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Am J Physiol Regul Integr Comp Physiol 276: R171-R177, 1999;
0363-6119/99 $5.00
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Vol. 276, Issue 1, R171-R177, January 1999

IgG-coated erythrocytes augment the lipopolysaccharidestimulated increase in serum tumor necrosis factor-alpha

Craig A. H. Richard, Paul W. Gudewicz, and Daniel J. Loegering

Department of Physiology and Cell Biology, Albany Medical College, Albany, New York 12208-3479

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Previous studies have shown that the injection of IgG-coated erythrocytes (EIgG) caused an increase in the mortality rate due to bacterial lipopolysaccharide (LPS). This observation led to the present evaluation of the effect of EIgG on the LPS-stimulated increase in serum tumor necrosis factor-alpha (TNF-alpha ) levels and TNF-alpha secretion by macrophages. The prior injection of EIgG augmented the increase in LPS-stimulated serum TNF-alpha levels ninefold at 1 h after LPS. Serum TNF-alpha levels were augmented when LPS was injected 2 or 6 h after EIgG but not at 0.5 or 12 h after EIgG. Complement activation caused by EIgG may contribute to the priming for TNF-alpha , because activation of complement with cobra venom factor caused a threefold augmentation of the LPS-stimulated serum TNF-alpha levels. Isolated macrophages that had ingested EIgG or were adherent to immobilized IgG showed augmented TNF-alpha secretion in response to LPS. Thus clearance of immune complexes from the blood can augment the LPS-stimulated increase in serum TNF-alpha levels that is due, in part, to complement activation and signaling via Fcgamma R.

splenic macrophages; RAW 264.7 cells; rats

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

TUMOR NECROSIS factor-alpha (TNF-alpha ) is a multifunctional cytokine that is capable of activating many of the cascades associated with inflammation, injury, and sepsis (2, 42, 43). The administration of TNF-alpha produces many of the responses to bacterial lipopolysaccharide (LPS), and passive immunization against TNF-alpha can prevent the effects of LPS, including lethality, in animals. Antibodies against TNF-alpha have also been shown to be remarkably therapeutic for rheumatoid arthritis (9, 10).

Although LPS is well known to cause TNF-alpha secretion, signaling via Fcgamma R will also stimulate the secretion of TNF-alpha by monocytes and macrophages. Secretion of TNF-alpha can be induced by incubation of macrophages with immune complexes, allowing the cells to adhere to immobilized IgG and cross-linking Fcgamma R with antibodies (5, 11, 15, 35). The ability of immune complexes containing low-density lipoproteins and rheumatoid factor to stimulate TNF-alpha secretion has been proposed to contribute to the pathogenesis of atherosclerosis and arthritis (14, 44). In addition, Fcgamma R signaling is capable of stimulating the secretion of other cytokines and chemokines, including interleukin (IL)-1beta , IL-6, IL-8, I-309 and macrophage inflammatory protein-1alpha (20-22, 34).

Either LPS or Fcgamma R can stimulate TNF-alpha secretion, but in most cases the amount of TNF-alpha secreted via Fcgamma R is much less than the amount when the cells are stimulated with LPS (15, 35). A few previous studies have shown a synergistic effect for the combination of Fcgamma R and LPS stimulation of cytokine and chemokine secretion. Kindt et al. (15) showed that preincubating monocytes with LPS for 30 min increased the TNF-alpha and IL-1beta secretion induced by Fcgamma R cross-linking. Marsh et al. (21) found that monocytes adherent to immobilized IgG had a 100-fold increase in sensitivity for LPS-stimulated IL-1beta secretion. Finally, Selvan et al. (34) demonstrated that stimulation of monocytes with immobilized IgG and LPS was synergistic for the secretion of I-309 and macrophage inflammatory protein-1alpha .

Immune complexes such as IgG-coated erythrocytes (EIgG) can activate complement in the blood and thereby generate ligands for complement receptors (CRs) and anaphylatoxins (C5a, C3a). Signaling via macrophage CR1 and CR3 can activate nuclear factor-kappa B, suggesting the possibility for increased TNF-alpha transcription (39). Also, monocytes attached to a surface coated with a CR3 ligand (fibrinogen) had augmented LPS-stimulated TNF-alpha secretion (8). C5a has been shown to be capable of augmenting the TNF-alpha secretion in response to LPS (1). Therefore, complement activation could contribute to the LPS-stimulated increase in serum TNF-alpha levels.

Previous studies have shown that the phagocytosis of EIgG can depress several macrophage functions, including in vivo clearance function and in vitro phagocytic function, respiratory burst capacity, arachidonate release, tumor cell killing, and bactericidal function (3, 7, 12, 16, 17, 32, 33). The possibility that the injection of EIgG augmented the LPS-stimulated increase in serum TNF-alpha levels was suggested by the increase in mortality due to LPS after EIgG phagocytosis (16, 17). The present study demonstrated that the injection of EIgG caused a large augmentation of the increase in serum TNF-alpha levels stimulated by a subsequent injection of LPS. A potential role for the complement activation caused by EIgG in the priming for TNF-alpha secretion was supported by the ability of cobra venom factor (CVF) to augment the LPS-stimulated increase in serum TNF-alpha levels. EIgG phagocytosis also augmented the LPS-stimulated TNF-alpha secretion by isolated macrophages. Therefore, this novel observation indicates that immune complexes can cause an exaggerated cytokine response to LPS and suggests a mechanism for the ability of bacterial infections to trigger or exacerbate conditions such as atherosclerosis, arthritis, and glomerular nephritis (4, 9, 19, 37, 38, 40).

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals. Male Sprague-Dawley rats weighing 250-300 g were used. Animals were anesthetized with halothane for all procedures. All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the Institutional Review Board for the care and use of laboratory animals at Albany Medical College.

Serum TNF-alpha levels after LPS. Salmonella enteriditis LPS B was obtained from Difco Labs. LPS suspended in PBS was stored at -70°C and sonicated for 1 min just before intravenous injection. Initial studies showed that serum TNF-alpha levels were maximal at 1 h after LPS injection, low at 2 h, and undetectable at 3 h. On this basis, blood samples were taken from the tail at 1, 2, and 3 h after LPS injection. LPS was injected intravenously 0.5, 2, 6, or 12 h after EIgG. CVF (Naja naja kaouthia; Sigma, St. Louis, MO) was injected intravenously (1 U/kg) 2 h before LPS. Serum C3 levels were determined by radial immunodiffusion, as previously described (16). Experiments were done on at least 3 separate days, with two or three animals in a group each day.

Preparation of EIgG for in vivo studies. EIgG for in vivo studies were prepared using washed rat erythrocytes and the IgG fraction of rabbit anti-rat erythrocyte antisera (US Biochemical), as previously described (16). The concentration of IgG was adjusted to achieve 70-80% liver localization of 51Cr-labeled EIgG (3 × 109 cells/kg) at 30 min after intravenous injection. Previous studies have shown that EIgG prepared from rat erythrocytes do not lyse when injected due to the low lytic activity of complement for homologous erythrocytes (16).

Macrophages. Splenic macrophages were isolated from rat spleens by teasing the tissue apart in DMEM containing 100 U/ml penicillin and 100 µg/ml streptomycin. The resulting cell suspension was incubated in polystyrene petri dishes for 2 h, and the adherent macrophages were then cultured in DMEM plus 10% heat-inactivated calf serum for 4 days. The resulting macrophages were adherent at 6 × 105 cells/well (24-well plates). All experiments with splenic macrophages were carried out in DMEM with 2.5% heat-inactivated calf serum.

RAW 264.7 cells, a murine macrophage-like cell line, were maintained and used for experiments in RPMI supplemented with 10% heat-inactivated calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were allowed to adhere overnight in 48-well plates (1.5 × 105 cells/well) and were washed 2 h before the experiment.

EIgG were prepared for use in vitro from sheep erythrocytes and anti-sheep erythrocyte IgG (Accurate) (3). EIgG phagocytosis was quantified using 51Cr-labeled EIgG, and the radioactivity associated with the macrophages was determined after hypotonic lysis of noningested erythrocytes (3). Wells were coated with IgG by incubation overnight at 4°C with nonimmune rabbit IgG (0.1, 1.0, and 10 µg/ml). These wells were washed three times just before adding the RAW 264.7 cells.

In vitro experimental protocol. Splenic macrophages or RAW 264.7 cells were incubated with EIgG, erythrocytes, or the amount of IgG used to prepare 1 × 107 EIgG for 2 h, washed, and covered with media containing 100 ng/ml LPS. RAW 264.7 cells (2 × 105 cells/well) were added to wells coated with IgG and allowed to adhere for 1 h before addition of LPS. Four hours after addition of LPS, media was removed for analysis of TNF-alpha levels by ELISA.

TNF-alpha bioassay. The determination of serum TNF-alpha activity was carried out using a modification of the method of Holobaugh and McChesney (13). L929 cells (murine fibrosarcoma; ATCC, Manassas, VA) were plated in 96-well plates at 5 × 104 cells/well in low-glucose DMEM containing 10% calf serum, 2 mM L-glutamine, 0.1 µg/ml nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µg/ml gentamicin. After incubation overnight, fresh medium containing 1 µg/ml actinomycin plus serially diluted samples or recombinant human TNF-alpha standards (R and D Systems) was added to the wells and incubated for 18 h. After washing, the remaining cells were stained with crystal violet and the absorbance was read at 595 nm. A unit of TNF-alpha activity was defined as the dilution of the sample that caused a 50% decrease in absorbance. Specificity of the bioassay was confirmed by the ability of an anti-murine TNF-alpha polyclonal antibody (Genzyme) to cause complete inhibition of the cytotoxicity of the samples.

TNF-alpha ELISA. The assay described by Tracy and Fox (41) was used. Wells were initially coated with monoclonal hamster anti-murine TNF-alpha (1 µg/ml) and then blocked with 1% BSA. Samples were incubated in the wells for 24 h at 4°C followed by polyclonal rabbit anti-murine TNF-alpha and horseradish peroxidase-conjugated goat anti-rabbit IgG. Horseradish peroxidase substrate was added for 5 min, and the reaction was stopped with phosphoric acid. Absorbance was read, and TNF-alpha concentration was determined from a standard curve of recombinant murine TNF-alpha (Genzyme). TNF-alpha results from the L929 assay and ELISA were compared by linear regression analysis of samples measured with both assays. Twenty serum samples and twenty-nine media samples had r values of 0.867 (P < 0.001) and 0.767 (P < 0.001), respectively.

Statistics. Data are expressed as means ± SE. Comparisons were analyzed using ANOVA plus the Newman-Keuls post hoc comparison for differences between groups. The level of confidence was placed at 95% for all experiments.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

EIgG augments LPS-stimulated increase in serum TNF-alpha levels. Serum TNF-alpha levels were increased at 1 and 2 h after the injection of LPS (2 mg/kg) into animals that received a prior injection of PBS (EIgG diluent) (Fig. 1). A similar increase in LPS-stimulated TNF-alpha levels was seen when rat erythrocytes that were not coated with IgG (2 × 1010 cells/kg) were injected. In contrast, the injection of EIgG (2 × 1010 cells/kg) 2 h before LPS (2 mg/kg) caused the increase in serum TNF-alpha levels to be augmented and protracted (Fig. 1). The augmentation was ninefold at 1 h after LPS and 27-fold at 2 h after LPS, and serum TNF-alpha levels were still detectable at 3 h after LPS. The injection of EIgG or PBS followed 2 h later by PBS in place of LPS did not cause a detectable increase in serum TNF-alpha levels. These results show that the prior injection of EIgG can augment the LPS-stimulated increase in serum TNF-alpha levels.


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  		Fig. 1.   Effect of IgG-coated erythrocytes (EIgG) on lipopolysaccharide (LPS)-stimulated serum tumor necrosis factor-alpha (TNF-alpha ) levels. PBS, EIgG (2 × 1010 cells/kg), or erythrocytes (2 × 1010 cells/kg) were injected intravenously 2 h before injection of LPS (2 mg/kg iv) or PBS. Serum samples for TNF-alpha analysis (bioassay) were taken at 1, 2, and 3 h after injection of LPS or PBS. Values are means ± SE, with 4-8 animals per group. Groups: open circle , PBS before LPS; bullet , EIgG before LPS; star , erythrocytes before LPS; , EIgG before PBS; down-triangle, PBS before PBS. * P < 0.05 compared with PBS before LPS group.

Effect of dose of EIgG. When the dose of EIgG was reduced to 0.5 × 1010 cells/kg, augmentation of the LPS-stimulated (2 mg/kg) serum TNF-alpha levels was not significantly less than that caused by 2 × 1010 cells/kg EIgG (Fig. 2). The augmentation was sixfold at 1 h and 17-fold at 2 h after LPS. However, when the dose of EIgG was decreased to 0.1 × 1010 cells/kg, no augmentation of serum TNF-alpha levels was observed.


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  		Fig. 2.   Effect of different doses of EIgG on LPS-stimulated serum TNF-alpha levels. EIgG were injected at doses of 2.0, 0.5, or 0.1 × 1010 cells/kg 2 h before LPS (2 mg/kg). Controls received PBS before LPS. Serum samples for TNF-alpha analysis (bioassay) were taken at 1, 2, and 3 h after injection of LPS. Values are means ± SE, with 6 animals per group. Groups: bullet , 2 × 1010 cells/kg EIgG; open circle , 0.5 × 1010 cells/kg EIgG; black-triangle, 0.1 × 1010 cells/kg EIgG; down-triangle, PBS. * P < 0.05 compared with PBS before LPS control group.

Time between the injection of EIgG and LPS. When the time between the injection of EIgG (2 × 1010 cells/kg) and LPS (2 mg/kg) was decreased from 2 h to 0.5 h, serum TNF-alpha levels were not significantly augmented after LPS (Fig. 3). Increasing the time between the injection of EIgG and LPS to 6 h resulted in an augmentation of TNF-alpha levels that was similar to that with the 2 h group (10-fold increase at 1 h after LPS). When the time between EIgG and LPS was increased to 12 h, the TNF-alpha response to LPS was not significantly augmented (Fig. 3).


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  		Fig. 3.   Effect of injecting EIgG at different times before LPS on LPS-stimulated increase in serum TNF-alpha levels. EIgG (2 × 1010 cells/kg) were injected at 0.5, 2, 6, or 12 h before injection of LPS (2 mg/kg). Controls received PBS 2 h before LPS. Serum samples for TNF-alpha analysis (bioassay) were taken at 1, 2, or 3 h after injection of LPS. Values are means ± SE, with 4-6 animals per group. Groups: open circle , EIgG 0.5 h; bullet , EIgG 2 h; black-triangle, EIgG 6 h; down-triangle, EIgG 12 h; , PBS. * P < 0.05 compared with PBS before LPS control group.

Effect of dose of LPS. These studies were carried out to determine if the injection of EIgG augments the TNF-alpha response to low levels of LPS that might occur after infection. Initial studies were carried out to determine the minimum dose of LPS that caused an increase in serum TNF-alpha levels, as determined by ELISA. As shown in Fig. 4, 1.0 and 0.3 µg/kg doses of LPS stimulated low levels of serum TNF-alpha , both of which were augmented by EIgG. EIgG without LPS resulted in minimally detectable levels of TNF-alpha , which was not seen with the bioassay. These results suggest that the injection of EIgG can prime macrophages for an exaggerated cytokine response to minimal TNF-alpha -eliciting levels of LPS.


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  		Fig. 4.   Effect of EIgG on serum TNF-alpha response to different doses of LPS. EIgG (2 × 1010 cells/100 g) or PBS was injected intravenously 2 h before injection of indicated doses of LPS. Serum samples for TNF-alpha analysis (ELISA) were taken at 1 h after injection of LPS. Values are means ± SE, with 3-6 animals per group. * P < 0.05 compared with PBS-LPS.

CVF augments LPS-stimulated increase in serum TNF-alpha levels. CVF was given to determine the effect of the complement activation caused by EIgG in the absence of phagocytosis. CVF (1 U/kg) given 2 h before LPS (2 mg/kg) caused an augmentation of the TNF-alpha response (Fig. 5). The augmentation was 2.6-fold at 1 h and 3.5-fold at 2 h after LPS. At this dose, CVF caused a 10% decrease in the C3 levels at the end of the experiments (5 h after CVF; data not shown). This degree of complement activation is similar to the 11% decrease in C3 levels previously shown to be induced by the injection of 1.7 × 1010 EIgG/kg (16). Thus complement activation may contribute to the priming for TNF-alpha secretion caused by EIgG.


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  		Fig. 5.   Effect of cobra venom factor (CVF) on LPS-stimulated increase in serum TNF-alpha levels. PBS or CVF (1 U/kg) was injected 2 h before injection of LPS (2 mg/kg iv). Serum samples were taken at 1, 2, and 3 h after injection of LPS, and serum TNF-alpha levels were determined by ELISA. Values are means ± SE, with 6 animals per group. * P < 0.05 compared with respective PBS-LPS value.

Effect of EIgG on LPS-stimulated TNF-alpha secretion by isolated macrophages. Splenic macrophages incubated with EIgG (1 × 107 cells/ml) ingested 1.3 EIgG per macrophage, and RAW 264.7 cells ingested 3.1 EIgG per macrophage. EIgG phagocytosis caused an augmentation of the LPS-stimulated secretion of TNF-alpha in both types of macrophage (Table 1). However, the augmentation was much less than that observed in vivo. LPS-stimulated TNF-alpha secretion was unaltered when macrophages were incubated with erythrocytes that were not coated with IgG. Similarly, incubation of RAW 264.7 cells with the amount of IgG used to prepare 1 × 107 EIgG had no effect on the LPS-stimulated TNF-alpha secretion (data not shown). Under these conditions, LPS alone stimulated the secretion of 346 ± 94 and 742 ± 52 pg/ml TNF-alpha by splenic macrophages and RAW 264.7 cells, respectively. Phagocytosis of EIgG without subsequent LPS caused the secretion of <5% of that amount of TNF-alpha .

                              
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  		Table 1.   Effect of EIgG on LPS-stimulated TNF-alpha production by splenic macrophages or RAW 264.7 cells

To determine if phagocytosis was required to prime macrophages for TNF-alpha secretion, RAW 264.7 cells were added to plates coated with IgG. Adherence to immobilized IgG caused a significant augmentation of LPS-stimulated TNF-alpha secretion when plates were coated with 1.0 or 10 µg/ml IgG (Fig. 6). The highest level of augmentation was similar to that seen with EIgG phagocytosis. Thus signaling via Fcgamma R without phagocytosis can prime macrophages for LPS-stimulated TNF-alpha secretion.


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  		Fig. 6.   Effect of IgG-coated wells on LPS-stimulated TNF-alpha secretion. Tissue culture wells were coated with indicated concentrations of IgG and washed. RAW 264.7 cells were added to these wells and incubated for 1 h. LPS was then added (100 ng/ml), and 4 h later media was removed for TNF-alpha analysis by ELISA. Values are means ± SE for 3 experiments. * P < 0.05 compared with LPS only.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The present study demonstrated that the increase in serum TNF-alpha levels due to LPS is augmented after the injection of EIgG. A substantial effect was observed when LPS was injected 2 or 6 h after EIgG, with no augmentation at 30 min and 12 h after EIgG. The lowest dose of EIgG tested that augmented the TNF-alpha response to LPS was 0.5 × 1010 cells/kg. EIgG augmented the response to the lowest dose of LPS that caused a detectable increase in serum TNF-alpha levels. A previous study by Satoh et al. (29) demonstrated that the injection of IgG-coated sheep erythrocytes into mice caused an augmentation of the increase in serum TNF-alpha levels stimulated by LPS. The ability of CVF to augment the LPS-stimulated increase in serum TNF-alpha suggests that activation of complement may also contribute to the priming effect of EIgG. These results show that immune complexes can substantially augment the serum TNF-alpha response to LPS.

Our previous studies have been oriented toward the effect of erythrocyte phagocytosis induced by thermal injury on macrophage function (16, 17, 32). It is proposed that erythrocyte phagocytosis depresses macrophage function, which contributes to the impaired host defense against bacterial infections that is associated with this form of injury. The injection of EIgG was used as a model for thermal injury-induced erythrocyte phagocytosis. The number of erythrocytes taken up by the liver and spleen after experimental thermal injury in rats was found to be 11.7 × 108 cells (18, 31). The lowest dose of EIgG that augmented the LPS-stimulated increase in serum TNF-alpha levels (0.5 × 1010 cells/kg) would be expected to cause the phagocytosis of ~7.6 × 108 EIgG, as extrapolated from our previous work (16). Thus burn injury can induce the phagocytosis of a sufficient number of erythrocytes to augment the cytokine secretion caused by LPS. In addition, the 0.5 × 1010 cells/kg dose of EIgG is close to the minimum dose of EIgG that caused an increase in mortality due to LPS (8.7 × 1010 cells/kg) (16).

The duration of the effect of EIgG on the LPS-stimulated increase in serum TNF-alpha levels was similar to other effects of injected EIgG (17). LPS-stimulated serum TNF levels returned to normal between 6 and 12 h after EIgG injection. We showed previously that Kupffer cell CR clearance function was depressed at 3 h after injection, returned nearly to normal at 6 h, and was at control levels at 12 h after injection (17). In addition, the mortality rate due to LPS was increased at 3 and 6 h after EIgG but returned to normal at 12 h after injection. Recovery of normal function was related to the digestion of EIgG by Kupffer cells. Electron microscopy demonstrated that there was a decrease in the number of intact EIgG within Kupffer cells at 6 h, and only a few remained at 12 h after injection (17). It is possible that signaling from Fcgamma R and/or CRs was terminated as the erythrocytes were disrupted within the phagosomes.

The reason EIgG did not prime for TNF-alpha secretion when LPS was injected 30 min after the EIgG is not clear at this time. Our previous work showed that the phagocytosis of EIgG by Kupffer cells is maximal at 30 min after injection, but splenic uptake continues for up to 3 h (17). Although splenic uptake of EIgG could be critical for the priming effect, other factors such as the generation of complement activation products, production of erythrocyte degradation products, maturation of signaling events, or the synthesis of new proteins may contribute.

Complement activation may contribute to the priming for TNF-alpha secretion caused by EIgG. We have previously shown that the injection of EIgG at a dose of 1.7 × 1010 cells/kg decreased serum C3 levels by 11% (16). The dose of CVF (1 U/kg) used in the present study caused a similar degree of complement activation and augmented the LPS-stimulated increase in serum TNF-alpha levels. A previous study found that a larger dose of CVF (1 U/mouse) augmented the serum TNF-alpha response to OK-432, a streptococcal preparation (30). The in vitro studies reported here show that the priming caused by Fcgamma R-mediated phagocytosis is much less than that observed in vivo. Thus complement activation, which does not occur in vitro, may be required to elicit the full priming effect caused by the injection of EIgG.

Although the exact mechanism for the augmentation of the LPS-stimulated increase in serum TNF-alpha levels caused by EIgG is unknown, it is possible that signaling by Fcgamma R and CRs mediates the priming for subsequent LPS stimulation of TNF-alpha secretion. With regard to Fcgamma R, it has been shown that phagocytosis of EIgG or cross-linking of Fcgamma R causes activation of NF-kappa B and a low level of TNF-alpha secretion (5, 25, 35). The injection of EIgG causes complement activation with deposition of CR ligands on the surface of the EIgG (16). These ligands are necessary for the binding of EIgG to Kupffer cells in vivo, with the subsequent phagocytosis being mediated by Fcgamma R. We showed previously that the injection of EIgG at a dose of 1.7 × 1010 cells/kg caused an 11% decrease in serum C3 levels (16). Recent studies have shown that 1) ligation of CD11b/CD18 (CR3) increases the LPS-stimulated TNF-alpha secretion by monocytes, 2) triggering CR1 or CR3 can activate NF-kappa B, 3) C5a and C3a can increase the LPS-stimulated TNF-alpha secretion by macrophages, and 4) antibodies against CD18 decrease the LPS-stimulated increase in serum TNF-alpha levels (1, 8, 24, 36, 39).

Perspectives

The present study has shown that the entrance of immune complexes into the circulation can prime macrophages for an augmented TNF-alpha response to LPS. Although Fcgamma R signaling is known to be capable of stimulating cytokine secretion (5, 11, 15, 35), a synergistic effect between immune complexes and LPS for increased serum TNF-alpha levels has not been previously appreciated. This observation may be relevant to thermal injury in which injury-induced erythrocyte phagocytosis may prime macrophages for an exaggerated inflammatory response to sepsis or endotoxemia. Indeed, several studies have shown that macrophages removed from animals after thermal injury are primed for cytokine secretion (6, 23, 26, 27). An exaggerated inflammatory response is considered to contribute to the development of multiple organ failure in burn patients (28). Our observation may also be relevant to diseases in which immune complexes could prime macrophages for augmented cytokine secretion in response to an infection and thereby initiate or exacerbate the condition. With atherosclerosis, immune complexes formed with antibodies against oxidized low-density lipoproteins may prime macrophages for an exaggerated response to infections with Chlamydia pneumoniae (4, 19). For arthritis, autoantibodies may prime synovial macrophages for infections that could initiate or exacerbate the condition (9, 37). This is supported by the observation that the administration of antibodies to collagen type II followed by LPS results in a 10-fold higher arthritic score in mice than LPS or antibodies alone (38). Similarly, experimental glomerular nephritis, induced with anti-glomerular basement membrane antibodies, is exacerbated with LPS (40). It is well known that clinical glomerular nephritis can be triggered by a streptococcal infection. Thus the combination of immune complexes and bacterial products may act synergistically to induce cytokine secretion and thereby augment disease severity.

    ACKNOWLEDGEMENTS

The authors thank Luis Rodriguez for technical assistance and Wendy Ward for editorial assistance.

    FOOTNOTES

This study was supported by National Institute of General Medical Sciences Grant GM-50368.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: D. J. Loegering, Dept. of Physiology and Cell Biology (mail code 134), Albany Medical College, 47 New Scotland Ave., Albany, NY 12208-3479.

Received 8 June 1998; accepted in final form 14 September 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Regul Integr Compar Physiol 276(1):R171-R177
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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