied. Following reperfusion, plasma IL-8 levels increased significantly from 58 pg/mL (pre-bypass) and 66 pg/mL (after 20 min of bypass) to 98 pg/mL (p = .02 and .04, respectively), but this was accompanied by a concomitant threefold decrease in the IL-8 binding affinity of circulating neutrophils (Dissociation constant (KL) post-reperfusion/KL pre-bypass = 3.2; KL post-reperfusion/KL after 20 min of bypass = 2.8). IL-8-triggered release of myeloperoxidase and elastase by peripheral blood neutrophils ex vivo was also down-regulated following reperfusion. There were no significant changes in beta 2 integrin expression or inositol polyphosphate metabolism of peripheral blood neutrophils. These changes in receptor affinity and neutrophil responsiveness to IL-8 may represent an important in vivo regulatory mechanism which serves to prevent excessive tissue injury from inflammatory triggers.

We have previously detailed the effects of exogenous cytokines (IL-1 beta, TNF-alpha, and IL-8) on polymorphonuclear leukocyte (PMN) bactericidal function during normoxia. In these studies, we have investigated the effects of hypoxia +/- reoxygenation independently and with IL-1 beta, TNF-alpha, and IL-8 on PMN bactericidal activity. Hypoxia in and of itself did not significantly alter PMN bactericidal activity against Escherichia coli or Staphylcoccus aureus; however, after 2 h of reoxygenation (H/R), PMN bactericidal activity against E. coli was significantly reduced compared with levels seen after 2 h of hypoxia. Similar to what was observed during normoxia, TNF-alpha, IL-1 beta, and IL-8 increased PMN bactericidal activity during hypoxia compared with buffer control PMN for S. aureus but not E. coli after 4 h of hypoxia. Following H/R, neither TNF-alpha, IL-1 beta, nor IL-8 reversed the decline in bactericidal activity induced by reoxygenation alone. Monoclonal antibodies that blocked the functional epitope of particular cytokine receptors demonstrated that during both hypoxia and H/R IL-1 beta R type I, IL-8R type A, and TNF-alpha R p60 were the predominant receptors responsible for mediating the bactericidal effect of the cytokines. During hypoxia and H/R, the addition of exogenous cytokines did prevent the fall in bactericidal activity seen as PMN: target ratios decreased. The decline in bactericidal activity following H/R was mediated in part by reduced phagocytosis of serum-opsonized bacteria following H/R. These results demonstrated that oxidative stress in the form of hypoxia +/- reoxygenation independently modulate the effect of exogenously added cytokines on PMN bactericidal capability.

We investigated the effects of exogenous cytokines (interleukin (IL)-8, tumor necrosis factor (TNF)-alpha, and IL-1 beta) on polymorphonuclear neutrophil (PMN) bactericidal activity against both Staphylococcus aureus and Escherichia coli. Both baseline and IL-8-stimulated PMN bactericidal activity against E. coli, but not against S. aureus, declined significantly from 0 to 240 min. The decline in bactericidal activity was prevented by TNF-alpha, but not IL-1 beta. Bactericidal activity against both E. coli and S. aureus declined as PMN:target ratios went from 20:1 to 5:1. TNF-alpha and IL-1 beta preserved bactericidal activity even at a 5:1 PMN:target ratio against E. coli, whereas all three cytokines preserved bactericidal activity at a 5:1 PMN:target ratio against S. aureus. Dose-response curves demonstrated significant increases in bactericidal activity with physiologically relevant concentrations of cytokines (IL-8: .1-10 ng/mL; TNF-alpha: 1-10(2) U/mL; and IL-1 beta: 0-10 ng/mL). Binding of cytokine receptors with monoclonal antibodies directed against IL-8R Type A, TNF-alpha R (p60) or (p80), and IL-1 beta R Type I significantly reduced the effect of individual cytokines on PMN bactericidal activity. Inhibition of terminal, but not proximal, products of the PMN oxidative burst significantly reduced the effect of exogenous cytokines on PMN bactericidal activity. These results demonstrate that individual cytokines at relatively low concentrations enhance PMN bactericidal activity via oxidant-dependent mechanisms and that inhibiting cytokine functions may not be advantageous at infectious foci in vivo.

Interleukin-8 (IL-8) has been associated with a variety of hyperinflammatory states and adverse clinical events. Circulating IL-8 levels correlate with the severity of tissue trauma, and excessive elevations of IL-8 are associated with postinjury adult respiratory distress syndrome and multiple organ failure. While IL-8 is a potent neutrophil (PMN) chemoattractant and activator and enhances PMN transendothelial migration, it also acts to inhibit PMN adhesion to stimulated endothelial cells (ECs). We hypothesized that IL-8 could interact directly with ECs to increase permeability independent of PMNs. Human umbilical vein ECs (HUVECs) were cultured on collagen-coated micropore filters, and integrity of the EC monolayer measured by albumin flux across the filter. Cytochalasin D was used as a positive control. IL-8 induced increased permeability at a concentration of 1000 ng/mL. This effect was abrogated by preincubation of HUVECs with a protein synthesis inhibitor (cycloheximide). These data suggest a role for IL-8 in promoting endothelial leak independent of PMNs, via a mechanism involving protein synthesis.

Interleukin-8 (IL-8) is a chemokine produced by a variety of cell types involved in atherogenesis and is chemotactic for neutrophils and lymphocytes. A recent study has shown that IL-8 is angiogenic and induces proliferation and chemotaxis of endothelial cells. The present study was undertaken to find out whether IL-8 is also mitogenic and chemotactic for vascular smooth muscle cells. IL-8 induced a concentration-dependent (0.1 to 10 nmol/L) stimulation of DNA synthesis and cell proliferation in both human and rat aortic smooth muscle cells. In addition, IL-8 stimulated smooth muscle cells to produce prostaglandin E2, which can inhibit IL-8-induced smooth muscle cell proliferation. In the presence of indomethacin (5 mumol/L), IL-8 (1 nmol/L) stimulated an increase in human and rat aortic smooth muscle cell number during a 3-day period of incubation by 61 +/- 16% and 59 +/- 7% (n = 4), respectively. IL-8 also increased DNA synthesis in human and rat aortic smooth muscle cells by 98 +/- 10% and 151 +/- 27% (n = 5), respectively. Moreover, IL-8 stimulated rat aortic smooth muscle cell migration by 20-fold over the control value, with an EC50 value of 0.83 nmol/L; this chemotactic activity of IL-8 was also potentiated by indomethacin. Exposure of smooth muscle cells to IL-8 caused rapid and transient expression of the immediate-early genes c-fos and zif268 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokine induction of intercellular adhesion molecule-1 (ICAM-1) in cardiac myocytes may be a critical step in inflammation associated with ischemia-reperfusion injury. We investigated the involvement of tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), and interleukin 8 (IL-8) on neutrophil-myocyte adhesion; These cytokines are increased in plasma of patients with acute myocardial infarction (AMI). ICAM-1 expression on cultured neonatal rat cardiac myocytes was determined through immunohistochemical and enzyme-linked immunosorbent assay (ELISA) analysis. ICAM-1 mRNA expression in myocytes was investigated by Northern blot hybridization. Rat neutrophils isolated from peripheral blood (PB) were used for adherence assay. In immunohistochemical study, cultured neonatal rat cardiac myocytes constitutively expressed ICAM-1 molecules. In ELISA analysis, ICAM-1 molecule expression on myocytes was significantly stimulated by TNF-alpha (100 U/ml), but not by IL-6 (100 U/ml) or IL-8 (100 ng/ml) dose dependently. The effect of TNF-alpha was observed as early as 6 h after stimulation. Levels of ICAM-1 mRNA were very low or almost undetectable in unstimulated myocytes, but its expression was markedly induced after exposure to TNF-alpha for 3 h. IL-6 and IL-8 showed no effect on ICAM-1 mRNA accumulation. Adhesion of rat neutrophils to myocytes was stimulated by TNF-alpha, and the effect of TNF-alpha on adherence was significantly inhibited by an anti-ICAM-1 monoclonal antibody (MoAb). These results show that TNF-alpha, but not IL-6 and IL-8, promotes neutrophil-myocyte adhesion through ICAM-1 expression, suggesting involvement of TNF-alpha in inflammation associated with ischemia-reperfusion injury.

Endothelin-1 (ET-1) is a vasoconstrictive peptide released by ischemic/injured endothelium which increases intracellular ionized calcium [Ca2+]i in vascular smooth muscle. Previous work from this lab has shown that ET-1 also increases human peripheral blood monocyte [Ca2+]i, and that 24 h incubation of monocytes with 10(-9) M ET-1 causes production of prostaglandin E2 and interleukin-6. In these studies, ET-1-stimulated monocyte supernatants were evaluated for their effect on neutrophil superoxide production. While ET-1 alone had no direct effect, incubation of neutrophils for 20 min in ET-1-stimulated monocyte supernatants resulted in a 10-fold increase in superoxide production over basal levels, 44% as much superoxide production as induced by peptide N-formyl-methionyl-leucyl-phenylalanine (N = 6, p < .001). Monocyte supernatants were analyzed for interleukin-8 (IL-8 or neutrophil activation protein) content by radioimmunoassay. ET-1-stimulation resulted in production of 54% as much IL-8 as lipopolysaccharide controls (N = 6, p < .001). While a number of monokines can activate neutrophils, IL-8 has been shown to be a potent neutrophil activator as well as a superoxide primer. Therefore, ET-1-treated monocytes probably upregulate neutrophil superoxide production via a mechanism which includes IL-8.

Interventions that inhibit neutrophil infiltration into myocardial tissue after ischaemia and reperfusion are reported to reduce the size of the infarct. We examined whether administration of trimetazidine, which is reported to reduce myocardial infarct size, affects this process. [111In]Neutrophils and [125I]albumin were administered intravenously (i.v.) to anaesthetized rabbits to allow measurement of cell accumulation and changes in microvascular plasma protein leakage. A 30-min period of coronary artery occlusion followed by 3-h reperfusion was used, and the area at risk (AR) myocardium was defined by dye exclusion. Twelve rabbits received 2.5 mg/kg trimetazidine i.v., 10 min before coronary artery occlusion; the 13 controls received saline. In the control group, the number of [111In]neutrophils/g tissue in the AR (30,591 +/- 6,725) was significantly greater than in the normal zone (NZ, 11,519 +/- 1,605, p < 0.01). In the trimetazidine-treated group, the number of [111In]neutrophils in the AR was significantly lower than in the control group (12,717 +/- 1,958 [111In]neutrophils/g, p < 0.01). There was no significant difference in neutrophil content of the NZ (7,832 +/- 1,117 [111In]neutrophils/g) in treated animals as compared with that in control. Accumulation of [111In]neutrophils in response to intradermal administration of leukotriene B4, interleukin-8 (IL-8), or zymosan-activated plasma was not affected by the drug. The effect of trimetazidine on neutrophil accumulation into post-ischaemic reperfused myocardium therefore does not appear to result from a direct action on the neutrophil.

The purpose of this study was to assess the influence of thermal injury and the inflammatory process on chemotactic responses of neutrophils to four attractants (N-formyl-methionyl-leucyl-phenylalanine, the complement fragment C5a, interleukin-8, and leukotriene B4) under agarose, expression of Mac-1 (CD11b/CD18) adherence receptors on the cell surface, and polymerization of actin in the cell cytoplasm. Circulating neutrophils were isolated from peripheral blood, and exudate neutrophils from fluid collecting under two different wound dressings applied to abrasion sites of healthy subjects and to skin graft donor sites of patients with burns. Burn injury reduced the chemotactic responses of circulating neutrophils to all four attractants, suggesting a "global" defect in chemotactic function. Patient-exudate neutrophils collected under Tegaderm exhibited further decrements in all chemotactic responses, and patient-exudate neutrophils collected under Biobrane were nonmotile. The exudate neutrophils collected under Biobrane expressed high levels of Mac-1 receptors and irreversibly polymerized actin, which may contribute to the nonmotility of these exudate cells.

Previous studies of myocardial ischemia suggest that complement activation may play a central role in the inflammatory response during reperfusion. Our previous work has demonstrated neutrophil chemotactic activity to be present in reperfusion canine cardiac lymph after myocardial ischemia and infarction. To evaluate the contribution of the complement-dependent anaphylatoxin C5a to this neutrophil chemotactic activity, rabbit antiserum to canine C5a was prepared. At dilutions > 1:500 but < 1:2,000, the antiserum abolished the ability of zymosan-activated dog serum to induce a ruffled, bipolar morphology in isolated neutrophils used as a bioassay of chemotactic stimulation. This antiserum did not affect similar morphological changes in neutrophils exposed to platelet activating factor (10(-7)-10(-6) M) or recombinant human interleukin-8 (10(-9)-10(-8) M); thus, we deemed it functionally specific for canine C5a. In a pattern similar to what we previously reported, cardiac lymph collected before a 1-hour ligation of the left circumflex coronary artery had little ability to alter the morphology of canine neutrophils (shape change index, 11.3 +/- 4.6, mean +/- SEM; n = 7), but by 1 hour of reperfusion, lymph activated neutrophils significantly in five of seven dogs (mean shape change index, 72.6 +/- 17.7; p < 0.01). At 2 hours of reperfusion, neutrophil activation by lymph occurred in six of seven dogs (mean shape change index, 103.1 +/- 22.2). At 3 hours of reperfusion, cardiac lymph of only three of six dogs caused neutrophil activation, and at 4 hours of reperfusion, this activity was evident in lymph from only two of five dogs.(ABSTRACT TRUNCATED AT 250 WORDS)