Activation of the alternative complement pathway: recognition of surface structures on activators by bound C3b.
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Animals↗
Biomedical subjects
D C Morrison
Publications and source records attributed to D C Morrison.
The use of polymyxin B and C3H/HeJ mouse spleen cells as criteria for endotoxin contamination.
We have performed experiments designed to evaluate the potential contribution of endotoxin contamination to lymphocyte reponses. Saline and EDTA extracts of 4 different strains of gram negative bacteria were examined for their capacity to initiate mitogenic responses in murine spleen cells. As compared to phenol extracts of these bacteria which contain primarily lipopolysaccharide-LPS, these saline and EDTA extracts were significantly less active in this assay. The mitogenic activity which was present was also manifest in spleen cells from the C3H/HeJ mouse, whereas phenol-extracted LPS preparations were inactive. In addition, mitogenic activity of saline and EDTA extracts was not blocked by polymyxin B, an agent known to abrogate LPS mediated responses. We conclude that LPS contamination may not normally be as significant a problem as had earlier been assumed. However, when endotoxin contamination is present, neither the use of C3H/HeJ spleen cells nor polymyxin B is an appropriate test to evaluate this possibility.
Animals↗
Bactericidal activity of the alternative complement pathway generated from 11 isolated plasma proteins.
Exposure of E. coli K12 W1485 to the cytolytic alternative pathway assembled from the 11 isolated pathway proteins resulted in killing of the bacteria, as evidenced by loss of viability. Lysis of the bacteria required introduction of lysozyme into the reaction mixture. The time-course and dose dependency of bacteriolysis in the isolated system were identical to those in C4-depleted serum. The bacteriolytic activity of the pathway was highly dependent on the concentration of the pathway proteins and became insignificant at 1:16 physiological concentration. Electron microscopic visualization of killed and of lysed bacteria revealed numerous complement membrane lesions and partial disintegration of the outer phospholipid membrane. Scanning electron microscopy showed that killed bacteria were enlarged, partially collapsed and exhibited irregular surface protrusions. Lysed bacteria were fragmented and appeared polymorphic. This study demonstrates that the alternative pathway, in absence of immunoglobulins, has the potential or eradicating gram-negative bacteria.
Blood Bactericidal Activity↗
Role of complement in endotoxin initiated lethality in mice.
We have examined the role of complement in eliciting a lethal response in C3H/HeJ and C3H/St mice. The results reported here indicate that endotoxin-initiated complement activation, leading to significant drops in circulating C3 levels, is not sufficient to cause lethality. The complement system in both strains was demonstrated to be responsive in vitro to activation both by E. coli 0111:B4 LPS I, an alternative pathway activator in other systems, as well as S. minnesota R595 LPS, which activates almost exclusively the classical pathway. In vivo injection of high (lethal) doses of E. coli 0111:B4 LPS I and S. minnesota R595 LPS causes a significant decrease in the circulating C3 levels of both strains after 4 hr. In contrast, circulating C3 levels were not significantly different from normal values in either strain following injection with minimal (lethal) amounts of E. coli 0111:B4 LPS II, a weakly anticomplementary LPS preparation. In all cases, lethality was observed in only the C3H/St mice, indicating that neither complement activation, nor the lack of it, is responsible for lethality in mice.
Animals↗
Bacterial endotoxins and host immune responses.
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Animals↗
Stimulation of T-independent antibody responses by hapten-lipopolysaccharides without repeating polymeric structure.
The murine immune response to a haptenated lipopolysaccharide (LPS) lacking repeating oligosaccharide determinants was studied. The LPS was extracted from a rough strain of bacteria (Salmonella minnesota R595) and chemically haptenated with either trinitrophenol or fluorescein isothiocyanate. These preparations of hapten-R595 LPS were shown to be immunogenic. Furthermore, the immune response to the hapten was demonstrated to occur independent of T cells and was not merely the result of enhanced polyclonal B-cell activation. The capacity of such hapten-LPS conjugates without repeating polymeric structures to stimulate T-independent antibody responses provides information on the molecular requirements for the activation of murine B lymphocytes.
Animals↗
Mechanisms of lipopolysaccharide-initiated rabbit platelet responses. II. Evidence that lipid A is responsible for binding of lipopolysaccharide to the platelet.
The mechanism of bacterial lipopolysaccharide-(LPS) initiated, complement-(C) mediated rabbit platelet lysis has been examined. The results of these studies support our previous observations that activation of the alternative C pathway is required for platelet lysis and that preparations of LPS that activate only the classical pathway (e.g., lipid A) do not cause lysis. The temporal relationship of the interaction of the LPS with the platelet before the addition of plasma suggests a time-dependent association of the LPS with the platelet. On the basis of a number of experiments, including inhibition with polymyxin B, treatment of LPS with alkali, and blocking experiments with polysaccharide-free LPS preparations, it is concluded that the lipid A region of the LPS molecule is responsible for attaching the LPS to the platelet. Finally, a comparison of the activity of lipid A-associated protein-LPS complexes with protein-free LPS demonstrated that an equivalent extent of platelet lysis was achieved with one-one hundredth the concentration of the former as that required for protein-free LPS. The data suggest that LAP facilitates attachment of the LPS to the platelet.
Animals↗
Macrophage stimulation by bacterial lipopolysaccharides. II. Evidence for differentiation signals delivered by lipid A and by a protein rich fraction of lipopolysaccharides.
Stimulation of macrophages to lyse tumor cells is a property common to lipopolysaccharide (LPS) extracted from a variety of smooth and rough bacterial strains by several different preparative procedures. The relationship between macrophage stimulation and the structural characteristics of LPS is defined. In protein-free LPS, lipid A bears the stimulatory signal which results in the differentiation of elicited macrophages into killer cells. The polysaccharide moiety is neither stimulatory itself nor does it block the activity of complete LPS on macrophages. Extraction of LPS by the butanol or Boivin procedures produces preparations in which LPS is complexed through its lipid A moiety to a protein rich component, LAP. Isolated LAP delivers a macrophage differentiation signal which is independent of lipid A. The presence of these two structurally distinct constituents in the cell walls of gram-negative bacteria broadens the biological environments in which they can stimulate macrophages in vivo.
Cell Differentiation↗
Two distinct mechanisms for the initiation of mast cell degranulation. II. A specific inhibition of amine release by serum proteins.
Our experiments have provided additional data in support of the concept that different mast cell activators follow distinct biochemical path-ways in the initiation of secretion and degranulation. To do this we have taken advantage of the observation that some serum proteins, and albumins in particular, have the capacity to inhibit selectively the release of amines from rat peritoneal mast cells initiated by some, but not all, stimuli. We show that the relative inhibition of release obtained is independent of the concentration of activator but dependent upon the concentration of albumin, indicating that the inhibitory process does not involve a direct activator--inhibitor interaction. Finally, our data demonstrate that the inhibition does not interfere with the ability of the acitivator to interact with the mast cell. Thus, cells incubated with activator in the presence of inhibitor become increasingly unresponsive, or desensitized, to subsequent challenge with activator in the absence of inhibitor. These combined data therefore provide evidence, that, in addition to a selectivity in the activation/desensitization process initiated by different mast cell stimuli, at least one of the biochemical steps subsequent to the activation step is also not shared by all mast cell stimuli.
Animals↗
Receptor modulation and mast cell secretion.
Co-workers and I examined the early events in the interaction of rat peritoneal mast cells with a number of noncytotoxic mast cell stimuli. These studies demonstrated that an interaction limited to the cytoplasmic membrane is sufficient to induce a secretory mast cell response. Additional experiments suggested that a continued interaction between the stimulus and the membrane receptor is required for maintenance of the secretory response. We have provided evidence to support the concept that different mast cell activators interact with different receptors on the cell membrane and initiate distinct biochemical pathways leading to secretion. Finally, we have shown that pretreatment of mast cells with concentrations of a stimulus that, by themselves, are incapable of initiating a secretory response can profoundly affect the ability of mast cells to respond fully to a subsequent challenge with an optional concentration of stimulus.
Anaphylatoxins↗
Mechanisms of lipopolysaccharide-initiated rabbit platelet responses: alternative complement pathway dependence of the lytic response.
Experiments were performed to examine the relationship of endotoxin-initiated complement activation and rabbit platelet lysis. The results of these experiments supported the concept that activation of the alternative pathway is required for endotoxin-initiated complement-dependent rabbit platelet lysis. Our data demonstrated that preparations of endotoxin or isolated lipid A, which activate selectively the classical pathway, are incapable of initiating platelet lysis. Essentially equivalent results were obtained in citrated or heparinized plasma, although the latter anticoagulated plasma appeared to be more efficient in supporting lysis. Additional data support the concept that natural antibody to either the polysaccharide or the lipid A region of the lipopolysaccharide, which might be present in rabbit plasma, probably did not play a prominent role in the complement-mediated lytic response.
Animals↗
Distribution of endotoxin (lipopolysaccharide) in the tissues of lipopolysaccharide-responsive and -unresponsive mice.
We examined the distribution of bacterial lipopolysaccharide (LPS) in LPS-responsive (C3H/St) and LPS-unresponsive (C3H/HeJ) mice. The results reported here demonstrate that the rates of removal of an immunological or a toxic dose of LPS from the circulation are the same in both strains of mice. C3H/St spleens accumulated significantly more LPS than C3H/HeJ spleens after the intravenous injection of either an immunogenic or a toxic dose of LPS. There was also a greater amount of LPS associated with cells teased from C3H/St spleens compared to those from C3H/HeJ spleens. After a toxic dose of LPS, there was more LPS in C3H/St lymph nodes, adrenals, lungs, kidneys, and heart than in the corresponding C3H/HeJ tissues. The accumulation of more LPS in tissues from C3H/St mice compared to C3H/HeJ mice suggests that these tissues are involved in the pathophysiological and, ultimately, the toxic effects of LPS. The differential accumulation of LPS in the tissues of these two strains may be the reason for the decreased responses of C3H/HeJ mice to LPS.
Animals↗
Binding and activation of the first component of human complement by the lipid A region of lipopolysaccharides.
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Bacterial Proteins↗
The effects of bacterial endotoxins on host mediation systems. A review.
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Animals↗
The selective binding of aggregated IgG to lipid A-rich bacterial lipopolysaccharides.
To explore the mechanism by which certain bacterial lipopolysaccharides (LPS) enhance platelet stimulation by aggregated IgG, we studied potential interactions between the two ligands. Lipid A or the lipid A-rich LPS from Salmonella minnesota R595 (LPS R595) selectively increased the sedimentation of aggregated rather than monomer IgG in sucrose density gradients. Insolubilized IgG aggregates adsorbed LPS R595 from solution. These two experiments suggested binding of IgG aggregates to LPS R595 or lipid A and this was confirmed by isopycnic density gradient ultracentrifugation studies. The presence of R595 LPS shifted the equilibrium density profile of aggregated IgG from its usual equilibrium density at 1.30 g/ml to a new position superimposable with that of the LPS R595. The possibility that a selective binding of IgG aggregates to LPS may represent a fundamental mechanism of the action of LPS on cellular mediation systems is proposed.
Antigen-Antibody Complex↗
Inhibition of the mitogenic response to lipopolysaccharide (LPS) in mouse spleen cells by polymyxin B.
The addition of low doses of the cationic polypeptide antibiotic, polymyxin B (PB), to cultures of mouse spleen cells inhibits lipopolysaccharide-(LPS) induced DNA synthesis but not that stimulated by PPD, PHA, or Con A. Inhibition is stoichiometric; the mitogenic response is suppressed by 50% at a weight ratio of PB:LPS of 0.055 to 1. Furthermore, PB-LPS complexes have a much reduced mitogenic capacity. These complexes inhibit the mitogenic response of spleen cells to unmodified LPS but not to PPD, Con A, or PHA. The inhibitory activity of PB is less effective when added after LPS is mixed with responding cells, achieving 50% inhibition when addition is made at 4 to 6 hr. Time course experiments indicate that partial inhibition is a reflection of a lower rate of DNA synthesis. Thus, PB inhibition of LPS mitogenesis apparently occurs as a result of formation of PB-LPS complexes with reduced mitogenic capacity. Specific inhibition by the complexes of mitogenesis induced by native LPS suggests that the inactive complex may bind to B cells but is unable to trigger them.
Animals↗
Activation of the classical and properdin pathways of complement by bacterial lipopolysaccharides (LPS).
Bacterial lipopolysaccharides (LPS) have been demonstrated to activate both the classical and the properdin pathways of complement. The lipid A region of the LPS is responsible for classical pathway activation and the polysaccharide region responsible for properdin pathway activation. Classical pathway activation by lipid A does not depend upon antibody to the lipid A and properdin pathway activation proceeds by a lipid A-independent mechanism. The polysaccharide portion of the LPS molecule exerts a modifying influence on the potential anticomplementary activity of the lipid A.
Complement System Proteins↗
Lipid A as the biologically active moiety in bacterial endotoxin (LPS)-initiated generation of procoagulant activity by peripheral blood leukocytes.
Preparations of rabbit or human leukocytes, when incubated with bacterial endotoxins (lipopolysaccharides, LPS) are stimulated to generate a procoagulant-tissue factor activity (TFa). As LPS has been shown to consist of specific repeating oligosaccharide side chains (O-antigen) linked to a central polysaccharide core region that is, in turn, linked to the lipid region of the molecule (lipid A), we have examined the biochemical requirement of the LPS necessary for generation of TFa. Using preparations of LPS from mutant strains of bacteria, which contain varying amounts of polysaccharide in relation to lipid A, we have demonstrated that activity is associated with the lipid A region of the LPS molecule. These observations have been confirmed using isolated lipid A, which is a potent stimulator of TFa, as well as a native protoplasmic polysaccharide that is both devoid of lipid A and without detectable TFa stimulatory activity. Modification of LPS by treatment with mild alkali abrogated its capacity to stimulate TFa generation. In addition, such altered preparations of LPS partially inhibit the stimulatory effect of native LPS. Similarly, treatment of LPS (or lipid A) with the antibiotic polymyxin B substantially inhibited the stimulatory effect of LPS.
Animals↗