C1q augments platelet activation in response to aggregated Ig

Immune complexes and aggregated IgG (agg-IgG) induce platelet aggregation and the release reaction. Immune complexes also activate the complement system and interact with the complement component C1q. Since platelets possess both Fc and C1q receptors capable of signal transduction, the present study focused on the interaction between these binding sites and platelet activation. Subaggregating doses of agg-IgG (20-400 microg/ml) were identified for washed platelets from each of 11 healthy donors, and platelet aggregation was monitored in the presence or the absence of increasing concentrations of C1q (5-100 microg/ml). C1q produced a dose-dependent potentiation of platelet alphaIIb/beta3 integrin activation, platelet aggregation, and granule secretion when combined with low doses of agg-IgG. C1q alone was without effect. Maximal enhancement of agg-IgG-induced platelet activation was noted at C1q concentrations ranging from 50 to 100 microg/ml. The observed C1q-induced potentiation of platelet aggregation in response to agg-IgG was blocked by polyclonal antibody F(ab')2 directed against platelet binding sites recognizing the collagen-like domain of C1q (cC1qR) or by mAb Fab (IV.3) directed against platelet FcgammaRII receptors. These data suggest a cooperative interaction between platelet FcgammaRII and cC1q receptors and support a potential role for platelet cC1q receptors in pathologic platelet activation by circulating immune complexes often associated with in vivo thrombosis and thrombocytopenia.     

Histochemical study on rat tear film and ocular surface epithelial cells

Fibroblasts of healthy and granulation gingiva are phenotypically heterogeneous with regard to binding C1q collagen-like (cC1qR) or C1q globular-heads (gC1qR) regions, respectively. Here, isolated fibroblast subsets, expressing either the cC1qR or the gC1qR phenotype, were stimulated with C1q, and assessed for changes in cytosolic free calcium [Ca2+]i, accumulation of inositol trisphosphate (IP3), and redistribution of Ca2+-dependent protein kinases-C (cPKCs) from cytosol to membranes. Changes in [Ca2+]i were determined using Indo-1 fluorescence in combination with adhering cell analysis and sorting (ACAS) cytometry. Accumulation of IP3 was quantified using a competitive radioreceptor binding assay. Redistribution of cPKCs was evaluated by immunoblotting with antibodies to PKCalpha/betaI-betaII/gamma. Subsets manifested different fluctuations in [Ca2+]i levels 20 seconds after C1q-stimulation in the presence of millimolar concentrations of external calcium. Whereas cC1qR fibroblasts responded with a 38% over baseline [Ca2+]i increase which was sustained for 20 to 30 minutes, gC1qR fibroblasts responded with a higher (264% over baseline) and more rapid (2 to 3 minutes) transient. Likewise, subsets exhibited different kinetics of IP3 accumulation. Whereas cC1qR fibroblasts responded with an IP3 increase of 32 +/- 3 pmol/10(4) cells over baseline after 5 seconds stimulation, gC1qR fibroblasts responded after 15 to 20 seconds with a lower increase (13 +/- 0.8 IP3 pmol/10(4) cells over baseline). Subsets differed in cPKCs redistribution which peaked in gC1qR-membranes 30 seconds after stimulation and remained sustained between 10 and 30 minutes. No cPKC redistribution was detectable in stimulated cC1qR-cells. We conclude that fibroblasts are heterogeneous in phosphoinositide-Ca2+ signaling and cPKC redistribution to C1q, and suggest that these differences may affect activities of normal and granulation gingiva.     

Borrelia burgdorferi and interleukin-1 promote the transendothelial migration of monocytes in vitro by different mechanisms

A prominent feature of Lyme disease is the perivascular accumulation of mononuclear leukocytes. Incubation of human umbilical vein endothelial cells (HUVEC) cultured on amniotic tissue with either interleukin-1 (IL-1) or Borrelia burgdorferi, the spirochetal agent of Lyme disease, increased the rate at which human monocytes migrated across the endothelial monolayers. Very late antigen 4 (VLA-4) and CD11/CD18 integrins mediated migration of monocytes across HUVEC exposed to either B. burgdorferi or IL-1 in similar manners. Neutralizing antibodies to the chemokine monocyte chemoattractant protein 1 (MCP-1) inhibited the migration of monocytes across unstimulated, IL-1-treated, or B. burgdorferi-stimulated HUVEC by 91% +/- 3%, 65% +/- 2%, or 25% +/- 22%, respectively. Stimulation of HUVEC with B. burgdorferi also promoted a 6-fold +/- 2-fold increase in the migration of human CD4(+) T lymphocytes. Although MCP-1 played only a limited role in the migration of monocytes across B. burgdorferi-treated HUVEC, migration of CD4(+) T lymphocytes across HUVEC exposed to spirochetes was highly dependent on this chemokine. The anti-inflammatory cytokine IL-10 reduced both migration of monocytes and endothelial production of MCP-1 in response to B. burgdorferi by approximately 50%, yet IL-10 inhibited neither migration nor secretion of MCP-1 when HUVEC were stimulated with IL-1. Our results suggest that activation of endothelium by B. burgdorferi may contribute to formation of the chronic inflammatory infiltrates associated with Lyme disease. The transendothelial migration of monocytes that is induced by B. burgdorferi is significantly less dependent on MCP-1 than is migration induced by IL-1. Selective inhibition by IL-10 further indicates that B. burgdorferi and IL-1 employ distinct mechanisms to activate endothelial cells.     

Complement-induced expression of chemokine genes in endothelium: regulation by IL-1-dependent and -independent mechanisms

Activation of complement in the vicinity of endothelium is thought to contribute to the tissue manifestations of inflammatory and immune responses. Endothelial cells contribute to these processes in part by the elaboration of chemokines that activate various leukocytes and direct their migration into tissues. We investigated the mechanisms by which activation of complement on endothelial cell surfaces might influence the expression of chemokine genes in endothelial cells. In a model for the immune reaction occurring in a xenograft, human serum, as a source of xenoreactive anti-endothelial Abs and complement, induced expression of the monocyte chemotactic protein-1 (MCP-1), IL-8, and RANTES genes. The MCP-1 and IL-8 genes were expressed within 3 h as a first phase and at > 12 h as a second phase. The RANTES gene was expressed in porcine endothelial cells only 12 h after exposure to human serum. The expression of these genes required activation of complement and assembly of membrane attack complex, as it was inhibited by soluble CR1 and did not occur in the absence of C8. The early phase of MCP-1 and IL-8 gene expression did not require de novo protein synthesis. The late phase of MCP-1, IL-8, and RANTES gene expression predominantly required the production of IL-1alpha as an intermediate step. The results indicate that the expression of chemokine genes in endothelial cells occurs as a function of differential responses to complement and may in part be conditioned by the availability of IL-1alpha.     

Cytokine secretion by human fetal membranes, decidua and placenta at term

Cytokines such as monocyte chemotactic peptide-1 (MCP-1), interleukin-8 (IL-8), RANTES (Regulated on Activation and Normally T-cells Expressed and presumably Secreted) and interleukin-10 (IL-10) are thought to play pivotal roles in immune recognition, acceptance of the fetal allograft, maintenance of pregnancy and parturition. Their secretion and regulation within the third trimester uterus is, however, less well defined. We therefore investigated the release of these cytokines by third trimester amnion, chorion, placenta and decidua, and studied the influence of prostaglandin E2 (PGE2) infusion on their release in a dynamic placental cotyledon perfusion system. MCP-1 was released predominately by the chorion (78.2 +/- 7.3 pg/mg wet tissue weight; mean +/- SEM), decidua (112.4 +/- 5.2 pg/mg) and placenta (101.8 +/- 5.0 pg/mg) with low amounts from the amnion (1.3 +/- 0.4 pg/mg). High concentrations of IL-8 were released by the amnion (39.9 +/- 5.3 pg/mg), chorion (52.8 +/- 1.9 pg/mg), decidua (42.2 +/- 1.5 pg/mg) and placenta (45 +/- 1.3 pg/mg). Release of RANTES was not detectable from the amnion but was detected in moderate amounts from the chorion (6.0 +/- 1.2 pg/mg), decidua (15.2 +/- 1.4 pg/mg) and placenta (26.9 +/- 1.6 pg/mg). Low concentrations of IL-10 were secreted by the chorion (6.8 +/- 0.8 pg/mg), decidua (9.0 +/- 0.9 pg/mg) and placenta (3.3 +/- 0.3 pg/mg) with none detectable from the amnion. MCP-1, IL-8, RANTES and IL-10 were all released by perfused placental cotyledons. PGE2 stimulated release of MCP-1, IL-8 and IL-10 into the maternal and of MCP-1 and IL-8 into the fetal circulation of the placenta but had no effect on RANTES release. It is suggested that MCP-1 and IL-8 may be involved in the inflammatory process of parturition and IL-10 in the protection of the fetal allograft. In addition, PGE2 may have an important immunomodulatory role within the uterus at term.     

Standardization of a questionnaire for neurotoxic symptoms

C1q receptor (C1qR/collectin receptor/cC1qR) has an almost complete amino acid sequence identity with calreticulin (CRT). C1qR/CRT is located on the surface of many cell types. Binding of C1q to C1q receptor elicits a range of immunological responses. C1qR also interacts with the collectins SP-A, MBL, CL43 and conglutinin via a cluster of charged residues on the collagen tails of the ligands. In order to localise C1q and collectin binding activity within C1qR/CRT, recombinant C1qR/CRT domains [N (residues 18-196), P (197-308) and C (309-417)] were produced. Both the N- and P-domains bound to C1q, demonstrating that the binding site spans the intersection of these domains. Amino acid alignment analysis identified a putative CUB module within this region. This S-domain (residues 160-283) was expressed and showed concentration-dependent binding to immobilised C1q, demonstrating that it contains the C1q binding site. Competitive inhibition studies of the S-domain-C1q interaction revealed that the S-domain binds to C1q collagen tails and to the collectin proteins, SP-A, MBL, CL43 and conglutinin. The C1q and collection binding site on C1qR/CRT has therefore been localised to the S-domain.     

Relationships between plasma levels of type-II phospholipase A2, PAF-acetylhydrolase, leukotriene B4, complements, endothelin-1, and thrombomodulin in patients with sepsis

We determined the plasma levels of type-II phospholipase A2 (type II PLA2), platelet-activating factor acetylhydrolase (PAFAH) leukotriene B4 (LTB4) and of several complements (C3a, C4a, and C5a), which are considered to be among the cytokines and eicosanoids involved in vascular endothelial disorders and that vary in concentration during sepsis. We investigated the relationship between those levels and those of ET-1 and TM levels in plasma. Plasma levels of type II PLA2, PAFAH, LTB4, C3a, C4a, ET-1, and TM at the time that sepsis was diagnosed in 30 patients were 218.3 +/- 179.9 ng/ml, 23.92 +/- 9.66 nmol/min/ml, 90.35 +/- 31.49 pg/ml, 838.73 +/- 2.30 pg/ml, 1951.46 +/- 1697.78 pg/ml, 6.98 +/- 4.08 pg/ml and 7.80 +/- 3.34 ng/ml, respectively. The C5a plasma level was below the limit of detection in all cases. There were significant correlations between type II PLA2 and ET-1 plasma levels (r = 0.39, p = 0.032) and C3a and ET-1 plasma levels (r = 0.60, p = 0.03). There were also significant correlations between type II PLA2 and TM levels in plasma (r = 0.76, p = 0.0017), PAFAH and TM plasma levels (r = 0.53, p = 0.037), LTB4 and TM plasma levels (r = 0.46, p = 0.016) and C4a and TM plasma levels (r = 0.58, p = 0.037). Results suggest that the elevation of type II PLA2, PAFAH, LTB4 and complement in plasma is involved in vascular endothelial disorders in patients with sepsis.     

The multiligand-binding protein gC1qR, putative C1q receptor, is a mitochondrial protein

A protein of 33 kDa (p33) that tightly binds to the globular domains of the first complement component, C1q, is thought to serve as the major C1q receptor (gC1qR) on B cells, neutrophils, and mast cells. However, the cellular routing and the subcellular localization of p33/gC1qR are unknown. We have performed confocal laser-scanning microscopy and found that p33/gC1qR is present in intracellular compartments, where it colocalizes with the mitochondrial marker protein, pyruvate dehydrogenase. No surface staining for p33/gC1qR on endothelial EA.hy926 cells was observed. A fusion protein of the p33/gC1qR presequence with green fluorescent protein translocated to the mitochondria of transfected COS-7 cells. Concomitantly, a 6-kDa portion of the fusion protein was proteolytically removed. The 33 amino-terminal residues of the presequence proved sufficient to direct reporter constructs to mitochondria. Association of p33/gC1qR with mitoplasts indicated that the mature protein of 209 residues resides in the matrix and/or the inner membrane of mitochondria. Immunocytochemistry of fetal mice tissues revealed a ubiquitous expression of p33/gC1qR, most prominently in tissues that are rich in mitochondria. Thus, the candidate complement receptor p33/gC1qR of intact cells cannot interact with plasma C1q due to mutually exclusive localizations of the components. The functional role of p33/gC1qR needs to be reconsidered.     

Reduced production of immunoreactive interleukin-1 by peripheral blood monocytes of patients with acute and chronic viral hepatitis

The in vitro production of interleukin-1 beta by peripheral blood monocytes derived from patients with various liver diseases was studied. An impaired production of immunoreactive interleukin-1 (IL-1) (mean +/- SEM) by monocytes stimulated with an optimal dose (100 ng/ml) of lipopolysaccharide was observed in patients with chronic hepatitis B (N = 13; 32 +/- 6 pg/ml) or chronic hepatitis C (N = 13; 61 +/- 12 pg/ml) as compared to those of healthy control individuals (N = 35; 166 +/- 24 pg/ml; P = 0.0003 and P = 0.015, respectively), whereas an unaltered IL-1 production was seen in patients with alcoholic cirrhosis (N = 23; 125 +/- 28 pg/ml) and primary biliary cirrhosis (N = 6; 111 +/- 33 pg/ml). Similar to the situation seen in chronic viral hepatitis, lipopolysaccharide-stimulated monocytes from patients with acute hepatitis also showed a decreased IL-1 production in the first week after onset of jaundice (N = 17; 55 +/- 20 pg/ml; P = 0.001) and a return to normal in the second and third week. An impaired production of IL-1 in chronic as well as acute viral hepatitis is a further example of the known disturbed immunoregulation in this disease.     

The role of oxidative stress in rhinovirus induced elaboration of IL-8 by respiratory epithelial cells

A direct correlation has been reported between the severity of symptoms associated with rhinovirus infection and the concentration of interleukin-8 in nasal secretions. The purpose of these studies was to examine the mechanism of rhinovirus-induced IL-8 elaboration. Rhinovirus infection induced oxidative stress in Beas-2b cells and the concentration of H2O2 in supernatant media from rhinovirus challenged cells was 12.5 +/- 6.1 microM 1 h after challenge compared to 0.7 +/- 0.3 microM in supernatant from control cells. N-acetyl cysteine inhibited RV-induced NF-kappaB activation and IL-8 elaboration. IL-8 concentrations were 36 +/- 2 pg/ml and 10 +/- 1 pg/ml 6 h after virus challenge in untreated and NAC-treated (30 mM NAC) cells, respectively. Despite the effects of NAC on IL-8 elaboration and NF-kappaB activation, RV stimulated increases in supernatant H2O2 were not altered by NAC. These data suggest that RV stimulation of IL-8 in respiratory epithelium is mediated through production of oxidative species and the subsequent activation of NF-kappaB.     

Diminished production of interleukin-6 in chronic lymphocytic leukaemia (B-CLL) cells from patients at advanced stages of disease. Tampere CLL Group

The production of the cytokines interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) in B-CLL cells from 24 patients at different stages of chronic lymphocytic B-cell leukaemia (B-CLL) was investigated in vitro. In the majority of these cases, low spontaneous IL-6 production was measured. Mitogenic stimulation with phorbol 12-myristate 13-acetate (PMA) or PMA plus interleukin-2 (IL-2) resulted in a tremendous increase in TNF-alpha and IL-6 production in cells representing early stage (Binet A) disease. In contrast, very little, if any, production took place in cells from patients with advanced stage (Binet C) B-CLL. The results from stage B patients were intermediate. The most remarkable difference was recorded in PMA-stimulated (1 ng/ml) IL-6 production. In stimulated 72 h cultures, IL-6 concentrations were 1280 +/- 1080 pg/ml for Binet A (n = 11), 757 +/- 597 pg/ml for Binet B (n = 8) and 46.0 +/- 84.0 pg/ml for Binet C (n = 5). The differences in IL-6 production between stage C v B and stage C v A were both statistically significant (P=0.025). Similar effects, but to a lesser extent, were observed in TNF-alpha production. These results suggest that the varying capacity to produce IL-6 and TNF-alpha may play a role in B-CLL progression and in clinical manifestations of the disease.     

Polymorphonuclear leukocytes induce PDGF release from IL-1beta-treated endothelial cells: role of adhesion molecules and serine proteases

Polymorphonuclear leukocytes (PMNs) and endothelial cells interact at sites of vascular injury during inflammatory response and during the development of atherosclerotic lesions. Such close proximity leads to the modulation of several of the biological functions of the 2 cell types. Because we have shown previously that PMNs enhance release of growth factors from resting endothelial cells, we decided to evaluate whether coincubation of PMNs with interleukin-1beta (IL-1beta)-stimulated human umbilical vein endothelial cells (HUVEC) could further modulate mitogen release from HUVEC. We found that PMN-HUVEC coincubation resulted in a 10-fold increase in mitogen release, compared with HUVEC alone (14+/-6 versus 1.3+/-0.1). When PMNs were incubated with IL-1beta-treated HUVEC, a further increase in mitogen release (up to 35-fold) was observed. The mitogenic activity was immunologically related to platelet-derived growth factor (PDGF) because the activity was abolished by an anti-PDGF antibody. PDGF-AB antigen, detected in low concentrations in conditioned medium from HUVEC alone, was increased 4-fold when IL-1beta or PMNs were incubated with HUVEC and dramatically upregulated (up to 40-fold) when PMNs were cocultured with IL-1beta-treated HUVEC. The presence of the protease inhibitor eglin C abolished mitogenic activity generation, suggesting a role for PMN-derived elastase and cathepsin G. Indeed, purified elastase and cathepsin G mimicked PMN-induced mitogen release from HUVEC. Because PMNs firmly adhered to IL-1beta-treated HUVEC, we investigated the role of cell-cell adhesion in mitogen release. Adhesion and PDGF release were inhibited by approximately 60% in the presence of anti-CD11a/CD18 and anti-intercellular adhesion molecule-1 monoclonal antibodies. This study suggests a new role for PMNs and their interaction with endothelium in pathological conditions in which intimal hyperplasia is a common feature.     

Production and regulation of monocyte chemoattractant protein-1 in lipopolysaccharide- or monosodium urate crystal-induced arthritis in rabbits: roles of tumor necrosis factor alpha, interleukin-1, and interleukin-8

The production of monocyte chemoattractant protein-1 (MCP-1) and its regulation by TNFalpha, IL-1, and IL-8 were investigated in two rabbit models of arthritis induced by intra-articular injection of lipopolysaccharide (LPS) or monosodium urate (MSU) crystals. We first prepared recombinant rabbit MCP-1 and antibodies and then developed an immunoassay. The immunoassay detected 3 pg/ml rabbit MCP-1 and did not cross-react with other rabbit chemokines such as IL-8 or GRO. MCP-1 was first detected in synovial fluid (SF) at 1 hour, and peaked at 4 or 2 hours after the injection of LPS or MSU crystals, respectively. Immunohistochemically, MCP-1 was detected in synovial lining cells and infiltrating neutrophils. The amounts of MCP-1 detected in SF from neutrophil-depleted rabbits were similar to those in normal rabbits, suggesting that synovial lining cells were the main source of MCP-1 detected in SF. The peak level of MCP-1 in SF after LPS-injection was inhibited by 57% with anti-TNFalpha mAb and by 41% with IL-1 receptor antagonist (IL-1Ra). Coadministration of anti-TNFalpha mAb and IL-1Ra inhibited 90% of MCP-1 production. In contrast, the peak level of MCP-1 in SF after MSU crystal-injection was not affected by any cytokine inhibitor, but was reduced by 52% with coadministration of anti-TNFalpha mAb and IL-1Ra. Anti-IL-8 IgG had no effect on the production of MCP-1 in either model. Thus, the production of MCP-1 in LPS-induced arthritis was mostly regulated by TNFalpha and IL-1, whereas half the extent of MCP-1 production in MSU crystal-induced arthritis was independent of TNFalpha or IL-1. IL-8 does not seem to regulate the production of MCP-1 in SF either directly or indirectly. Finally, administration of neutralizing anti-MCP-1 antibody inhibited LPS- and MSU crystal-induced monocyte infiltration by 58.4% and 44.9%, respectively, suggesting that synovial production of MCP-1 plays an important role in the recruitment of monocytes in these arthritis models.     

Bronchoalveolar lavage neutrophilia is associated with obliterative bronchiolitis after lung transplantation: role of IL-8

Obliterative bronchiolitis (OB) is a devastating complication in lung transplantation. We postulated that the pathogenesis of OB is mediated, in part, by neutrophils. We serially collected bronchoalveolar lavage (BAL) fluid from lung transplant recipients. Patients were divided into two groups depending on the presence or absence of OB. Samples from patients who never developed OB were further divided according to whether rejection was present. These samples were labeled healthy or rejection. Samples from patients who developed OB were divided according to whether the sample was obtained before (future OB) or at the time of diagnosis of OB (OB). The OB group, as compared with the healthy and rejection group, had significantly elevated neutrophil counts (3.9 x 10(5) +/- 1.8 x 10(5) vs 0.3 x 10(5) +/- 0.07 x 10(5) and 0.4 x 10(5) +/- 0.1 x 10(5), respectively, p < 0.01 for both) and levels of IL-8 (3131 +/- 1468 pg/ml vs 240 +/- 62 pg/ml and 172 +/- 47 pg/ml, p < 0.01 for both). Furthermore, we demonstrated immunolocalization of IL-8 associated with alpha smooth muscle actin-positive cells in the peribronchial region of OB. To confirm that the IL-8 present in BAL fluid from patients with OB was bioactive, we performed neutrophil chemotaxis experiments that showed that IL-8 accounted for a significant amount of the neutrophil chemotactic activity. We also found a trend toward higher levels of neutrophils and IL-8 in BALs from the future OB as compared with the healthy group (7.1 x 10(4) +/- 4.2 x 10(4) vs 3.4 x 10(4) +/- 0.7 x 10(4) and 500 +/- 306 pg/ml vs 240 +/- 62 pg/ml). In conclusion, we have provided the novel observation that in lung transplant recipients with OB, neutrophilia is present and highly correlated with the presence of IL-8.     

Clinical study of anaerobic respiratory infection

Interleukin-2 (IL-2) therapy is dose limited by a severe vascular leak with resulting systemic and pulmonary toxicity. Although recognized as a mediator of septic shock and vascular leak, the relative role of IL-1 in IL-2 toxicity is unclear. We evaluated the effect of IL-1 receptor antagonist (IL-1ra) on IL-2 lethality, pulmonary vascular leak, and treatment of pulmonary metastases in a murine model. In vivo induction of mRNA for IL-1 alpha was evaluated in liver by Northern blots after 0, 5, 8, and 11 doses of IL-2 in C3H/HEN mice. The expression index for the IL-1 alpha gene increased from 0.16 to 0.74 after 5 doses of IL-2, and further increased to 1.04 after 11 doses of IL-2. C3H/HEN mice (n = 56) were randomized to receive phosphate-buffered saline (PBS), IL-1ra high dose (HD), or IL-1ra low dose (LD) by continuous subcutaneous infusion via Alzet mini-pumps. The biologic effectiveness of the dose and administration of IL-1ra was determined by the ability to block IL-1-induced IL-6 production in vivo. Mean serum IL-6 levels 3 hr after intraperitoneal IL-1 alpha (10 micrograms/kg) were: PBS, 3730 +/- 526 (mean +/- SEM pg/ml); IL-1ra (LD), 1156 +/- 398; and IL-1ra (HD), 594 +/- 30 (P < 0.01, IL-1ra HD or LD vs PBS). Pulmonary vascular leak was measured by iv I125 albumin after 8 doses of IL-2 (100,000 U ip q 8 hr).(ABSTRACT TRUNCATED AT 250 WORDS)     

The up-regulation of IL-6 and IL-8 in human endothelial cells by activated protein C

The protein C/protein S anticoagulant pathway has been proposed to be a common link between coagulation and inflammation. Studies have suggested that a component of the anticoagulant pathway, activated protein C (APC), may play a role in the inflammatory response by modulating the effects of cytokines such as TNF and by blocking neutrophil activation. Cytokines are known to be intimately involved in the inflammatory response and to function in part to restore hemostatic balance. To begin to delineate what role APC may have in the inflammatory response, we have investigated the effect of APC on the production of the proinflammatory cytokines IL-6 and IL-8 in primary HUVEC, human microvascular endothelial cells, and human coronary artery endothelial cells. Our results have demonstrated that physiologic concentrations of APC significantly up-regulated the production of both IL-6 and IL-8. This increase, which was seen at both the RNA and protein level, was not due to either thrombin or LPS contamination of the APC preparation. Additional studies also showed that the APC-mediated up-regulation of IL-6 and IL-8 was IL-1 independent. Although neither purified protein C nor protein S alone had an effect on cytokine production, protein S, the cofactor for APC, significantly enhanced the ability of APC to up-regulate IL-6/IL-8 production. These results provide further evidence for a role for APC in the inflammatory response.