Detection of the complement fragment C5a in inflammatory exudates from the rabbit peritoneal cavity using radioimmunoassay

We describe a radioimmunoassay for rabbit C5a and its use to obtain evidence of extravascular C5a generation in two inflammatory reactions in the peritoneal cavity. These observations, together with the potent activity of C5a in inducing increased microvascular permeability involving circulating PMN leukocytes, strengthen the case for considering C5a an important inflammatory mediator. These findings offer an explanation for the many different experimental inflammatory reactions where oedema formation can be suppressed either by systemic depletion of complement or by depletion of circulating PMN leukocytes.     

Enhancement of Neutrophil Function as a Result of Prior Exposure to Chemotactic Factor

Exposure of human polymorphonuclear leukocytes (PMN) to chemotactic factor, as well as the migration of PMN through a 5-mum pore-size membrane, results in a PMN population with enhanced chemiluminescence, enhanced capacity for superoxide anion production, and increased Escherichia coli bactericidal activity. The enhanced PMN response resulting from exposure to chemotactic factor was observed with several chemotactic stimuli, including a mixture of casein and autologous serum, chemotactic C5 fragment, and formyl-l-methionyl-l-leucine-l-phenylalanine (f-Met-Leu-Phe). Enhanced levels of chemiluminescence were observed with both soluble stimuli (concanavalin A and phorbol myristate acetate) as well as particulate stimuli (opsonized zymosan).Once activated by chemotactic factor, PMN retained their enhanced stimulated chemiluminescence in the absence of chemotactic factor for at least 2.5 h. Enhanced activity could not be correlated with a shift in the number of immunoglobulin (Ig)G Fc receptor positive or complement receptor positive PMN. In vivo studies with guinea pigs indicated that PMN attracted to an intraperitoneal injection of casein, like those attracted through a chemotaxis membrane in vitro in response to casein, showed markedly enhanced stimulated chemiluminescence when compared with peripheral blood PMN from the same animal. Such a mechanism to stimulated PMN function may enhance the effectiveness of PMN in host defense at inflammatory foci.     

INTERACTIONS OF THE COMPLEMENT SYSTEM WITH ENDOTOXIC LIPOPOLYSACCHARIDE : GENERATION OF A FACTOR CHEMOTACTIC FOR POLYMORPHONUCLEAR LEUKOCYTES

Endotoxic lipopolysaccharide has recently been shown to fix large amounts of the complement components related to the biologic activities mediated by that system. The present study sought to determine whether the generation of chemotactic factor by endotoxin in serum was dependent upon complement system activation. Preheating serum, incubating at 0°C, or incubating in the presence of EDTA, all prevented chemotactic factor generation as well as complement fixation by endotoxin. "Endotoxoids" deficient in complement-firing activity were also deficient in chemotactic factor generation. Chemotactic factor could not be generated by endotoxin in sera of mice congenitally deficient in the C'S component of complement, while chemotactic factor was generated by endotoxin in the sera of coisogenic mice with normal complement levels for that species. The chemotactic factor induced by endotoxin was heat stable and nondialyzable. Molecular sieve chromatography and sucrose density gradient ultracentrifugation demonstrated that the chemotactic factor was a relatively low molecular weight product (15,000–30,000) and as such different from previously scribed C' system-derived chemotactic factors. These experiments demonstrate that generation of chemotactic factor by endotoxin in serum is dependent upon C' system activation involving at least C'5. Furthermore, the relatively low molecular weight of this factor suggests that it might be derived from activation of a single complement component rather than from complexing of multiple complement components.     

Mitochondrial N-formylmethionyl proteins as chemoattractants for neutrophils

Mitochondria synthesize several hydrophobic proteins. Like bacteria, mitochondria initiate protein synthesis with an N-formylmethionine residue. Because N-formylmethionyl peptides have been found to be chemotactic for polymorphonuclear leukocytes (PMN), mitochondria isolated from cultured human cells and purified bovine mitochondrial proteins were tested for PMN chemotactic activity in vitro. Nondisrupted mitochondria were not chemotactic. However, intact mitochondria that had been incubated with a lysosomal lysate did stimulate PMN migration. Antibodies directed against two mitochondrial enzymes, cytochrome oxidase and ATPase, (both of which contain mitochondrially synthesized subunits) but not anti-C3 or anti-C5 decreased mitochondrially derived chemotactic activity. In addition, purified bovine mitochondrial N-formylmethionyl proteins stimulated PMN migration in vitro, whereas nonformylated mitochondrial proteins did not. Furthermore, the chemotactic activity of purified mitochondrial proteins and disrupted mitochondria was decreased by the formyl peptide antagonist butyloxycarbonyl-phenylalanine-leucine-phenylalanine-leucine-phenylalanine. Finally, disrupted mitochondria and purified mitochondrial proteins stimulated PMN-directed migration (chemotaxis), according to accepted criteria. In addition to other chemotactic factors, release of N-formylmethionyl proteins from mitochondria at sites of tissue damage, may play a role in the accumulation of inflammatory cells at these sites.     

PLATELET-DEPENDENT GENERATION OF CHEMOTACTIC ACTIVITY IN SERUM

A protein fraction extracted from the lysosomal granules of human platelets generated chemotactic activity for polymorphonuclear leukocytes when incubated with fresh serum. The platelet factor was also released during platelet aggregation with collagen or epinephrine and appeared to be released during blood clotting. Heated serum did not support the platelet-dependent generation of chemotactic activity. Treatment of fresh serum with antibody to the fifth component of complement also prevented development of activity. Purified human C5 but not C3 yielded chemotactic activity upon incubation with the platelet factor. Thus, human platelets are capable of stimulating chemotaxis via complement activation in a manner similar to leukocytes, and may therefore participate in the early stages of inflammation.     

Bioglass attenuates a proinflammatory response in mouse peritoneal endotoxicosis

Bioglass is a bioactive, resorbable ceramic particle that was developed to assure binding to living tissues. Bioglass is currently employed to fill osseus defects in oral surgery, and it possesses both unique anti-inflammatory and antimicrobial properties. In an effort to determine whether Bioglass may be useful as an adjunct anti-inflammatory device in local inflammatory processes, we examined whether exposure of the peritoneal cavity to Bioglass would induce a pro- or anti-inflammatory response, and then modulate a subsequent proinflammatory response to endotoxin. Three- to fifty-milligram doses of 5 pm Bioglass were administered intraperitoneally in C57BL/6 mice. Total leukocyte, myeloperoxidase, and cytokine levels in the peritoneal wash fluid were determined. In addition, the peritoneal cavity was preexposed to Bioglass, and was then subjected to a subsequent endotoxin administration. All doses of Bioglass were found to induce a significant peritoneal IL-6 response; however, Bioglass did not induce a TNF-alpha, IL-1alpha, IL-10, or a white cell recruitment into the peritoneal lavage fluid. Pretreatment of the peritoneal cavity with Bioglass produced a transient reduction in the proinflammatory response to endotoxin. We conclude that exposure to Bioglass produces an IL-6 response without concurrent expression of TNF-alpha or IL-1alpha. Bioglass appears to transiently suppress the inflammatory response to endotoxin, possibly through the early induction of IL-6. These findings suggest that Bioglass may offer a unique approach in modifying the inflammatory response in local tissue compartments.     

Coagulant Activity of Leukocytes. TISSUE FACTOR ACTIVITY

Peritoneal leukocytes harvested from rabbits which have received two spaced doses of endotoxin have significantly greater (10-fold) coagulant activity than leukocytes from control rabbits. The coagulant activity accelerates the clotting of normal plasma and activates factor X in the presence of factor VII and calcium and is therefore regarded as tissue factor. A total of 40-80 mg tissue factor activity was obtained from the peritoneal cavity of single endotoxin-treated rabbits. In leukocyte subcellular fractions, separated by centrifugation, the specific tissue factor activity sedimented mainly at 14,500 g and above. The procoagulant activity was destroyed after heating for 10 min at 65 degrees C but was preserved at lower temperatures. Polymyxin B, when given with the first dose of endotoxin, reduced both the number of peritoneal leukocytes and their tissue factor activity by two-thirds. When given immediately before the second dose of endotoxin, polymyxin B had no inhibitory effect.     

Production of a Low Molecular Weight Eosinophil Polymorphonuclear Leukocyte Chemotactic Factor by Anaplastic Squamous Cell Carcinomas of Human Lung

A peptide of approximately 300-400 daltons exhibiting in vitro chemotactic activity for human polymorphonuclear (PMN) leukocytes, with a preference for the eosinophil series, was isolated from extracts of anaplastic lung carcinomas of the large squamous cell type obtained from three patients with marked peripheral blood hypereosinophilia and eosinophilic infiltration of the tumors and surrounding normal pulmonary tissues. This chemotactic factor was termed ECF-LSC (eosinophil chemotactic factor of lung squamous cell carcinoma). ECF-LSC appeared in the urine of two of the patients in increasing quantities late in the course of their disease and was also elaborated by long-term cultures of dispersed tumor cells from the same two patients. Three anaplastic large cell bronchogenic carcinomas which were not associated with tumor tissue or peripheral blood eosinophilia, a bronchogenic adenocarcinoma from a patient with only peripheral eosinophilia, and a renal cell carcinoma metastatic to the lungs and associated with transient pleural tissue and fluid eosinophilia were all devoid of ECF-LSC. ECF-LSC from tumor tissue extracts, urine, and tumor cell culture medium was comparable to the mast cell-associated tetrapeptides of the eosinophil chemotactic factor of anaphylaxis (ECF-A) in size, but eluted from Dowex-1 at pH 5.0-3.5 in contrast to the more acidic ECF-A tetrapeptides which eluted at pH 3.2-2.2 ECF-LSC, like the tetrapeptides of ECF-A, had a secondary chemotactic activity for neutrophil PMN leukocytes, but not mononuclear leukocytes, and deactivated both eosinophil and neutrophil PMN leukocytes so that they would not respond to a subsequent in vitro chemotactic stimulus. Eosinophils from the two patients with urinary excretion of ECF-LSC and the highest concentrations in tumor extracts were hyporesponsive in vitro to homologous and heterologous chemotactic stimuli, suggesting that ECF-LSC had deactivated the eosinophils in vivo.     

THE ROLE OF SERUM COMPLEMENT IN CHEMOTAXIS OF LEUKOCYTES IN VITRO

By the use of chambers containing two compartments with an interposed micropore filter, chemotaxis of polymorphonuclear leukocytes (PMN's) in vitro was studied employing various agents that fixed serum complement (C'). Antigen-antibody complexes, zymosan, and aggregated human gamma globulin, in the presence of fresh rabbit, guinea pig, or mouse serum resulted in the migration of PMN's through the micropore filter. Pepsin-degraded rabbit antibody or unaltered duck serum containing antibody did not exhibit such activity after addition of antigen. Heating of the serum before treatment or the presence of EDTA prevented the generation of the chemotactic factor. The chemotactic factor could not be generated in whole serum from rabbits genetically deficient in C'. However, the defect in this rabbit serum could be corrected by addition of rabbit or human C'6. Serum of B10·D2 mice deficient in hemolytic C' also yielded poor chemotactic activity. Interaction of the first four reacting components of guinea pig C' did not result in significant chemotactic activity unless guinea pig euglobulin with heat labile components was also present. In rabbit serum, C'5 and C'6, when "activated" by interaction with the first four reacting components, behaved like a protein-protein complex and exhibited marked chemotactic activity. By employing conditions favoring dissociation of the complex, the individual components were isolated and shown to be chemotactically inactive. Upon recombination of the two components, however, activity reappeared. Using another approach, the C'5–C'6 complex was isolated intact, and shown to be chemotactically active while other fractions not containing these components were not active. It is postulated that the C'5–C'6 complex is the active chemotactic factor generated in serum after the addition of C'-fixing agents.     

Antibacterial activity of human neutrophil defensins in experimental infections in mice is accompanied by increased leukocyte accumulation.

Neutrophil defensins (or human neutrophil peptides-HNP) are major constituents of the azurophilic granules of human neutrophils and have been shown to display broad-spectrum antimicrobial activity. Other activities of these defensins, which are released from stimulated neutrophils, include cytotoxic, stimulatory, and chemotactic activities toward a variety of target cells. We studied the potential use of HNP-1 for antibacterial therapy of experimental bacterial infections in mice. In experimental peritoneal Klebsiella pneumoniae infections in mice, HNP-1 injection was shown to markedly reduce bacterial numbers in the infected peritoneal cavity 24 h after infection. This antibacterial effect was found to be associated with an increased influx of macrophages, granulocytes, and lymphocytes into the peritoneal cavity. These leukocytes appeared to be a requirement for the antibacterial effect, since in leukocytopenic mice administration of HNP-1 did not display antibacterial activity. HNP-1 treatment also reduced bacterial numbers in experimental K. pneumoniae or Staphylococcus aureus thigh muscle infections. In this model, radiolabeled HNP-1 was found to accumulate at the site of infection, whereas most of the injected HNP-1 was rapidly removed from the circulation via renal excretion. These results demonstrate that neutrophil defensins display marked in vivo antibacterial activity in experimental infections in mice and that this activity appears to be mediated, at least in part, by local leukocyte accumulation.     

Specific Inhibition of the Polymorphonuclear Leukocyte Chemotactic Response to Hydroxy-Fatty Acid Metabolites of Arachidonic Acid by Methyl Ester Derivatives

The human polymorphonuclear (PMN) leukocyte chemotactic activity of the hydroxy-fatty acid metabolites of arachidonic acid, 12-l-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-l-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), is eliminated by methylation. Both methyl esters are specific competitive inhibitors of the PMN leukotactic responses to the parent stimuli, and exert no effect on the responses to formyl-methionyl peptides or chemotactic fragments of the fifth component of complement. 50% inhibition of the in vitro chemotactic responses of PMN leukocytes to HETE and HHT was achieved by an equimolar concentration of the corresponding methyl esters, whereas reciprocal cross-inhibition was observed at molar ratios of HETE methyl ester to HHT and HHT methyl ester to HETE which reflected the three- to fivefold greater chemotactic potency of HETE relative to HHT. Methyl esters of structurally related, but nonchemotactic, fatty acids did not competitively inhibit the chemotaxis elicited by HETE or HHT. The intraperitoneal injection of HETE in guinea pigs evoked an eosinophil response at 30 min and a neutrophil response at 5 h, which were prevented by a one-to twofold molar ratio of HETE methyl ester. The competitive inhibition of the in vitro chemotactic activity and the in vivo leukotactic effect of the unsaturated hydroxy-fatty acids by homologous methyl ester derivatives suggests that the cellular component of natural inflammatory reactions may be susceptible to specific regulation by receptor-directed modulation of the activity of the predominant chemotactic principles.     

CHANGES IN THE TITRE OF SERUM OPSONINS AND PHAGOCYTIC PROPERTIES OF MOUSE PERITONEAL MACROPHAGES FOLLOWING INJECTION OF ENDOTOXIN

A study has been made of the changes in the opsonic properties of mouse serum and bactericidal activity of mouse peritoneal macrophages at different times following the injection of various doses of lipopolysaccharide. It has been found that changes in the percentage of bacteria phagocytosed and killed within the mouse peritoneal cavity in a given time could be correlated with changes in the opsonic titre of the serum. Macrophages harvested from the peritoneal cavities of mice injected with endotoxin appear to be more efficient in phagocytosing bacteria in the presence of serum opsonins than macrophages obtained from normal mice. The relative importance of these changes in determining an animal's resistance to infection is discussed.     

Effect of bacterial endotoxin on lipoxygenase and cyclo-oxygenase metabolites by rat neutrophils and correlation with cellular functional parameters

The effect of Salmonella enteritidis endotoxin on in vitro rat neutrophil cyclo-oxygenase and lipoxygenase metabolism, phagocytic activity, superoxide (O2-) generation, and microbicidal activity was investigated. Incubation of polymorphonuclear leukocytes (PMN) with 5, 25, and 50 micrograms of endotoxin significantly enhanced synthesis of immunoreactive (i) leukotriene (LT)C4/D4 and thromboxane (Tx)B2 (P less than 0.001) as compared to control cells. Endotoxin 5 micrograms/ml produced optimal stimulation of the arachidonic acid metabolites. Calcium ionophore, A23187, significantly enhanced iLTC4/D4 and iTxB2 synthesis more than that elicited with endotoxin. Although phagocytic function was not significantly altered by endotoxin, intracellular killing of C. albicans demonstrated enhanced microbicidal activity at 5 micrograms/ml of endotoxin. Superoxide generation was significantly enhanced in neutrophils stimulated with phorbol myristate acetate (PMA). Endotoxin (5 micrograms/ml) further potentiated superoxide generation by these cells when stimulated by PMA. These findings demonstrate that endotoxin directly enhances neutrophil iLTC4/D4 and iTxB2 synthesis. The enhanced arachidonic acid metabolism elicited by endotoxin in these cells parallels increased microbicidal activity and superoxide generation.     

IN VITRO AND IN VIVO ACTIVITY OF A LYMPHOCYTE AND IMMUNE COMPLEX-DEPENDENT CHEMOTACTIC FACTOR FOR EOSINOPHILS

When cultured in the presence of specific antigen, lymphocytes from delayed-hypersensitive guinea pigs release a number of biologically active substances into the culture medium. Such active supernatants can react with immune complexes in vitro to generate a factor which is chemotactic for eosinophils. The factor involved is unique, since previously described chemotactic factors for other cell types require for their generation either immune complexes or substances released into lymphocyte culture, but not both. In the case of the eosinophil chemotactic factor, the interaction between the substance elaborated by the lymphocytes and the immune complexes appears to be specific in that the immune complexes must contain the same antigen as that used to activate the lymphocyte cultures. Although this factor was generated in an in vitro system, it has been shown to possess in vivo as well as in vitro activity. It is therefore possible that this factor may be of biological significance in situations where eosinophils are participants in inflammatory or immunologic reactions.     

Chemotaxis of the peritoneal cells and the detection of a chemo-attractant in the effluent from peritoneal dialysis patients

The migration of peritoneal cells from 25 continuous ambulatory peritoneal dialysis patients and eight healthy women undergoing laparoscopy were studied. Peritoneal cells of continuous ambulatory peritoneal dialysis patients migrated to commonly used chemoattractants, like N-formyl-methionyl-leucyl-phenyl-alanine-methyl- ester and casein, but they also migrated to high concentrations of recombinant interleukin-1 alpha and to endotoxin (lipopolysaccharide). In the peritoneal effluent from continuous ambulatory peritoneal dialysis patients a rather heat stable chemoattractant was found with a molecular weight of 40-200 kDa with an optimal activity at approximately 125 kDa. The chemoattractant is a protein and is not complement factor 5a or interleukin-1 and was only found in peritoneal effluent from continuous ambulatory peritoneal dialysis patients, but not in peritoneal fluid from healthy women undergoing laparoscopy. Therefore, peritoneal dialysis might induce the generation of a chemoattractant. The optimal chemotactic response of peritoneal cells from continuous ambulatory peritoneal dialysis patients to N-formyl-methionyl-leucyl-phenyl-alanine-methyl- ester in medium could be enhanced by replacing the medium by peritoneal effluent. So the chemotaxis of peritoneal cells to the factor in the peritoneal effluent is caused by another mechanism, which might involve different cell surface receptor populations, than the chemotactic response to N-formyl-methionyl-leucyl-phenyl-alanine-methyl-ester.     

THE RELATION OF INFLAMMATION TO THE MOLECULAR STRUCTURE OF CARBON COMPOUNDS SOLUBLE IN THE FLUIDS OF THE BODY

The peritoneal, like the pleural cavity, gives opportunity to measure with adequate accuracy the activity of inflammatory reactions defined by movement of fluid within the cavity, by migration of leucocytes into it, and by exudation of proteins from the plasma. The activity of inflammatory reactions caused by saccharides or by alcohols that were tested varied in accord with their molecular weight, the osmotic pressure maintained by solutions of corresponding concentration, their boiling point, or by other colligative properties. Blood serum or globulin in the concentration with which it occurs in blood serum injected into the peritoneal cavity caused changes which differed little from those caused by physiological salt solution. Protein with molecular weight as low as that of cytochrome C (12,000) or ovalbumin (45,000) when in dilute solution (1 per cent) were rapidly absorbed, whereas trypsin and chymotrypsin under the same conditions caused very active inflammatory reactions because they set free amino acids and perhaps polypeptides with amino acids in short chains. The activity of inflammatory reactions caused by carbon compounds soluble in body fluids varied in accord with their colligative properties.     

ACUTE DESTRUCTION BY HUMORAL ANTIBODY OF RAT SKIN GRAFTED TO MICE : THE ROLE OF COMPLEMENT AND POLYMORPHONUCLEAR LEUKOCYTES

A study has been made of the roles played by complement and polymorphonuclear leukocytes (PMN) in the acute destruction of xenografts of rat skin that follows injection of their hosts with antisera specifically reactive with graft antigens. The rat skin was grafted onto mice whose immune responses were suppressed by removal of the thymus and a brief course of treatment with rabbit antimouse lymphocyte serum. At about 2 wk after grafting the mice were injected intravenously or intraperitoneally with mouse antirat serum (MARS). This time interval was chosen because it avoided the complications that might be associated with either the process of healing in or with incipient rejection. Signs of graft damage were evident as early as 10 min after the injection of MARS, and in most animals so injected the grafts were completely destroyed within 24–48 h. The role of complement (C) in this acute destructive process is indicated by the results of three lines of experimentation. (a) Non-C-fixing antibodies or antibody fragments failed to cause damage to the grafts. Indeed, both chicken antirat serum and F(ab')₂ fragments from rabbit antirat serum completely protected the grafts against the effects of MARS that was administered 24 h later. (b) When mice were depleted of hemolytic C by treatment with cobra venom factor or heat-aggregated gamma globulin, the damage caused by MARS was greatly reduced or completely inhibited. (c) In mice with a genetically determined absence of C5 much greater quantities of MARS were required to cause graft damage; the tempo of the destructive process was consistently slower; and a greater number of grafts recovered from the initial inflammatory process than was the case for animals with an intact complement system. The participation of PMN in serum-mediated destruction of grafts was initially suggested by the results of microscope examination of fixed tissues. The essential role of these cells in the process is indicated by the failure of MARS to cause tissue damage in mice whose circulating PMN have been reduced to very low levels by treatment with nitrogen mustard or more specifically with an anti-PMN serum. The absence of tissue damage when circulating PMN are reduced but C levels are normal suggests that C-mediated cytolysis is unimportant in graft destruction and that the role of C lies in its ability to generate chemotactic factors. The latter may then attract the PMN that provide mediators of tissue damage.     

Mechanisms of increased survival after lipopolysaccharide-induced endotoxic shock in mice consuming olive oil-enriched diet

We examined the impact of dietary fatty acid intake on lipopolysaccharide (LPS)-induced endotoxic shock. C57Bl/6J mice were fed for 6 weeks with a commercial laboratory chow (CC) or with test chows containing 7% (w/w) canola oil (CO), sesame oil (SeO), soybean oil (SO), or virgin olive oil (OO). The increase in body weight and energy consumption were similar for all diets tested. In the sixth week, mice were injected intraperitoneally with 400 microg of bacterial LPS to induce endotoxic shock. LPS induced a massive neutrophil infiltration into the peritoneal cavity and an increase in lipid body (LB) formation in leukocytes recovered from the peritoneal fluid of mice fed with CC, CO, SeO, or SO. In addition, there were increases in prostaglandin E(2) (PGE(2)), leukotriene B4 (LTB(4)), and cytokines IL-6, IL-10, and MCP-1 in peritoneal lavage, as well as in plasma TNF-alpha. In contrast, mice fed with OO exhibited reduced neutrophil accumulation and LB formation, and also had lower levels of PGE(2), LTB(4), MCP-1, and TNF-alpha. All mice fed with CC, CO, SeO, or SO died within 48 to 72 h after LPS injection. Interestingly, mice fed with the OO diet were resistant to endotoxic shock, with 60% survival at 168 h. These data indicate that intake of OO may have a beneficial role, reducing the magnitude of the inflammatory process triggered by endotoxic shock through modulation of LB formation and of the production of inflammatory mediators.     

Motility and Adhesiveness in Human Neutrophils: EFFECTS OF CHEMOTACTIC FACTORS

Human peripheral blood neutrophils (PMN) obtained from healthy adults were examined in vitro with techniques adapted to assess the effects of chemotactic factors (CF) on cellular configuration and adhesiveness. The results were compared with those that use certain conventional techniques for assessing chemotaxis and chemokinesis. Exposure of PMN to N-formyl-l-methionyl-l-phenylalanine (f-Met-Phe), zymosan-activated serum, bacterial chemotactic factor, or a low molecular weight chemotactic factor from activated serum (C5a) in the absence of a gradient resulted in a change in cellular shape from a spherical to a polarized configuration in a high percentage of cells. This occurred rapidly in suspension, under conditions designed to exclude a role for cell adhesiveness, and was reversible upon removal of the CF. Restimulation of cells with the CF resulted in reappearance of the polarized configuration to the same extent as on initial stimulation with one exception: f-Met-Phe pretreated cells failed to respond to f-Met-Phe, though they responded fully to the other CF. Each CF caused a significant increase in PMN attachment to protein-coated glass. This enhanced adhesiveness was not reversible upon removal of the CF when the cells were treated under conditions shown to produce chemotactic deactivation. Cells treated under these conditions also exhibited significantly reduced motility on glass and in micropore filters in the absence of a gradient of CF. Bacterial chemotactic factor, even at high concentrations, failed to produce deactivation and did not cause a sustained enhancement of adhesiveness.     

Low molecular weight heparin improves peritoneal ultrafiltration and blocks complement and coagulation.

OBJECTIVES: Clinical studies have demonstrated that the intraperitoneal (IP) complement and coagulation systems are activated in peritoneal dialysis (PD) patients. In animal models, low molecular weight heparin (LMWH) was seen to inhibit peritoneal angiogenesis, and related compounds have increased ultrafiltration volumes after repeated administration to PD patients. The present study evaluated the effects of LMWH on ultrafiltration, coagulation, and complement activation during a single PD dwell. DESIGN: Rats were exposed to a single dose of 20 mL 2.5% glucose-based, filter-sterilized PD fluid, with or without supplementation with LMWH. The PD fluid was administered either as an IP injection or as an infusion through an indwelling catheter. The dwell fluid was analyzed 2 hours later concerning activation of the complement and coagulation cascades, chemotactic activity, neutrophil recruitment, ultrafiltration volume, and glucose and urea concentrations. RESULTS: Exposure to PD fluid induced activation of IP complement [formation of C3a (desArg) and increase of C5a-dependent chemotactic activity] and coagulation (formation of thrombin-antithrombin complex) and recruitment of neutrophils. In the case of IP injection, neutrophil recruitment and complement activation were inhibited by LMWH. In both models, LMWH inhibited thrombin formation, reduced complement-dependent chemotactic activity, and increased the IP fluid volume, indicating an improved ultrafiltration. CONCLUSIONS: The acute inflammatory reaction to PD fluid involves the complement and coagulation cascades. Addition of LMWH to the PD fluid improves ultrafiltration, inhibits formation of thrombin, and potentially blocks C5a activity. The present results motivate further investigations of the IP cascade systems in PD.