Unidirectional heterologous receptor desensitization between both the fMLP and C5a receptor and the IL-8 receptor.

During inflammation neutrophils receive multiple signals that are integrated, allowing a single modified response. One mechanism for this discrimination is receptor desensitization, a process whereby ligand-receptor binding is disassociated from cell activation. We examined the effect of heterologous receptor desensitization on neutrophil chemotaxis, calcium mobilization, and arachidonic acid production, using interleukin-8 (IL-8), C5a, and N-formyl-methionyl-leucyl-phenylalanine (fMLP). We observed reciprocal inhibition with respect to chemotaxis. We demonstrated that homologous desensitization, with respect to the mobilization of intracellular calcium stores, lasted approximately 15 min. Heterologous desensitization between the fMLP receptor and the C5a receptor was reciprocal; either stimulant would diminish the cells' response to stimulation by the other for approximately 3-5 min. However, we observed a unidirectional heterologous desensitization of the IL-8 receptor by both the fMLP and the C5a receptor. This unidirectional heterologous desensitization was observed with respect to both calcium mobilization and arachidonic acid production (i.e., prestimulation of the IL-8 receptor had no effect on subsequent stimulation by either fMLP or C5a).

Interleukin-8 is a major component of pleural liquid chemotactic activity in a rabbit model of endotoxin pleurisy.

Gram-negative endotoxin induces production of the potent chemotactic factor interleukin-8 (IL-8) in vitro; however, the importance of IL-8 in endotoxin-induced inflammation in vivo is unknown. We asked whether IL-8 is an important contributor to chemotactic activity in acute inflammatory liquids formed in response to endotoxin, and, if present, what concentrations of IL-8 antigen are generated. For these studies, we cloned and expressed rabbit recombinant IL-8 (rrIL-8), developed specific anti-rabbit IL-8 monoclonal antibodies (mAb), and then used these reagents to develop assays to detect rabbit IL-8 bioactivity and measure rabbit IL-8 antigen. Escherichia coli endotoxin (20 ng/ml, n = 4, or 2,000 ng/ml, n = 4) was instilled into the pleural space of eight rabbits for 6 h. Rabbit IL-8 bioactivity in the endotoxin pleurisy samples was assayed by measuring the migration of rabbit neutrophils toward the pleural liquid under two different conditions: 1) after addition of an anti-IL-8 neutralizing mAb and 2) after desensitization of the neutrophils to rrIL-8. Addition of the anti-IL-8 mAb decreased neutrophil migration toward the pleural liquid by 65 +/- 13 and 75 +/- 22% (mean +/- SE, after 20 and 2,000 ng/ml endotoxin, respectively; P < 0.01 compared with a control mAb). Desensitization of neutrophils to rrIL-8 decreased their migration toward the pleural liquid by 72 +/- 5% (P = 0.03, compared with exposure of neutrophils to buffer alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of polymorphonuclear leucocyte functions in vivo by Yersinia enterocolitica lipopolysaccharide.

A single intravenous injection of 5 micrograms of Yersinia enterocolitica lipopolysaccharide (LPS) inhibits rabbit polymorphonuclear leucocyte (PMN) chemotaxis, enzyme secretion, and respiratory burst activation in response to partially purified rabbit C5a and leucotriene B4 (LTB4). Respiratory burst activation is also inhibited in response to platelet activating factor (PAF). In contrast, all these responses to n-formyl-methionyl-leucyl-phenylalanine (FMLP) remain unaltered. This LPS does not modulate PMN activation in vitro or activate the respiratory burst. Thus Y enterocolitica LPS acts in vivo by inhibiting PMN responses to endogenous mediators of inflammation. This inhibition presumably impairs the elimination of pathogens and might, therefore, provide favourable conditions for induction by bacteria of further immunological consequences.

Lack of correlation between calcium mobilization and respiratory burst activation induced by chemotactic factors in rabbit polymorphonuclear leukocytes.

Low concentrations of FMLP, partially purified rabbit C5a, leukotriene B4 and platelet activating factor induced a rapid rise of intracellular free Ca2+ in rabbit polymorphonuclear leukocytes. However, the four factors differed markedly in their ability to activate the respiratory burst. The peptides FMLP and C5a induced a single, strong chemiluminescence response whereas the lipids leukotriene B4 and platelet activating factor induced a markedly less intense response with a two-peak profile. Respiratory burst activation by the peptides was dependent on extracellular Ca2+ whereas the lipids required both Mg2+ and Ca2+. The results indicate that mobilization of intracellular Ca2+ is insufficient by itself to induce respiratory burst activation and that the intracellular pathways leading to activation differ depending on the nature of the stimulus.

Endotoxin-induced selective dysfunction of rabbit polymorphonuclear leukocytes in response to endogenous chemotactic factors.

To assess the mechanism and specificity of polymorphonuclear leukocyte (PMN) dysfunction induced by endotoxin, rabbits were injected intravenously with 100 micrograms of Escherichia coli endotoxin, and PMN function was studied 18 to 24 h later. Compared to PMN from normal rabbits, peripheral blood PMN from rabbits injected with endotoxin showed diminished chemotactic responsiveness to two endogenous peptides, C5a (complement) and platelet-derived growth factor, and to two endogenous lipids, leukotriene B4 and platelet-activating factor. The chemotactic response to the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), was unimpaired. In contrast to migration, endotoxin injection resulted in inhibition of the secretory response to the two endogenous peptides but not to the lipids or to FMLP. At a 1:4 (vol/vol) dilution, the plasma either 1 or 24 h after the endotoxin injection inhibited normal PMN chemotactic responses to C5a but not to FMLP. Similarly, at a 1:10 dilution, this plasma inhibited normal PMN chemotactic responses to leukotriene B4. The factor responsible for inhibiting responses to leukotriene B4 was anionic, specific for leukotriene B4 responses, and greater than 12,000 daltons. These data may be relevant to understanding PMN dysfunction during gram-negative sepsis.

Endotoxin tolerance diminishes certain antiinflammatory effects of endotoxin.

Endotoxin (bacterial lipopolysaccharide, LPS) is paradoxically both inflammatory and antiinflammatory. A single intravenous injection of 100 micrograms Escherichia coli LPS markedly inhibits the inflammatory changes associated with cutaneous reversed passive Arthus (RPA) reactions in New Zealand white rabbits. Polymorphonuclear (PMN) leukocytes from LPS-treated rabbits exhibit diminished responsiveness in vitro to complement (C5) -derived peptides. Repeated injections of LPS render animals "tolerant", that is, refractory to the toxic and inflammatory effects of LPS. We examined whether tolerance would enhance the ability of LPS to inhibit inflammation not attributable to LPS. Surprisingly, as compared with rabbits receiving a single dose of LPS, tolerant rabbits demonstrated greater inflammatory changes (i.e., PMN exudation, vascular permeability) associated with RPA reactions. PMNs from LPS-tolerant rabbits responded in vitro to C5-derived peptides significantly more than PMNs from rabbits that received a single dose of LPS. We speculate that some antiinflammatory effects of LPS require the toxic or inflammatory effects of LPS itself. These observations might relate to the limited efficacy of fever therapy and the variable effects of gram-negative sepsis on functions of human PMNs.

Complement and polymorphonuclear leukocytes do not determine the vascular permeability induced by intraocular LPS.

The intravitreous injection of an endotoxin of Escherichia coli 055:B5 (LPS; 0.1-0.5 microgram/50 microliters of saline) induces ocular inflammation in rabbits that is maximal 20-24 hours later and disappears by 4 days. The inflammation is characterized by an alteration in ocular vascular permeability (OVP) measured by the ocular extravasation of 125I-albumin and an outpouring of leukocytes, most of which are polymorphonuclear leukocytes (PMNs), as determined by histopathologic study. Nitrogen mustard (mechlorethamine, 1.75 mg/kg) administered 3 days prior to LPS virtually eliminates PMNs in the circulation and those infiltrating ocular tissues 20 hours after intravitreous LPS, and yet the average increase in vascular permeability is not different from that of controls. Cobra venom factor (CVF; 300-400 units) 7 hours before intravitreous LPS produces a greater than 90% decrease in both hemolytic complement activity and zymosan-inducible serum chemotactic activity; yet 20 hours after LPS, the OVP is the same in CVF-treated rabbits and controls. For comparison, an ocular passive Arthus reaction (ovalbumin-anti-ovalbumin) was significantly affected by CVF pretreatment. Chemotactic activity in the aqueous humor is found in both CVF-treated and control rabbits 20 hours after intravitreous LPS. This activity attracts rabbit, but not human, PMNs, is partially heat-sensitive, and is not inhibited when PMNs are preincubated with C5a. These results indicate that neither PMNs nor circulating complement determine the OVP following intravitreous LPS, and that the chemotactic activity present in aqueous humor at the height of the inflammatory response is not primarily C5a.

Complement (C5)-derived chemotactic activity accounts for accumulation of polymorphonuclear leukocytes in cerebrospinal fluid of rabbits with pneumococcal meningitis.

Experiments were performed to identify the chemoattractant for polymorphonuclear leukocytes that appears in the cerebrospinal fluid of rabbits with experimental pneumococcal meningitis. Meningitis was induced in anesthetized New Zealand white rabbits by injecting 10(4) cells of stationary-phase Streptococcus pneumoniae type III intracisternally. Before bacteria were injected, cerebrospinal fluid contained neither polymorphonuclear leukocytes nor chemotactic activity. Significant chemotactic activity for rabbit polymorphonuclear leukocytes was detected 12 h after inoculation with bacteria and was maximal after 18 to 20 h. Chemotactic activity appeared in cerebrospinal fluid while concentrations of pneumococci and total protein were increasing but before there was any accumulation of polymorphonuclear leukocytes. The chemotactic activity in cerebrospinal fluid was heat stable (56 degrees C for 30 min), eluted from Sephadex G-75 with a profile identical to that of the chemotactic activity in zymosan-activated rabbit serum, and was inhibited by treatment with antibodies to native human C5. In addition, preincubation of polymorphonuclear leukocytes with partially purified rabbit C5a selectively inhibited their subsequent chemotactic responses to cerebrospinal fluid. These data indicate that complement (C5)-derived chemotactic activity appears in cerebrospinal fluid during the course of experimental pneumococcal meningitis in rabbits and suggest that this activity accounts for the accumulation of polymorphonuclear leukocytes observed in this infection.

Antiinflammatory effects of endotoxin. Inhibition of rabbit polymorphonuclear leukocyte responses to complement (C5)-derived peptides in vivo and in vitro.

Although capable of provoking a variety of inflammatory effects, endotoxin (bacterial lipopolysaccharide) paradoxically has been reported to be antiinflammatory. The authors have found that single intravenous injections of Escherichia coli endotoxin, 24 hours before challenge, inhibit almost completely the vascular permeability changes and exudation of polymorphonuclear leukocytes induced in rabbit skin by reversed passive Arthus reactions. Whereas intravenous injections of endotoxin also caused modest inhibition of the vascular permeability changes induced in rabbit skin by the synthetic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP), exudation of polymorphonuclear leukocytes was unaffected. Polymorphonuclear leukocytes from rabbits given single injected doses of endotoxin exhibited markedly diminished chemotactic and degranulation responses to complement (C5)-derived peptides in vitro. Responses of these cells to FMLP, however, were normal. These data suggest that selective suppression of polymorphonuclear leukocyte responses to C5-derived peptides accounts, in part, for the antiinflammatory effects of endotoxin.