Modification of lipid A reduces endotoxin-induced eye effects.

Intravenous endotoxin produces an acute toxic ocular reaction in rabbits. The core component of endotoxin, lipid A, can be modified by acid hydrolysis. This results in a detoxified ET that is relatively ineffective in inducing fever or lethal effects but which retains activity as a mitogen or as a cofactor in inducing tumor necrosis. We report that detoxified endotoxin was relatively ineffective in inducing iris hyperemia, increased ocular vascular permeability, a rise in aqueous humor prostaglandin E2, or the generation of aqueous humor neutrophil chemotactic activity. Chemotactic activity was not increased in aqueous humor even though detoxified endotoxin effectively generated chemotactic activity from serum in vitro. These observations indicate the critical role of lipid A structure in producing ET-induced ocular effects and show that the ability of ET to act as a mitogen, induce tumor necrosis, or generate serum chemotactic activity can be dissociated from its ocular toxicity.

Naturally occurring antibodies against Mycobacterium avium complex.

Serum obtained from 57 healthy individuals and patients admitted to the hospital owing to diverse pathological causes, as well as serum from seven patients with AIDS and disseminated Mycobacterium avium complex (MAC) infection, were studied to determine the prevalence of IgG and IgM antibodies against surface proteins of M. avium complex. Immunodot assay to detect serum positivity against MAC and Western Blot technique in order to determine the MAC antigens eliciting antibody production were performed. Sera from 89 percent and 81 percent of the non-AIDS population had IgG and IgM antibodies against MAC antigens, respectively. In contrast, 43 percent and 71 percent of the AIDS population had IgG and IgM antibodies against MAC antigens, respectively, in the serum. To define further the antigens recognized by these naturally occurring antibodies, the serum of 14 non-AIDS and four acquired immunodeficiency syndrome (AIDS) patients were studied. Multiple antigens of MAC, with molecular weight ranging from 10 to 95 kilodaltons were recognized by IgG and IgM antibodies present in the sera. The IgM type antibodies were shown to react mainly against 10 kilodalton and 31 kilodalton antigenic protein, while the IgG type antibodies were produced mainly against the 10, 31, and 65 kilodalton proteins. Although the pattern of reaction was consistent between non-AIDS and AIDS populations, IgM antibodies were not detected against the 10 kilodalton protein nor were IgG antibodies detected against the 31 kilodalton protein in the AIDS population.

Stimulus-specific effects of endotoxin on superoxide production by rabbit polymorphonuclear leukocytes.

The release of superoxide (O2-) by polymorphonuclear leukocytes (PMN) is an important function that contributes to microbial death. Controversy exists as to the effect of bacterial endotoxin (lipopolysaccharide, or LPS) on the production of O2-. We have injected rabbits with 25 micrograms Escherichia coli LPS intravenously and studied PMN function 18 to 24 hours later. Relative to PMN from saline-injected controls, PMN from LPS-treated rabbits released markedly greater amounts of O2- in response to 10 ng/ml phorbol myristate acetate (PMA) as measured by nmol cytochrome C reduced in 20 minutes (40.8 +/- 7.8 for LPS-treated PMN versus 10.1 +/- 1.6 for control, p less than 0.01). LPS injection, however, significantly reduced O2- release in response to C (complement) 5a (1.4 +/- 0.6 nmole/20 minutes for LPS-treated PMN versus 5.6 +/- 1.3 nmole/20 minutes for control, p less than 0.01). O2- release in response to a third stimulus, n-formyl-methionyl-leucyl-phenylalanine (10(-7) to 10(-9) M), was not affected by LPS. O2- release in response to PMA was enhanced over a wide range of PMA concentrations (10 to 300 ng/ml). Kinetic studies over 30 minutes indicated that, after a brief initial latency in measurable response, LPS enhanced responsiveness to PMA at all time points observed. The reduced responsiveness to C5a corresponds to a previously reported down regulation of receptors for this ligand after intravenous LPS. The observations indicate that intravenous LPS can alter a critical function of PMN for at least 24 hours in a stimulus-specific manner.

Interaction of Mycobacterium avium complex with human macrophages: roles of membrane receptors and serum proteins.

Invasive, disease-associated members of the Mycobacterium avium complex are facultative intracellular pathogens of mammalian macrophages. The mechanism(s) by which M. avium is ingested by mononuclear phagocytes is unknown. We examined the role of membrane receptors on macrophages as well as the role of opsonic components of the serum (complement, fibronectin, C-reactive protein and fibrinogen in the attachment and ingestion of M. avium by human monocyte-derived macrophages. Preincubation of serum with antibodies against C3 and fibronectin, in contrast to preincubation of serum with antibodies against complement-reactive protein and fibrinogen, significantly reduced the binding of M. avium to macrophages in concentration-dependent manner (57 to 93% and 35 to 61% inhibition by anti-C3 and anti-fibronectin antibody, respectively, in a concentration range of 25 to 100 micrograms/ml). We also observed that incubation of macrophages with OKM1 anti-complement receptor type 3 (CR3) antibody in the presence of serum decreased the binding of M. avium to macrophages by 86% +/- 6%, while the same antibody inhibited binding by 63% +/- 7% in the absence of serum. In contrast, incubation of macrophages with anti-LFA-1, anti-p 150.95, anti-CR1, or anti-Mac-1 had no effect on the ability of M. avium to bind to the cell. In addition, incubation of macrophages with alpha-methyl-D-mannoside was also associated with decreased attachment of M. avium to macrophages. These results provide evidence for the role of three macrophage receptors (CR3, fibronectin, and mannosyl-fucosyl receptors) in the uptake of M. avium by human macrophages.

Lipopolysaccharide modulates receptors for leukotriene B4, C5a, and formyl-methionyl-leucyl-phenylalanine on rabbit polymorphonuclear leukocytes.

Peripheral blood polymorphonuclear leukocytes (PMNL) isolated from rabbits after an i.v. injection of endotoxin exhibited decreased chemotactic migration in response to leukotriene B4 (LTB4) and C5a, but not N-formyl-methionyl-leucyl-phenylalanine (fMLP), after endotoxin treatment. The binding of radiolabeled LTB4, fMLP, and C5a to isolated PMNL was assessed in order to determine whether altered receptor expression could account for the observed functional changes. Control PMNL expressed binding sites for fMLP, LTB4, and C5a similar to those previously characterized from human PMNL. Control PMNL expressed a single class of 14,600 +/- 2700 receptors for fMLP with a mean dissociation constant (Kd) of 2.0 +/- 0.6 nM at 0 degrees C, whereas two subclasses of binding sites were expressed for LTB4: 10,300 +/- 6800 high-affinity and 85,600 +/- 53,000 low-affinity binding sites per PMNL with mean Kd for LTB4 of 0.75 +/- 0.43 nM and 70 +/- 58 nM (mean +/- SD, n = 5), respectively. Control PMNL bound [125I]-C5a in a dose-dependent and saturable manner at 24 degrees C. At saturating concentrations of C5a, PMNL obtained from control rabbits bound 270,000 +/- 50,000 molecules of [125I]-C5a with half-maximal binding occurring at [125I]-C5a concentrations of 5.5 +/- 1.9 nM. The binding of LTB4 and C5a to PMNL obtained 24 hr after an i.v. injection of endotoxin was markedly decreased compared with control PMNL. PMNL from endotoxin-treated rabbits exhibited 68% fewer high-affinity binding sites per PMNL for LTB4 and a 51% decrease in the amount of [125I]-C5a bound at saturating concentrations compared with control PMNL. There was no significant change in the Kd of the high-affinity binding sites for LTB4, no change in the Kd and number of the low-affinity binding sites for LTB4, and a small decrease in the apparent Kd for C5a to 3.3 +/- 1.1 nM. Even though the pretreatment with i.v. endotoxin did not alter chemotactic or degranulation responses elicited by fMLP, the endotoxin pretreatment induced an eightfold increase in the receptor density without altering the Kd for fMLP. Decreased receptor expression could account in large part for the decreased chemotactic responsiveness towards C5a and LTB4 induced by LPS. The finding that a substantial increase in receptors for fMLP need not be accompanied by a comparable functional change suggests that decreased efficiency in receptor coupling to intracellular biochemical events may also result from i.v. endotoxin.

The effect of corticosteroids or nitrogen mustard on aqueous humor chemotactic activity induced by intravitreal endotoxin.

In rabbits intravitreal injection of endotoxin induces potent chemotactic activity for monocytes in aqueous humor. We have assessed the effect of methylprednisolone (30 mg/kg intramuscularly) or nitrogen mustard (1.75 mg/kg intravenously) on the generation of this chemotactic activity. Histologic changes, anterior chamber protein extravasation, and aqueous humor cellular infiltration were reduced by corticosteroids to a variable degree. However, even in animals with a marked reduction in protein extravasation or histologic change, chemotactic activity was substantially preserved. Similarly, nitrogen mustard induced a leukopenia without affecting the ability of endotoxin to generate chemotactic activity in aqueous humor. In contrast, corticosteroids reduced both protein extravasation and the generation of chemotactic activity induced by intravenously injected endotoxin. The results suggest that the chemotactic activity induced in the eye by intravitreal endotoxin may be locally synthesized and may be present without a substantial leukocytic infiltrate.