Anti-endotoxin monoclonal antibodies inhibit secretion of tumor necrosis factor-alpha by two distinct mechanisms.

OBJECTIVE: To determine whether monoclonal antibodies (mAbs) directed against lipopolysaccharide (LPS, endotoxin) act by promoting LPS neutralization, LPS uptake by macrophages, or both processes, the authors assessed the effects of these agents on LPS-induced cytokine secretion and cellular uptake of LPS. SUMMARY BACKGROUND DATA: MAbs directed against LPS have been shown to attenuate LPS-induced macrophage tumor necrosis factor-alpha (TNF-alpha) secretion, a process that may contribute to protective capacity. The mechanisms by which this process occurs have not been established. METHODS: MAbs directed against LPS were evaluated in vitro for their capacity to (1) inhibit TNF-alpha secretion, and (2) alter fluorescein isothiocyanate-labeled LPS uptake (employing flow cytometry analysis and fluorescence microscopy) by the macrophage-like cell line RAW 264.7. RESULTS: MAb 8G9, an IgG3 directed against the O-antigen polysaccharide region of Escherichia coli 0111:B4 LPS, significantly reduced LPS-induced TNF-alpha secretion and promoted a more than 40-fold increase in LPS uptake by macrophages. The authors established that this was mediated by a Fc receptor-mediated process because 8G9 F(ab')2 fragments that lack the Fc portion of the IgG molecule were capable of inhibiting TNF-alpha secretion, but did not promote increased LPS uptake to the same degree. Cross-reactive, anti-deep core/lipid A mAb 1B6, an IgG2a, also promoted uptake of E. coli 0111:B4 LPS and O-antigen polysaccharide-deficient E. coli J5 LPS, but only inhibited TNF-alpha secretion induced by E. coli J5 LPS to which it binds most efficiently. MAb 3D10, an IgM also directed against the O-antigen polysaccharide region of E. coli 0111:B4 LPS, inhibited TNF-alpha secretion but did not increase cellular uptake of LPS, presumably acting solely due to LPS neutralization. Polymyxin B, an antibiotic that binds stoichiometrically to the lipid A portion of LPS, inhibited TNF-alpha secretion and prevented cellular LPS uptake. CONCLUSIONS: These results suggest that IgG and IgM anti-LPS mAbs exert protective capacity by extracellular neutralization of LPS, while IgG Fc receptor-mediated cellular uptake also may serve to bypass macrophage activation and TNF-alpha secretion by promoting internalization and intracellular neutralization.

Anti-lipopolysaccharide monoclonal antibodies inhibit macrophage TNF messenger RNA synthesis in vitro.

Gram-negative bacterial lipopolysaccharide (LPS, endotoxin) directly stimulates macrophages to produce tumor necrosis factor (TNF). TNF, in turn, produces a constellation of adverse effects that includes hypotension, systemic acidosis, arterial hypoxemia, and death. Transcription of the TNF gene occurs within minutes of LPS stimulation and appears to be a critical control point in the synthesis and secretion of TNF protein by macrophages. We hypothesized that murine monoclonal antibody (mAb) 8G9 directed against Escherichia coli 0111:B4 LPS would provide protective capacity against an E. coli 0111:B4 bacterial challenge in vivo and would concurrently inhibit LPS-induced synthesis of TNF mRNA and secretion of TNF protein in vitro. E. coli 0111:B4 LPS was used to stimulate a macrophage-derived cell line (RAW 264.7) to produce TNF in the presence or absence of mAb 8G9. Media alone and LPS without 8G9 mAb served as controls against which the effect of 8G9 mAb was compared. Total cellular RNA was purified and analyzed by a Northern blotting technique utilizing a radiolabeled cDNA probe specific for TNF mRNA. TNF mRNA levels from each sample were quantitated by autoradiograph densitometry. Pretreatment with mAb 8G9 provided protective capacity against an intraperitoneal E. coli 0111:B4 bacterial challenge in vivo when compared with saline pretreatment alone (22% versus 90% mortality respectively, P < 0.05). Preincubation of LPS with mAb 8G9 resulted in a significant inhibition of LPS-induced TNF mRNA synthesis (63 +/- 20%, P < 0.01) and TNF protein secretion (88 +/- 10%, P < 0.001) in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-endotoxin monoclonal antibodies protect by enhancing bacterial and endotoxin clearance.

In this study, we sought to determine the mechanism(s) by which a type-specific anti-lipopolysaccharide monoclonal antibody (an IgG directed against the O-antigen polysaccharide region of Salmonella minnesota lipopolysaccharide) and its F(ab')2 fragments protect during gram-negative bacterial peritonitis and endotoxemia in mice. During peritoneal infection, (1) IgG significantly decreased mortality, bacteremia, and endotoxemia at all time points compared with saline solution pretreatment and (2) F(ab')2 fragments reduced mortality at 24 hours but not thereafter, and had no effect on bacteremia but reduced endotoxemia compared with saline solution pretreatment. In the endotoxin model, IgG pretreatment significantly reduced mortality compared with saline solution pretreatment, while F(ab')2 fragments had no significant effect on mortality. No difference in endotoxemia was observed in mice that received IgG, F(ab')2 fragments, or saline solution pretreatment during endotoxemia. These results suggest that type-specific anti-lipopolysaccharide monoclonal antibodies protect by Fc-mediated clearance of both bacteria and endotoxin.

Protective anti-lipopolysaccharide monoclonal antibodies inhibit tumor necrosis factor production.

Elevated systemic levels of tumor necrosis factor (TNF) have been directly correlated with increased mortality during experimental gram-negative bacterial sepsis. Although monoclonal antibodies (mAbs) directed against gram-negative bacterial lipopolysaccharide (endotoxin, LPS) decrease TNF production in vitro and enhance survival in vivo, the precise relationship between inhibition of TNF secretion and protective capacity has not been defined. We hypothesized that protective anti-LPS mAbs inhibited LPS-stimulated TNF production. To test this hypothesis, we first produced and characterized three anti-LPS mAbs. We then examined the ability of these mAbs to decrease TNF secretion in an in vitro assay using cells from the murine macrophage cell line RAW 264.7. Subsequently, we assessed the protective capacities of these anti-LPS mAbs in a murine mucin peritonitis model of sepsis using live Escherichia coli 0111:B4 bacterial challenge. Our results demonstrated that those anti-LPS mAbs that decreased LPS-stimulated TNF secretion in vitro were protective in vivo. We concluded that inhibition of TNF secretion in vitro reflected protective capacity and that anti-LPS mAbs may confer protection via abrogation of macrophage TNF secretion. Inhibition of TNF production in vitro may provide a valuable test that may facilitate the selection of protective anti-LPS mAbs.

Impaired chemotaxis and neutrophil (polymorphonuclear leukocyte) function in glycogenosis type IB.

Polymorphonuclear leukocyte (PMN) function was investigated in two patients with glycogen storage disease type IB and neutropenia. Glycogen storage disease type IB was documented by liver biopsy and a normal amount of latent glucose-6-phosphatase activity. Patient A had stomatitis, skin infections, and septicemia; patient B had respiratory infections, periodontitis, and oral candidiasis. Absolute neutrophil counts ranged from 114 to 2580/mm3. Diminished and delayed migration of PMN into a skin "window" occurred in B. Random and directed PMN migration under agarose toward f-Met-Leu-Phe, pepstatin A, and zymosan-activated serum were severely diminished in both patients. At 10(-7) M f-Met-Leu-Phe, mean random and directed migration were 52 and 23% (A, n = 3) and 48 and 13% (B, n = 4) of controls. These results were independent of incubation time and chemoattractant concentration. Patients' PMN had diminished quantitative nitroblue tetrazolium reduction compared to controls. B had a significant defect in PMN bactericidal activity with Escherichia coli with less than 0.2 log killing at 2 h. These results further characterize the defect in PMN migration reported by Beaudet et al. (J Pediatr 97:906, 1980). The finding of other abnormalities of PMN function suggests a metabolic defect in the neutrophil which may be related to the microsomal membrane defect in hepatocytes in glycogen storage disease type IB.

Diminished bactericidal capacity for group B Streptococcus in neutrophils from "stressed" and healthy neonates.

This study compared the bactericidal capacity of polymorphonuclear leukocytes (PMNs) from neonates and adults for type Ic group B Streptococcus (GBS), and examined the effect of severe stress on the bactericidal capacity of PMNs from newborn infants. PMNs were obtained from three study groups: 26 adults, 13 healthy neonates (cord blood), and 29 stressed neonates. Stress was defined as an acute respiratory illness or bacterial infection requiring assisted ventilation. Bacterial killing was assessed using a fluorochrome microassay and PMNs adherent to glass coverslips. PMNs from stressed infants killed significantly fewer GBS than PMNs from adults (P less than 0.001 at both time points). PMNs from healthy infants also demonstrated reduced killing compared with adults (P less than 0.01 at 60 min; P less than 0.001 at 90 min). There was no significant difference in bacterial killing between stressed and healthy neonates and no correlation between bactericidal capacity and age at time of study, gestational age, birth weight, peripheral leukocyte count, or Apgar scores. Therefore, the bactericidal capacity for GBS by PMNs from neonates is diminished; however, it is not further compromised by stress.

Phagocytosis of group B streptococcus by neutrophils from newborn infants.

The purpose of this study was to investigate the effect of severe "stress" on neonatal neutrophil (PMN) phagocytosis, and to compare the ability of neonatal and adult PMNs to ingest [3H]-labeled type Ic group B streptococcus (GBS). Three patient populations were studied: healthy and "stressed" newborn infants and healthy adults. Severe "stress" was defined as an acute, noninfectious respiratory illness, sepsis or severe birth asphyxia; 94% of these infants required assisted ventilation. Radiolabeled GBS was opsonized with 5% adult serum, and incubated with PMN monolayers adherent to glass coverslips. Bacterial uptake was determined at 0, 40, 60, and 80 min and expressed as counts per min/10(6) PMNs. Neutrophils from healthy and "stressed" newborn infants demonstrated a significant increase in bacterial uptake with time. Adult controls also showed a significant increase in phagocytosis through 60 min, but not during the 60--80 min time period. PMNs from "stressed" and healthy infants demonstrated enhanced bacterial uptake when compared to adults at 40, 60, and 80 min. There were no significant differences in bacterial ingestion between "stressed" and healthy infants. Radiolabeled GBS uptake did not correlate with birth weight, study age, Apgar scores, or peripheral leukocyte count for the "stressed" newborn population. Neutrophils from healthy and "stressed" newborn infants thus demonstrate increased phagocytosis of GBS when compared to healthy adults.

Diminished bactericidal capacity for group B streptococci of neutrophils from children with chronic granulomatous disease.

The bactericidal capacity of neutrophils from healthy adults, male children with chronic granulomatous disease, and obligate heterozygote mothers for type Ic group B streptococci was assessed, using a fluorochrome microassay. Neutrophils from patients with chronic granulomatous disease had impaired bactericidal capacity for group B streptococci when compared with adults (P less than 0.01). Carriers demonstrated intermediate killing capacity.

In vitro lymphocyte proliferation in response to type III group B streptococci.

The in vitro cell-mediated responses to group B streptococci (GBS) and the relationship of cell-mediated immunity to specific humoral immunity to type III GBS were investigated. Blood was obtained from 20 adult volunteers, and lymphocytes were isolated and cultured in microtiter plates. Each well contained 2 x 10(5) lymphocytes, 15% autologous serum, and either GBS (cell-to-organism ratio of 1:10, 1:1, or 1:0.1), phytohemagglutinin, streptokinase-streptodornase, or RPMI 1640. Cells were harvested at 5, 6, or 7 days, and DNA synthesis was quantitated. Serum antibody titers were determined with an enzyme-linked immunosorbent assay. Maximal lymphocyte responses occurred at 6 days of culture and at a cell-to-organism ratio of 1:1. Individuals with significant antibody titers to type III GBS, as well as those with undetectable antibody, responded to GBS (stimulation index greater than 10). There was a significant difference (P less than 0.001) between mean antibody concentrations in responders (stimulation index greater than 10) and nonresponders (stimulation index less than 10). Thus, the in vitro responses to GBS may be both to a specific antigen and to a nonspecific mitogen and may be important in host immunity to GBS.