The Complement Pathway Is Activated in People With Human Immunodeficiency Virus and Is Associated With Non-AIDS Comorbidities.

Unbiased plasma proteomics in a matched case-control study of treated people with human immunodeficiency virus (PWH) revealed the complement cascade as being among the top pathways enriched in PWH. Specific complement components, namely C5, associated significantly with non-AIDS comorbidity prevalence, and did so more strongly than previously established predictive biomarkers.

Abnormal DNA methylation patterns in patients with infection­caused leukocytopenia based on methylation microarrays.

The present study aimed to investigate the association between gene methylation and leukocytopenia from the perspective of gene regulation. A total of 30 patients confirmed as having post­infection leukocytopenia at People's Hospital of Xinjiang Uygur Autonomous Region between January 2016 and June 2017 were successively recruited as the leukocytopenia group; 30 patients with post­infection leukocytosis were enrolled as the leukocytosis group. In addition, 30 healthy volunteers who received a health examination at the hospital during the same period were included as the normal control group. In each group, four individuals were randomly selected for whole genome methylation screening. After selection of key methylation sites, the remaining samples in each group were used for verification using matrix­assisted laser desorption/ionization­time of flight mass spectrometry. The levels of serum complement factors C3 and C5 in the leukocytopenia group were significantly lower than those in the other two groups (P<0.05). According to whole­genome DNA methylation detection, 66 and 27 methylation loci may be associated with leukocytopenia and leukocytosis, respectively. Most of these abnormal loci are located on chromosomes 2, 6, 7, 1, 17 and 11. The rates of WW domain containing E3 ubiquitin protein ligase 2 gene methylation at cytosine­phosphate­guanine (CpG)_1, CpG_5/6 and CpG_7 in the leukocytopenia group were higher than in the other two groups (P<0.05); the rate of AKT2 CpG_1 methylation was higher in the leukocytopenia group than in the other two groups (P<0.05); the rate of calcium­binding atopy­related autoantigen 1 gene CpG_2 methylation was higher in the leukocytosis group than in the normal control group (P<0.05); and the rate of NADPH oxidase 5 gene CpG_3 methylation was higher in the leukocytosis group than in the normal control group (P<0.05). Chemotactic factor secretion and cell migration abnormalities, ubiquitination modification disorders and reduced oxidative burst may participate in infection­complicated leukocytopenia. The results of this study shed new light on the molecular biological mechanisms of infection­complicated leukocytopenia and provide novel avenues for diagnosis and treatment.

iTRAQ reveals candidate pancreatic cancer serum biomarkers: influence of obstructive jaundice on their performance.

BACKGROUND: The aims of our study were to identify serum biomarkers that distinguish pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) patients from benign pancreatic disease patients and healthy subjects, and to assess the effects of jaundice on biomarker performance. METHODS: Isobaric tags for relative and absolute quantification were used to compare pooled serum and pancreatic juice samples from a test set of 59 and 25 subjects, respectively. Validation was undertaken in 113 independent subjects. RESULTS: Candidate proteins Complement C5, inter-α-trypsin inhibitor heavy chain H3, α1-ß glycoprotein and polymeric immunoglobulin receptor were elevated in cancer, as were the reference markers CA19-9 and Reg3A. Biliary obstruction had a significant effect on the performance of the markers, in particular within the PDAC group where the presence of jaundice was associated with a significant increase in the levels of all six proteins (P<0.01). Consequently, in the absence of jaundice, proteins showed reduced sensitivity for PDAC patients over benign subjects and healthy controls (HCs). Similarly, in the presence of jaundice, markers showed reduced specificity for PDAC patients over benign subjects with jaundice. Combining markers enabled improved sensitivity for non-jaundiced PDAC patients over HCs and improved specificity for jaundiced PDAC patients over jaundiced benign disease subjects. CONCLUSIONS: The presence-absence of jaundice in the clinical scenario severely impacts the performance of biomarkers for PDAC diagnosis and has implications for their clinical translation.

Complement activation in pediatric patients with recurrent acute otitis media.

OBJECTIVE: Otitis media (OM) is one of the most common childhood diseases. The relative contribution of complement activation in protection and pathogenesis during OM remains largely unknown. The purpose of this study was to investigate the beneficial and pathogenic contributions of complement activation in the middle ear of pediatric patients with recurrent acute otitis media (rAOM), and therefore to provide a rational approach to prevent sequelae of OM such as hearing loss. METHODS: Twenty children undergoing pressure equalization tube placement with or without adenoidectomy for rAOM were enrolled in the study. Bacterial cultures, enzyme-linked immunosorbent assay (ELISA) for complement components and cytokines and western blot for complement activation were performed on middle ear effusion (MEE) and serum samples. The levels of complement C3a, C5a and sC5-b9 in MEEs and serum samples were compared. The levels of these factors were also examined in regards to length of episode. Pearson's correlation coefficients were calculated on variables between C5a and IL-6 or IL-8. Complement gene expression in human middle ear epithelial (HMEE) cells induced by otopathogens was evaluated. Data were analyzed with Student's t test or the Mann-Whitney rank sum test. In all cases, a P value of <0.05 was set as the measure of significance. RESULTS: Our data demonstrated that the complement classical/lectin, alternative and terminal pathways were activated in the middle ear of children with rAOM. Increased complement components of C3a, C5a and sC5-b9 in MEEs were detected in patients with the episode lasting more than six weeks. There was a strong correlation between C5a and IL-6 or IL-8 in the MEEs. Additionally, otopathogens induced enhanced gene expression of factor B and C3 in HMEE cells, which is beneficial for host defense against invading pathogens. CONCLUSION: Our studies provided important new insights on how complement activation contributes to inflammatory process during rAOM. Knowledge of the activity of the complement pathway in patients with rAOM may stimulate the development of new strategies to prevent middle ear inflammatory tissue destruction by directing treatment to specific pathways within the complement cascade.

Innate immunity mediating inflammation secondary to endotracheal intubation.

OBJECTIVE: To investigate the inflammatory markers associated with short-term endotracheal intubation in healthy surgical patients. DESIGN: An observational and prospective study of subjects scheduled for same-day surgery procedures. SETTING: Level I trauma center. PATIENTS: Fourteen healthy patients intubated for same-day surgery procedures. The median duration of surgery was 3 hours. INTERVENTIONS: Serial lavages above the tracheal cuff were obtained at the beginning of surgery, at 1 hour, and at the end of surgery; samples were assayed for cellular counts and levels of cytokines and complement 5a (C5a). RESULTS: The total number of polymorphonuclear cells (PMNs) increased almost 10-fold from intubation to extubation (P < .01). The levels of 3 of the cytokines measured in tracheal lavage supernatants were significantly elevated over the time of intubation: tumor necrosis factor (TNF) (P < .01), interleukin 6 (IL-6) (P < .01), and IL-1ß (P < .025). Levels of IL-8 showed an upward trend over time but were not significantly increased; C5a levels were significantly elevated over time (P < .05). CONCLUSIONS: Short-term intubation in healthy patients resulted in significant tracheal inflammation. Involvement of the innate immune system as documented in the present study provides information that may help to better understand the pathophysiologic characteristics of subglottic stenosis and other endotracheal injuries secondary to endotracheal intubation.

Platelet-rich plasma stimulates cytokine expression and alkaline phosphatase activity in osteoblast-derived osteosarcoma cells.

OBJECTIVE: The aim of this study was to investigate the effects of PRP on SAOS-2 cells in terms of cytokine expression, cell activity and oxidative stress. DESIGN: Cell line SAOS-2 (1×10(5)cells/mL) were grown in culture medium α-MEM with 10% FBS for 24h and stimulated (or not) with PRP at concentrations of 3, 10 and 20%, LPS (E. coli, 10g/mL) and IL-1ß (1mg/mL) for 24h. The supernatant was collected and analyzed for the expression of cytokines in a panel array, ALP using a commercial kit and NO(2)(-) with Griess reaction method. Also, the cells were analyzed using Western blot for RANKL and slot blotting for nitrotyrosine expression. RESULT: There were no significant differences amongst the groups in terms of NO(2)(-), protein nitrotyrosine content and RANKL expression. However, all stimuli increased ALP activity and in case of PRP, it was in a dose-dependent manner (p<0.001). Also, all stimuli induced an increase in cytokines and chemokines expression, but only PRP promoted an increase of component C5, sICAM-1 and RANTES expression. Whilst IL-1 receptor antagonist (IL-1ra) expression was down-regulated by PRP, both LPS and IL-1ß caused up-regulation of this cytokine. CONCLUSIONS: PRP can stimulate osteoblast activity and cytokine/chemokine release, as well as indicate some of the mediators that can (and cannot) be involved in this activation.

Group A streptococcal cysteine protease degrades C3 (C3b) and contributes to evasion of innate immunity.

A relative lack of neutrophils around Streptococcus pyogenes is observed in streptococcal toxic shock syndrome (STSS). Because the bacteria spread rapidly into various organs in STSS, we speculated that S. pyogenes is equipped with molecules to evade the host innate immune system. Complement C3b opsonizes the pathogen to facilitate phagocytosis, and a complex of C3b converts C5 into anaphylatoxin. Because we found that C3 (C3b) is degraded in sera from patients with STSS, we investigated the mechanism of C3 (C3b) degradation by S. pyogenes. We incubated human C3b or serum with recombinant SpeB (rSpeB), a wild-type S. pyogenes strain isolated from an STSS patient or its isogenic DeltaspeB mutant and examined the supernatant by Western blotting with anti-human C3b. Western blot and Biacore analyses revealed that rSpeB and wild-type S. pyogenes rapidly degrade C3b. Additionally, C3 (C3b) was not detected in sera collected from infected areas of STSS patients. Furthermore, the survival rate in human blood and in mice was lower for the DeltaspeB mutant than the wild-type strain. Histopathological observations demonstrated that neutrophils were recruited to and phagocytosed the DeltaspeB mutant, whereas with the wild-type strain, few neutrophils migrated to the site of infection, and the bacteria spread along the fascia. We observed the degradation of C3 (C3b) in sera from STSS patients and the degradation of C3 (C3b) by rSpeB. This suggests that SpeB contributes to the escape of S. pyogenes from phagocytosis at the site of initial infection, allowing it to invade host tissues during severe infections.

Complement activation products in plasma after heart transplantation in humans.

BACKGROUND: Complement activation has recently been implicated as a contributing factor to early and late allograft dysfunction in cardiac transplantation. The current study was designed to determine whether measurement of plasma complement fragments C4d and SC5b-9 would be useful in detecting acute rejection or accelerated graft atherosclerosis (AGA) in cardiac allograft recipients. METHODS: We measured complement activation products, C4d (classical pathway) and SC5b-9 (terminal pathway), at the time of routine endomyocardial biopsy in heart transplant recipients. Ten patients in the immediate posttransplantation period (0-100 days) and 19 patients more than 6 months after transplantation were studied. RESULTS: No correlation was found between plasma levels of complement activation fragments and the presence of biopsy-proven acute allograft rejection or AGA (assessed by coronary angiography). However, plasma C4d and SC5b-9 were significantly elevated in 9 of 10 and 7 of 10 patients, respectively, in the immediate posttransplantation period. This was followed by progressive decrease in the levels of C4d and SC5b-9 fragments during the first 4-6 weeks after transplantation. CONCLUSION: We conclude that measuring plasma levels of fragments C4d and SC5b-9 is not a useful noninvasive method for detecting acute rejection or AGA after heart transplantation. However, this study provides further evidence that early complement activation after heart transplantation may play a pathogenic role in allograft injury.

Pemphigus with pemphigoid-like presentation, associated with squamous cell carcinoma of the tongue.

A 53-year-old woman presented with an inoperable squamous cell carcinoma of the tongue associated with tense large bullae consistent with bullous pemphigoid, preceded by a prodrome of urticarial plaques. The histological findings showed a regenerating subepidermal blister with eosinophils and no acantholysis. Direct immunofluorescence study, however, showed positive staining for IgG and C3 throughout the epidermis consistent with pemphigus. The blistering eruption had no mucosal involvement and responded to low dose corticosteroids. Our patient may represent another presentation of a 'paraneoplastic pemphigus spectrum'.

Paramesangial glomerular deposits in membranoproliferative glomerulonephritis type II correlate with hypocomplementemia.

To gain support for a previously proposed hypothesis that nephritic factors predispose to chronic glomerulonephritis, the glomerular deposits of patients with membranoproliferative glomerulonephritis type II have been studied by electron microscopy and immunofluorescence and the results correlated with the C3 level at the time of biopsy. If, as hypothesized, circulating convertase predisposes to nephritis, finding that the glomeruli of patients hypocomplementemic at biopsy, presumably with nephritic factor-stabilized convertase in their circulation, differ from those of patients normocomplementemic at biopsy would suggest that circulating convertase in some way alters the glomerulus. Among 25 biopsy specimens from 12 patients, hypocomplementemia did not correlate with capillary loop deposits, but there was strong correlation with deposits in the paramesangial region as detected by electron microscopy. Of 11 patients who were normocomplementemic at biopsy, none had paramesangial deposits in their glomeruli. Of 14 patients who were hypocomplementemic at biopsy, deposits were present in the paramesangium in 12 patients (P < 0.001). The deposits were either on both sides of the paramesangial segment of the basement membrane (waist basement membrane related) or in apposition to the paramesangial basement membrane in a subepithelial position only. The detection of paramesangial deposits in the ultrastructure correlated with the detection of C3-containing mesangial granules by immunofluorescence; immunoglobulin G, C5, properdin, and factor B could not be demonstrated in these granules. The study identifies the mesangial deposits described by others in membranoproliferative glomerulonephritis type II as paramesangial deposits and, more importantly, demonstrates that their presence correlates closely with hypocomplementemia. It is likely that these deposits in some way result from the presence in the circulation of convertase stabilized by the nephritic factor of the amplification loop.

Complement inhibition with soluble complement receptor type 1 in cardiopulmonary bypass.

Although complement activation during cardiopulmonary bypass (CPB) is well documented, its pathogenic role in postperfusion organ injury is unproven. In this study, soluble human complement receptor type 1 (sCR1), a potent inhibitor of complement activation, was used to determine the contribution of complement activation to pulmonary injury in a porcine model of CPB. In vitro experiments demonstrated that sCR1 inhibits both classic and alternative complement pathways in the pig. Seven control piglets and 6 piglets treated with sCR1 (12 mg/kg intravenously) underwent 2 hours of hypothermic (28 degrees C) CPB followed by 2 hours of observation. In control piglets, total hemolytic complement activity and functional activities of C3 and C5 declined to 61.3%, 67.8%, and 61.4% of prebypass values, respectively, after 2 hours of CPB. Plasma from animals treated with sCR1 had virtually no hemolytic activity (total hemolytic complement activity < 5% of baseline), demonstrating effective complement inhibition. Similar degrees of neutropenia developed in the two groups during CPB, and there was no difference in post-CPB lung tissue myeloperoxidase level. Two hours after CPB, pulmonary vascular resistance increased 338% in control piglets but only 147% in piglets pretreated with sCR1 (p < 0.05); the alveolar-arterial gradient was not significantly different between controls (331 +/- 52 mm Hg) and piglets receiving sCR1 (290 +/- 85 mm Hg). Histologic examination revealed similar degrees of pulmonary edema in both groups. These data constitute direct evidence that complement activation plays a pathogenic role in lung injury after CPB.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of self antigen: implications for antigen presentation and induction of tolerance.

The fifth component of complement (C5) is a self antigen expressed in serum of normal mice at a concentration of about 50 micrograms/ml. We have previously shown that C5 is constitutively processed and presented by antigen-presenting cells (APC) in normal mice to induce and maintain complete tolerance in major histocompatibility complex (MHC) class II-restricted T cells. This report addresses the question of whether C5 presentation involves exogenous antigen which has been internalized for processing or whether intracellular, biosynthesized C5 is being presented with MHC class II. Macrophages were found to synthesize, but not secrete C5 in bone marrow chimeras made from irradiated C5-deficient [C5(-)] hosts reconstituted with C5-sufficient [C5(+)] bone marrow [C5(+)-->C5(-)]. In these mice, macrophages are the only source of C5. [C5(+)-->C5(-)] chimeras are not tolerant of C5 and generate C5-specific T and B cell responses upon immunization indistinguishable from those of C5(-) mice. Macrophages from [C5(+)-->C5(-)] chimeras are unable to activate C5-specific T cell hybrids in vitro unlike macrophages from a C5(-) strain that has matured in a C5-expressing environment [C5(-)-->C5(+) chimeras]. This shows that under physiological conditions in vivo intracellular C5 does not get access to the class II presentation pathway and thus, does not induce tolerance in class II-restricted T cells.

Persistent complement activation on tumor cells in breast cancer.

The neoantigens of the C5b-9 complement complex, IgG, C3, C4, S-protein/vitronectin, fibronectin, and macrophages were localized on 17 samples of breast cancer and on 6 samples of benign breast tumors using polyclonal or monoclonal antibodies and the streptavidin-biotin-peroxidase technique. All the tissue samples with carcinoma in each the TNM stages presented C5b-9 deposits on the membranes of tumor cells, thin granules on cell remnants, and diffuse deposits in the necrotic areas. When chemotherapy and radiation therapy preceded surgery, C5b-9 deposits were more intense and extended. The C5b-9 deposits were absent in all the samples with benign lesions. S-protein/vitronectin was present as fibrillar deposits in the connective tissue matrix and as diffuse deposits around the tumor cells, less intense and extended than fibronectin. IgG, C3, and C4 deposits were present only in carcinoma samples. The presence of C5b-9 deposits is indicative of complement activation and its subsequent pathogenetic effects in breast cancer.

Fluid-phase activation of the alternative pathway of complement by excess factor D in regularly dialyzed patients.

We examined the effect of excess factor D on the alternative pathway of complement (APC). First, we demonstrated that the production of C3a is accelerated in the fluid-phase with the addition of purified factor D. Analysis by sodium dodecylsulfate polyacrylamide gel electrophoresis under reducing conditions showed that the serum iC3b level was elevated when incubated with excess factor D. Secondly, we demonstrated, by measuring the C5a-des-Arg level, that the generation of C5a was promoted in the fluid-phase with the addition of purified factor D. We then studied whether activation of APC is elevated in the blood of patients on maintenance hemodialysis whose sera contained a high concentration of factor D. First, we detected, by fluorescence activated cell sorter analysis, greater amounts of C3d on erythrocytes from the patients (mean fluorescence intensity +/- SD: 7.7 +/- 1.7 arbitrary units) than those from healthy individuals (5.4 +/- 0.5 arbitrary units; p less than 0.001). Secondly, serum C3 level was significantly lower (p less than 0.001) in patients (mean +/- SD: 63.3 +/- 8.2 mg/dl) than in healthy individuals (84.8 +/- 9.5 mg/dl), whereas there was no difference in serum C4 level between patients (32.4 +/- 6.9 mg/dl) and healthy individuals (33.0 +/- 7.4 mg/dl). Serum C5 level was almost the same in patients (10.5 +/- 1.5 mg/dl) and in healthy individuals (11.2 +/- 1.3 mg/dl). These results provide supportive evidence of elevated APC activation in patients with high serum factor D.

Studies on complement deposits in epidermolysis bullosa acquisita and bullous pemphigoid.

Epidermolysis bullosa acquisita (EBA) is an inflammatory subepidermal blistering disease characterized by circulating and tissue-bound autoantibodies specific for type VII collagen of the basement membrane zone. The antibodies consist of both complement- and noncomplement-binding populations and belong to all four subclasses of IgG. We investigated the presence of the membrane attack complex C3b, C5, and S protein in EBA and compared C3b and C5 in EBA and bullous pemphigoid. In 10 patients with EBA, these components were detected at the basement membrane zone as follows: membrane attack complex, 90%; S protein, 90%; direct C5, 90%; C3b, 100%; and C5 binding, 90%. In the patients with bullous pemphigoid, the results were as follows: direct C5, 58%; C3b, 33%; and C5 binding, 19%. These results provide additional evidence for complement activation at the basement membrane zone in EBA, show that complement activation in EBA proceeds to activation of terminal complement components, and suggest that EBA antibodies are more potent activators of C5 than are bullous pemphigoid antibodies.

Complement activation during prolonged extracorporeal membrane oxygenation.

Short-term cardiopulmonary bypass activates the complement system, possibly resulting in pulmonary dysfunction from granulocyte aggregation and pulmonary endothelial damage. These effects may be inhibited by steroids. Prolonged extracorporeal membrane oxygenation (ECMO) is used for newborn respiratory failure, but the effects of ECMO on complement activation are unknown. Twenty-one newborn infants with respiratory failure treated with ECMO were randomly assigned to group I (control, no steroids) or group II (30 mg/kg intravenous methylprednisolone before ECMO). Depletion assays of C3 and C5 were performed in each group at intervals before and during ECMO (declining values indicate complement activation). The groups were compared for complement levels, survival, time on ECMO and on the ventilator, and total hospitalization time. Steroids significantly shortened the time on ECMO and time on the ventilator after ECMO but did not affect survival or total hospitalization time. Steroids also enhanced activation of C3 and C5. Complement activation occurs during ECMO. Steroid administration paradoxically causes earlier complement activation but shortens ECMO and ventilator times. Complement activation during ECMO is of questionable significance. The benefits of steroids during ECMO may be mediated through other mechanisms.

Regulatory control of the terminal complement proteins at the surface of human endothelial cells: neutralization of a C5b-9 inhibitor by antibody to CD59.

Functionally inhibitory antibody to the plasma membrane complement inhibitor CD59 has been used to investigate control of the terminal complement proteins at the endothelial cell surface. Antibodies against purified human erythrocyte CD59 (polyclonal anti-CD59 and monoclonal antibodies [MoAbs] 1F1 and 1F5) were found to bind specifically to monolayers of cultured human umbilical vein endothelial cells, and by Western blotting to recognize an 18- to 21-Kd endothelial protein. When bound to the endothelial monolayer, anti-CD59 (immunoglobulin G or Fab fragment) potentiated membrane pore formation induced upon C9 binding to C5b-8, and augmented the C5b-9-induced cellular responses, including stimulated secretion of von Willebrand factor and expression of catalytic surface for the prothrombinase enzyme complex. Although potentiating endothelial responses to the terminal complement proteins, anti-CD59 had no effect on the response of these cells to stimulation by histamine. Taken together, these data suggest that human endothelial cells express the CD59 cell surface inhibitor of the terminal complement proteins, which serves to protect these cells from pore-forming and cell-stimulatory effects of the C5b-9 complex. These data also suggest that the inactivation or deletion of this cell surface regulatory molecule would increase the likelihood for procoagulant changes in endothelium exposed to complement activation in plasma.

Neutrophil recruitment in the respiratory tract of a patient with plasma cell granuloma of the lung.

A 69-year-old woman had plasma cell granuloma of the left middle lobe of the lung. Her symptoms and roentgenologic findings improved with antibiotic treatment. Before treatment, the number of neutrophils and NCA were markedly increased in BAL fluid obtained from the affected region of the left lung and moderately increased in BAL fluid obtained from the nonaffected region of the right lung. The number of neutrophils, the NCA as well as the contents of C5 and C5a des Arg (neutrophil chemotactic factors) in the BAL fluids from both these regions decreased during treatment. These findings suggest that plasma cell granuloma was due to chronic immune and inflammatory reactions in the lung, that neutrophils are involved in development of the symptoms and signs of this disease, and that neutrophil chemotactic factors, including complement-derived factors, are important in neutrophil recruitment at the lesion and in nonaffected parts of the lung.

[Serum complement composition of myasthenia gravis].

Serum complements of C1q C1, C4, BF, C1-INH and C5 were measured by single immunodiffusion in 54 patients with myasthenia gravis (MG), 46 normal control (NC) and 42 cases of other neurological disorders. It was found that C1-INH, C5 levels in MG were significantly higher than that in other groups (P less than 0.01). The serum levels of C1-INH, C5 were not related to the clinical type and the stage of the illness. These data suggest that endplate receptors might be disrupted through complement mediated immunoreaction.

Neutrophil chemotactic factors in bacterial pneumonia.

The influx of neutrophils into the lung is a prominent feature in patients with bacterial pneumonia. Since neutrophils migrate in response to chemotactic factors, chemotactic activity was evaluated in bronchoalveolar lavage (BAL) fluid obtained from 12 patients with bacterial pneumonia and ten normal control subjects. Chemotactic activity was greatly elevated in the BAL fluid of the pneumonia patients compared with control subjects (p less than 0.01). To partially characterize the chemotactic factors present in the lavage fluid of the patient group, molecular sieve chromatography was performed on the lavage fluid, and at least three peaks of chemotactic activity were identified. Since the molecular weight of the smaller peaks approximated the molecular weight of two known chemotactic factors, C5a and leukotriene B4, these factors were measured in lavage fluid by radioimmunoassay. C5a was detectable in none of the normal subjects but was detectable in four of 14 BAL samples obtained from the patients. Leukotriene B4 was detectable in all subjects and was significantly elevated in the pneumonia patients (552 +/- 95 vs 81 +/- 16 pg/ml, p less than 0.01). These findings demonstrate that elevated neutrophil chemotactic activity is present in the lungs of patients with bacterial pneumonia and suggest that C5a and leukotriene B4 may account, at least in part, for this increase.