C5a aggravates dysfunction of the articular cartilage and synovial fluid in rats with knee joint immobilization.

Degenerative alterations in articular cartilage are involved in the pathogenesis of osteoarthritis. The present study aimed to evaluate the role of complement component 5a (C5a) in osteoarthritic alterations in the articular cartilage and synovialis via a joint immobilization (IM) rat model. Rats were assigned to three groups: Control, IM and IM+anti­C5a antibody (IM+anti­C5a) groups. A terminal deoxynucleotidyl transferase dUTP nick end labeling assay and hematoxylin and eosin staining were used to evaluate the morphological alterations in the articular cartilage and synovialis. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis, immunohistochemical analysis and western blotting were used to evaluate C5a expression in the articular cartilage and synovialis. An ELISA was used to evaluate C5a­induced alterations in interleukin (IL)­1ß, IL­17A and tumor necrosis factor (TNF)­α levels in the serum and joint fluid. The results demonstrated that knee joint immobilization induced destruction of knee joint synovial fluid and cartilage in the IM and IM+anti­C5a antibody groups. Immobilization significantly increased the expression levels of C5a in serum and joint fluid in the IM group. Immunohistochemistry, western blotting and RT­qPCR analysis illustrated markedly increased expression of C5a in the IM group. Immobilization markedly increased the IL­1ß, IL­17A and TNF­α expression levels in the serum and joint fluid in the IM group. Anti­C5a was able to decrease immobilization­induced alterations in morphology and cytokines compared with the IM group. The expression of C5a was increased in synoviocytes and joint cartilage in the IM model. Pro­inflammatory cytokines, including TNF­α and IL­1ß were released in the activated synoviocytes via the induction of C5a, suggesting that C5a serves an important role in joint inflammatory processes.

Complement drives glucosylceramide accumulation and tissue inflammation in Gaucher disease.

Gaucher disease is caused by mutations in GBA1, which encodes the lysosomal enzyme glucocerebrosidase (GCase). GBA1 mutations drive extensive accumulation of glucosylceramide (GC) in multiple innate and adaptive immune cells in the spleen, liver, lung and bone marrow, often leading to chronic inflammation. The mechanisms that connect excess GC to tissue inflammation remain unknown. Here we show that activation of complement C5a and C5a receptor 1 (C5aR1) controls GC accumulation and the inflammatory response in experimental and clinical Gaucher disease. Marked local and systemic complement activation occurred in GCase-deficient mice or after pharmacological inhibition of GCase and was associated with GC storage, tissue inflammation and proinflammatory cytokine production. Whereas all GCase-inhibited mice died within 4-5 weeks, mice deficient in both GCase and C5aR1, and wild-type mice in which GCase and C5aR were pharmacologically inhibited, were protected from these adverse effects and consequently survived. In mice and humans, GCase deficiency was associated with strong formation of complement-activating GC-specific IgG autoantibodies, leading to complement activation and C5a generation. Subsequent C5aR1 activation controlled UDP-glucose ceramide glucosyltransferase production, thereby tipping the balance between GC formation and degradation. Thus, extensive GC storage induces complement-activating IgG autoantibodies that drive a pathway of C5a generation and C5aR1 activation that fuels a cycle of cellular GC accumulation, innate and adaptive immune cell recruitment and activation in Gaucher disease. As enzyme replacement and substrate reduction therapies are expensive and still associated with inflammation, increased risk of cancer and Parkinson disease, targeting C5aR1 may serve as a treatment option for patients with Gaucher disease and, possibly, other lysosomal storage diseases.

Down-regulation of complement receptors on the surface of host monocyte even as in vitro complement pathway blocking interferes in dengue infection.

In dengue virus (DENV) infection, complement system (CS) activation appears to have protective and pathogenic effects. In severe dengue fever (DF), the levels of DENV non-structural-1 protein and of the products of complement activation, including C3a, C5a and SC5b-9, are higher before vascular leakage occurs, supporting the hypothesis that complement activation contributes to unfavourable outcomes. The clinical manifestations of DF range from asymptomatic to severe and even fatal. Here, we aimed to characterise CS by their receptors or activation product, in vivo in DF patients and in vitro by DENV-2 stimulation on monocytes. In comparison with healthy controls, DF patients showed lower expression of CR3 (CD11b), CR4 (CD11c) and, CD59 on monocytes. The DF patients who were high producers of SC5b-9 were also those that showed more pronounced bleeding or vascular leakage. Those findings encouraged us to investigate the role of CS in vitro, using monocytes isolated from healthy subjects. Prior blocking with CR3 alone (CD11b) or CR3 (CD11b/CD18) reduced viral infection, as quantified by the levels of intracellular viral antigen expression and soluble DENV non-structural viral protein. However, we found that CR3 alone (CD11b) or CR3 (CD11b/CD18) blocking did not influence major histocompatibility complex presentation neither active caspase-1 on monocytes, thus probably ruling out inflammasome-related mechanisms. Although it did impair the secretion of tumour necrosis factor alpha and interferon alpha. Our data provide strategies of blocking CR3 (CD11b) pathways could have implications for the treatment of viral infection by antiviral-related mechanisms.

Aluminum hydroxide adjuvant differentially activates the three complement pathways with major involvement of the alternative pathway.

Al(OH)3 is the most common adjuvant in human vaccines, but its mode of action remains poorly understood. Complement involvement in the adjuvant properties of Al(OH)3 has been suggested in several reports together with a depot effect. It is here confirmed that Al(OH)3 treatment of serum depletes complement components and activates the complement system. We show that complement activation by Al(OH)3 involves the three major pathways by monitoring complement components in Al(OH)3-treated serum and in Al(OH)3-containing precipitates. Al(OH)3 activation of complement results in deposition of C3 cleavage products and membrane attack complex (MAC) and in generation of the anaphylatoxins C3a and C5a. Complement activation was time dependent and inhibited by chelation with EDTA but not EGTA+Mg(2+). We thus confirm that Al(OH)3 activates the complement system and show that the alternative pathway is of major importance.

Anaphylatoxin C5a creates a favorable microenvironment for lung cancer progression.

The complement system contributes to various immune and inflammatory diseases, including cancer. In this study, we investigated the capacity of lung cancer cells to activate complement and characterized the consequences of complement activation on tumor progression. We focused our study on the production and role of the anaphylatoxin C5a, a potent immune mediator generated after complement activation. We first measured the capacity of lung cancer cell lines to deposit C5 and release C5a. C5 deposition, after incubation with normal human serum, was higher in lung cancer cell lines than in nonmalignant bronchial epithelial cells. Notably, lung malignant cells produced complement C5a even in the absence of serum. We also found a significant increase of C5a in plasma from patients with non-small cell lung cancer, suggesting that the local production of C5a is followed by its systemic diffusion. The contribution of C5a to lung cancer growth in vivo was evaluated in the Lewis lung cancer model. Syngeneic tumors of 3LL cells grew slower in mice treated with an antagonist of the C5a receptor. C5a did not modify 3LL cell proliferation in vitro but induced endothelial cell chemotaxis and blood-vessels formation. C5a also contributed to the immunosuppressive microenvironment required for tumor growth. In particular, blockade of C5a receptor significantly reduced myeloid-derived suppressor cells and immunomodulators ARG1, CTLA-4, IL-6, IL-10, LAG3, and PDL1 (B7H1). In conclusion, lung cancer cells have the capacity to generate C5a, a molecule that creates a favorable tumor microenvironment for lung cancer progression.

Generation of complement component C5a by ischemic neurons promotes neuronal apoptosis.

C5a receptors are found in the central nervous system (CNS), on both neurons and glia. However, the origin of the C5a, which activates these receptors, is unclear. In the present study, we show that primary cultured mouse cortical neurons constitutively express C5, the precursor of C5a, and express the classical receptor for C5a, CD88. With cell ischemia caused by 12 h glucose deprivation, or oxygen-glucose deprivation (OGD), neurons demonstrated increased apoptosis, up-regulation of CD88, and increased levels of C5a in the media. Exogenous murine C5a (100 nM) added to the neuronal cultures resulted in apoptosis, without affecting cell necrosis. Pretreatment of the cells with the specific CD88 receptor antagonist PMX53 (100 nM) significantly blocked ischemia-induced apoptosis (∼50%), and neurons from CD88(-/-) mice were similarly protected. In a murine model of stroke, using middle cerebral artery occlusion (MCAO), we found that C5a levels in the brain increased; this also occurred in cerebral slice cultures exposed to OGD. CD88(-/-) mice subjected to MCAO had significantly reduced infarct volumes and improved neurological scores. Taken together, our results demonstrate that neurons in the CNS have the capability to generate C5a following ischemic stress, and this has the potential to activate their C5a receptors, with deleterious consequences.

Hemorrhage-induced intestinal damage is complement-independent in Helicobacter hepaticus-infected mice.

With more than half of the world population infected, Helicobacter infection is an important public health issue associated with gastrointestinal cancers and inflammatory bowel disease. Animal studies indicate that complement and oxidative stress play a role in Helicobacter infections. Hemorrhage (HS) induces tissue damage that is attenuated by blockade of either complement activation or oxidative stress products. Therefore, we hypothesized that chronic Helicobacter hepaticus infection would modulate HS-induced intestinal damage and inflammation. To test this hypothesis, we examined HS-induced jejunal damage and inflammation in uninfected and H. hepaticus-infected mice. Helicobacter hepaticus infection increased HS-induced midjejunal mucosal damage despite attenuating complement activation. In addition, infection alone increased chemokine secretion, changing the HS-induced neutrophil infiltration to a macrophage-mediated inflammatory response. The HS-induced macrophage infiltration correlated with increased secretion of tumor necrosis factor-α and nitric oxide in the infected mice. Together, these data indicate that Helicobacter infection modulates the mechanism of HS-induced intestinal damage and inflammation from a complement-mediated response to a macrophage response with elevated tumor necrosis factor-α and nitric oxide. These data indicate that chronic low-level infections change the response to trauma and should be considered when designing and administering therapeutics.

Locally produced C5a binds to T cell-expressed C5aR to enhance effector T-cell expansion by limiting antigen-induced apoptosis.

Our recent studies have shown that immune cell-produced complement provides costimulatory and survival signals to naive CD4(+) T cells. Whether these signals are similarly required during effector cell expansion and what molecular pathways link locally produced complement to T-cell survival were not clarified. To address this, we stimulated monoclonal and polyclonal T cells in vitro and in vivo with antigen-presenting cells (APCs) deficient in the complement regulatory protein, decay accelerating factor (DAF), and/or the complement component C3. We found that T-cell expansion induced by DAF-deficient APCs was augmented with diminished T-cell apoptosis, whereas T-cell expansion induced by C3(-/-) APCs was reduced because of enhanced T-cell apoptosis. These effects were traced to locally produced C5a, which through binding to T cell-expressed C5aR, enhanced expression of Bcl-2 and prevented Fas up-regulation. The results show that C5aR signal transduction in T cells is important to allow optimal T-cell expansion, as well as to maintain naive cell viability, and does so by suppressing programmed cell death.

Complement C5a anaphylatoxin is an innate determinant of dendritic cell-induced Th1 immunity to Mycobacterium bovis BCG infection in mice.

During acquired immunity to Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection in mice, dendritic cells (DCs) present mycobacterial antigens to naive T cells to prime an immune response. Complement C5a (anaphylatoxin) secreted by mycobacteria-infected macrophages regulates IL-12p70 production. As IL-12p70 regulates Th1 immunity against mycobacteria in mice, we examined the effects of C5a on IL-12p70 secretion by murine DCs and Th1 immunity. DCs cultured from C5-deficient (C5(-/-)) and -sufficient (C5(+/+)) mice were infected with BCG in the presence or absence of the C5a peptide. ELISA showed that C5(-/-) DCs secreted less IL-12p70 (600 pg/mL vs. 100 pg/mL) than C5(+/+) DCs, and they secreted more IL-10. Using immunophenotyping, reduced CD40 expression was found on C5(-/-) DCs after BCG infection. BCG-primed DCs were then cocultured with naive or BCG-immune T cells to differentiate them into IFN-gamma-secreting Th1 T cells. Coincident with increased IL-12p70 levels, BCG-primed C5(+/+) DCs cocultured with naive or immune C5(+/+) T cells showed a larger increase in CD4+ IFN-gamma/CD8+ IFN-gamma+ T cells compared with cocultured DCs and T cells from C5(-/-) mice. Thus, BCG-primed C5(+/+) DCs were better able to drive a Th1 response. Furthermore, BCG aerosol-infected C5(-/-) mice showed reduced CD4 and CD8 IFN-gamma-secreting T cells in the lungs, concurrent with an increased growth of BCG. Thus, C5a, an innate peptide, appears to play an important role in the generation of acquired immune responses in mice by regulating the Th1 response through modulation of IL-12p70 secretion from DCs.

Expression of complement factor H by lung cancer cells: effects on the activation of the alternative pathway of complement.

The complement system is important in immunosurveillance against tumors. However, malignant cells are usually resistant to complement-mediated lysis. In this study, we examine the expression of factor H, an inhibitor of complement activation, and factor H-like protein 1 (FHL-1), its alternatively spliced form, in lung cancer. We also evaluate the potential effect of factor H/FHL-1 in the protection of lung cancer cells against the activation of the complement cascade. By Northern blot analysis we demonstrate a high expression of factor H and FHL-1 in most non-small cell lung cancer cell lines, although neuroendocrine pulmonary tumors (small cell lung carcinoma and carcinoid cell lines) had undetectable levels. Western blot analysis of conditioned medium showed the active secretion of factor H and FHL-1 by cells that were positive by Northern blot. Expression of factor H/FHL-1 mRNA was also shown in a series of non-small cell lung cancer biopsies by in situ hybridization. Interestingly, many cultured lung cancer cells were able to bind fluorescence-labeled factor H to their surfaces. Deposition of C3 fragments from normal human serum on H1264, a lung adenocarcinoma cell line, was more efficient when factor H/FHL-1 activity was blocked by specific antibodies. Blocking factor H/FHL-1 activity also enhanced the release of anaphylatoxin C5a and moderately increased the susceptibility of these cells to complement-mediated cytotoxicity. In summary, we demonstrate the expression of factor H and FHL-1 by some lung cancer cells and analyze the contribution of these proteins to the protection against complement activation.

Neutrophil adhesion molecule expression and serum concentration of soluble adhesion molecules during and after pediatric cardiovascular surgery with or without cardiopulmonary bypass.

BACKGROUND: Increased neutrophil activation by cardiopulmonary bypass (CPB) during cardiovascular surgery is thought to be responsible for postoperative complications. In children, the contribution of cardiovascular surgery alone to this response is not well-characterized. METHODS: Children undergoing surgery with CPB (CPB group, n = 35) and without CPB (control, n = 22) were studied (age, 3-17 yr). Blood was drawn 24 h preoperatively before medication, after anesthesia, after connection to CPB, at reperfusion, 4 h to 2 days after surgery, at discharge, and months after surgery. Neutrophil antigen expression and serum concentration of adhesion molecules, interleukin 8, and C5a (fragment of C5 complement) were analyzed by flow cytometry and enzyme-linked immunosorbent assay, respectively. RESULTS: With and without CPB, anesthesia and surgery induced decreased LFA-1 (CD11a-CD18), Mac-1 (CD11b-CD18), CD45, and CD54 (intercellular adhesion molecule 1) surface expression and sICAM-1 serum concentrations (all P < 0.001). sL-selectin serum concentration decreased with CPB (P < 0.001) but was not significantly altered in the control. In contrast, CD62L expression increased during CPB (P < 0.001). The time course of all analyzed markers was not significantly different between CPB and control, with the exception of sL-selectin (P = 0.017). One-day preoperative baseline values were reached days to months after surgery. Interleukin 8 and C5a serum concentrations increased after surgery in both the CPB group and the control group. CONCLUSIONS: Pediatric cardiovascular surgery leads to reduced adhesiveness and activity of circulating neutrophils. This reduction is more pronounced and sustained with CPB. These data may be useful in the assessment of novel therapeutic strategies.

Concentrations of leucocyte-associated chemotaxin C5a correlate with the mobilisation of polymorphonuclear leucocytes during operations for rectal cancer.

OBJECTIVE: To describe the profile of potential chemotaxins C5a, interleukin 8 and tumour-necrosis factor alpha, and the peripheral blood leucocyte (PBL) response in five patients having uncomplicated operations for rectal cancer. DESIGN: Prospective study. SETTING: University hospital, Norway. SUBJECTS: Five patients, four men and one woman, median age 66 years (range 48-77) who were operated on for rectal cancer. INTERVENTION: Four had low anterior resections and total mesorectal excision (TME) and one patient had a diverting end sigmoidostomy because of local perirectal spread of the cancer and liver metastases. Blood samples were taken at the start of the operation; peroperatively after 1, 2, and 3 hours; postoperatively at 5, 8, and 24 hours, for analysis of potential chemotaxins. RESULTS: The number of PBL tripled between the start and end of the operation and declined to a slightly lower plateau between 5 and 24 hours. Peroperatively, the association of C5a with PBL increased six-fold resulting in a doubled concentration of C5a/PBL, whereas the corresponding concentration of C5a in plasma remained relatively constant. Postoperatively, the concentration of C5a associated with the PBL and in plasma fluctuated with the maximums being at 8 and 24 hours, respectively. In contrast to C5a in plasma, the concentration of cell-associated C5a correlated with number of PBL or polymorphonuclear leucocytes (PMN). The plasma concentration of IL-8 doubled during the operation, reached a six-fold maximum at 5 hours, and declined after 24 hours to twice the initial concentration. There were no correlations between plasma IL-8 and either number of PBL, plasma C5a, or PBL-associated C5a. No TNF alpha was detected in the plasma. CONCLUSION: Tissue trauma caused by uncomplicated excision of rectal cancer leads to leucocyte mobilisation peroperatively, probably partly by complement activation and subsequent generation and binding of the chemotaxin C5a to PBL. However, other chemotaxins may also play a role.

Differential induction of complement fragment C5a and inflammatory cytokines during intramammary infections with Escherichia coli and Staphylococcus aureus.

The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection. Escherichia coli is known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereas Staphylococcus aureus induces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied during S. aureus mastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited by E. coli and S. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and IL-8 in milk after inoculation of E. coli or S. aureus in lactating bovine udders. The low levels of C5a and the absence of cytokines in milk from S. aureus-infected cows, compared to the high levels found in milk from E. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk after S. aureus inoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.

Bothrops asper snake venom and its metalloproteinase BaP-1 activate the complement system. Role in leucocyte recruitment.

The venom of the snake Bothrops asper, the most important poisonous snake in Central America, evokes an inflammatory response, the mechanisms of which are not well characterized. The objectives of this study were to investigate whether B. asper venom and its purified toxins--phospholipases and metalloproteinase--activate the complement system and the contribution of the effect on leucocyte recruitment. In vitro chemotaxis assays were performed using Boyden's chamber model to investigate the ability of serum incubated with venom and its purified toxins to induce neutrophil migration. The complement consumption by the venom was evaluated using an in vitro haemolytic assay. The importance of complement activation by the venom on neutrophil migration was investigated in vivo by injecting the venom into the peritoneal cavity of C5-deficient mice. Data obtained demonstrated that serum incubated with crude venom and its purified metalloproteinase BaP-1 are able to induce rat neutrophil chemotaxis, probably mediated by agent(s) derived from the complement system. This hypothesis was corroborated by the capacity of the venom to activate this system in vitro. The involvement of C5a in neutrophil chemotaxis induced by venom-activated serum was demonstrated by abolishing migration when neutrophils were pre-incubated with antirat C5a receptor antibody. The relevance of the complement system in in vivo leucocyte mobilization was further demonstrated by the drastic decrease of this response in C5-deficient mice. Pre-incubation of serum with the soluble human recombinant complement receptor type 1 (sCR 1) did not prevent the response induced by the venom, but abolished the migration evoked by metalloproteinase-activated serum. These data show the role of the complement system in bothropic envenomation and the participation of metalloproteinase in the effect. Also, they suggest that the venom may contain other component(s) which can cause direct activation of C5a.

Generation of anaphylatoxins through proteolytic processing of C3 and C5 by house dust mite protease.

BACKGROUND: The group 3 allergen of Dermatophagoides species (Der p 3 and Der f 3) has been identified as a 30 kd trypsin-like protease of the house dust mite. We previously showed that the 30 kd protease from Dermatophagoides farinae (Df-protease) could activate the bradykinin-generating cascade and exacerbate inflammatory reactions. OBJECTIVE: The purpose of this study was to determine whether Df-protease could enzymatically generate anaphylatoxins from complement components C3 and C5. METHODS: Df-protease was incubated with human serum C3 or C5 in a purified system, and the anaphylatoxin activity produced was assayed by measuring enhancement of vascular permeability and release of histamine from mast cells triggered by C3a and by assessing chemotaxis of polymorphonuclear cells caused by C5a. We also attempted to determine whether protease isolated from house dust could cause release of C5a from C5. RESULTS: Df-protease showed strong activation of C3 and C5, producing C3a and C5a by proteolytic cleavage of the complements. An appreciable amount of Df-protease was recovered in the house dust extract, and the house dust protease caused C5a release from C5. CONCLUSION: Df-protease activated the complement system to produce anaphylatoxins. Thus it is suggested that house dust mite proteases may contribute to the pathogenesis of allergic and inflammatory diseases caused by house dust allergens.

Generation of the complement activation product C5a precedes interleukin-2-induced capillary leakage syndrome.

Capillary leakage syndrome (CLS) is a severe side effect of intravenous interleukin-2 (IL-2) therapy. Twenty-seven cycles of IL-2 therapy [six (day 1), nine (day 2), and 12 >( 10(6) U/m(2) body surface (days 3 to 5), given as continuous infusion] were analyzed in children and adolescents. The anaphylatoxin C5a was assessed as an early predictor for CLS. CLS developed in 11 of 27 cycles of IL-2 infusion. C5a at day 2 of IL-2 infusion (0.8-9.43 mu g/L; median, 1.8 mu g/L) was increased in CLS patients when compared with baseline values (0.21-0.74 mu g/L; median, 0.40 mu g/L; p = 0.01) and when compared with C5a at day 2 in non-CLS patients (0.44-1.2 mu g/L; median, 0.62 mu g/L; p <0.01). Ten of 11 CLS patients showed C5a levels >1.0 mu g/L, whereas 14 of 16 patients who did not develop CLS showed C5a <1.0 mu g/L (predictive value positive 83% for CLS).

OK-432 induces production of neutrophil chemotactic factors in malignant pleural effusion.

We investigated the changes in cellular components and neutrophil chemotactic factors in pleural fluid from 19 lung cancer patients who received intrapleural injection of OK-432 to treat malignant pleurisy. Not only neutrophil chemotactic activity (NCA) but also neutrophil count and percentage were increased significantly at 6 hours after OK-432 injection. The neutrophil count was significantly correlated with NCA level. The levels of C5a and IL-8 in pleural fluid were increased significantly after OK-432 injection. The increased IL-8 level was associated with a increase of both NCA and neutrophil count. OK-432 treatment also induced a marked increase of IL-1 beta and IL-6 in pleural fluid. Thus, intrapleural injection of OK-432 induced production of neutrophil chemotactic factors (IL-8 and C5a) and cytokines (IL-1 beta and IL-6), which eventually attracted neutrophils into the pleural space. These observations suggest that neutrophil migration mediated by these factors and cytokines may contribute to the sclerosing effects of OK-432 treatment.

Permeability of dialyzer membranes to TNF alpha-inducing substances derived from water bacteria.

Pro-inflammatory cytokine-inducing substances derived from cultured E. coli have previously been shown to pass across low-flux regenerated cellulosic dialyzer membranes. In the present study, a sterile filtrate of Pseudomonas maltophilia grown from standard bicarbonate dialysis fluid was used to test the permeability of various dialyzer membranes (regenerated cellulose, cellulose triacetate, polyacrylonitrile, polysulfone and polyamide) to TNF alpha-inducing bacterial substances. Pyrogen-free tissue culture medium (MEM) was recirculated for 60 minutes in the dialysate compartment of a closed-loop dialysis system, then P. maltophilia filtrate was added and recirculation was continued for a further hour. Samples from the dialysate (MEM) and the blood side (containing 10% human plasma in MEM) were incubated with donor mononuclear cells (MNC) for 18 hours and TNF alpha release was measured in MNC supernatants by radioimmunoassay. Five minutes after the addition of P. maltophilia filtrate, mean TNF alpha-inducing activity in the dialysate increased from (mean +/- SEM) 0.10 +/- 0.02 to 18.2 +2- 1.5 (ng/2.5 x 10(6) MNC/18 hr). TNF alpha-inducing activity in the blood side increased with regenerated cellulose from 0.10 +/- 0.01 to 4.57 +/- 1.55 (N = 8; P less than 0.001); with cellulose triacetate from 0.20 +/- 0.05 to 0.44 +/- 0.10 (N = 5; P less than 0.05), and with polyacrylonitrile from 0.10 +/- 0.02 to 1.16 +/- 0.45 (N = 5; P less than 0.03). No increased TNF alpha-inducing activity was observed in the blood side of polysulfone (N = 5) or polyamide dialyzers (N = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokine-stimulated human mesothelial cells produce chemotactic activity for neutrophils including NAP-1/IL-8.

To test the hypothesis that mesothelial cells play a role in regulating inflammatory responses within the pleural space, we examined neutrophil chemotactic activity released by cytokine-stimulated mesothelial cells. Human mesothelial cells were isolated from patients with transudative pleural effusions and cultured. The purity of the cell population was assessed by morphologic, immunocytochemical, and biochemical characteristics. Confluent fourth passage mesothelial cell plates were exposed to varying concentrations of the recombinant human cytokines IL-1 alpha, TNF-alpha, or IFN-gamma, or Escherichia coli endotoxin (LPS). Polymorphonuclear neutrophil (PMN) chemotactic activity in the conditioned media was measured in microchemotaxis chambers. Although none of the cytokines demonstrated inherent chemotactic activity, each stimulated mesothelial cells to produce PMN chemotactic activity in a dose-dependent manner. TNF-alpha stimulated the release of the greatest quantity, whereas stimulation with IFN-gamma and IL-1 alpha resulted in the release of lesser but still significant quantities of PMN chemotactic activity. By contrast, LPS did not increase the basal level of chemotactic activity produced by the cells. The cytokine-induced chemotactic activity was proteinaceous, required de novo synthesis, and had a predominant m.w. of 10,000. Significant quantities of immunoreactive neutrophil-activating peptide-1 (NAP-1)/IL-8 were detected in mesothelial cell supernatants after stimulation with each of the cytokines. The neutrophil chemotactic activity of supernatants from mesothelial cells stimulated with either IL-1 alpha or IFN-gamma was completely neutralized with rabbit anti-human NAP-1/IL-8 polyclonal antiserum. The same antiserum neutralized the majority, but not all, of the neutrophil chemotactic activity in supernatants from TNF-stimulated mesothelial cells. Stimulated mesothelial cells also expressed an inducible mRNA transcript that hybridized with a specific oligonucleotide probe for human NAP-1/IL-8. These observations provide a mechanism whereby mesothelial cells could respond to inflammatory stimuli in the underlying lung and regulate inflammatory responses within the pleural space.