Nitric oxide promotes the internalization and passage of viable bacteria through cultured Caco-2 intestinal epithelial cells.

Nitric oxide (NO) may play an important role in the pathophysiology of intestinal barrier disruption. Our purpose was to investigate the effects of NO donors on the internalization and passage of bacteria through cultured intestinal epithelial cells. Human intestinal epithelial cell line Caco-2 cells were grown on microtiter plastic plates. The cells were incubated with Escherichia coli and sodium nitroprusside (SNP) or S-nitroso-N-acetyl-penicillamine (SNAP), as NO donors, at several concentrations. The numbers of viable bacteria internalized into the epithelial cells were measured. Caco-2 cells were also grown to confluency on membranes of bicameral systems. The cells were incubated with E. coli and SNP. The numbers of viable bacteria passed through the epithelial layer were determined. Viability of the bacteria and the intestinal epithelial cells after culture with SNP or SNAP were also determined. Both SNP and SNAP at .1 or 1 mmol/L increased the number of viable bacteria internalized into the enterocytes. Both 1 or 10 mmol/L SNP promoted bacterial passage through the intestinal epithelial layer. However, 10 mmol/L SNP decreased the number of viable Caco-2 cells and failed to increase the bacterial internalization into Caco-2 cells. Incubation of E. coli with SNAP at 10 mmol/L slightly decreased the number of viable bacteria and failed to increase the bacterial internalization into Caco-2 cells. We conclude that NO donors promote both the viable bacterial uptake and passage through the intestinal epithelial layer.

Bone marrow and splenocyte coculture-generated cells enhance allograft survival.

BACKGROUND: Protocols that incorporate donor-specific cell infusions using bone marrow, spleen, or blood transfusion continue to enhance allograft survival and often lead to tolerance in experimental models. Clinical benefits from these modalities have not been as striking, leading to ongoing study in this field. We have explored culture techniques for the in vitro selection and development of cellular effectors capable of enhancing allograft survival. METHODS: Rat bone marrow or spleen cells cultured under a variety of conditions were screened for suppressor function. Bone marrow cells, nonadherent to plastic, cultured for 7 days with granulocyte-macrophage colony-stimulating factor, lipopolysaccharide, and with or without splenocytes were found to contain predominantly myeloid lineage cells and had the ability to suppress phytohemagglutinin or mixed lymphocyte reaction-induced splenocyte proliferation. Standard donor-specific peripheral blood transfusion was compared with cultured donor-specific bone marrow cells, splenocytes, or marrow cells cultured with splenocytes (cocultured) administered intravenously at 1 x 10(7) cells/kg the day before an ACI to Lewis heterotopic heart transplant. Cyclosporine was administered at 10 mg/kg on day -1 and 2.5 mg/kg on days 0-6 relative to transplantation. RESULTS: Mean allograft survival in cyclosporine-treated animals was 8.5 days without and 16.6 days with a donor-specific blood transfusion. Cocultured cells extended allograft survival (39.5 days), whereas bone marrow or splenocytes cultured alone did not. With Percoll gradient separation, two predominant culture subfractions, one with potent suppressor function and another with stimulator function, were identified. Flow cytometric analysis showed mixed populations enriched for macrophages but also including dendritic cells in both subfractions. The suppressive fraction extended allograft survival to 20.8 days and the stimulatory fraction was less effective, yet remixing of both fractions regained the full allograft survival advantage. CONCLUSIONS: In this model, the coculture of bone marrow cells and splenocytes with granulocyte-macrophage colony-stimulating factor and lipopolysaccharide produced functionally divergent subpopulations that synergistically enhanced allograft survival. The development of cellular effectors with enhanced ability to prolong allograft survival using in vitro culture techniques is possible, and provides a new therapeutic option in the use of cell infusion-based therapies.

Induction of endotoxin tolerance in rat bone marrow cells by in vivo infusion of tumor necrosis factor.

OBJECTIVE: To determine in a rat model whether a low-dose infusion of tumor necrosis factor (TNF) affects the production of the inflammatory cytokines TNF and interleukin (IL)-6, the immunosuppressive factor prostaglandin E2 (PGE2), and complement component C3 (C3) by isolated bone marrow-adherent and -nonadherent cells, cultured in the presence of lipopolysaccharide, a component of bacterial endotoxin. DESIGN: Randomized, controlled animal study. SETTING: Research laboratory of a university medical center. SUBJECTS: Sprague-Dawley rats (n = 18), 250 to 275 g. INTERVENTIONS: Animals received a continuous infusion of one of the following three treatments for 4 days: a) TNF in saline containing bovine serum albumin; b) saline containing bovine serum albumin; and c) saline alone. MEASUREMENTS AND MAIN RESULTS: After infusion, isolated bone marrow cells were cultured for 1 day and 3 days, with and without lipopolysaccharide (1 microgram/mL); culture supernatants were assayed for TNF, IL-6, PGE2, and C3. TNF infusion caused a decrease in the in vitro production of TNF, IL-6, and PGE2 by the lipopolysaccharide-stimulated adherent and nonadherent bone marrow cells. This tolerance to lipopolysaccharide stimulation was present after both 1 day and 3 days of culture. TNF infusion caused an increase in C3 production by the nonadherent cells. The production of TNF by adherent cells from saline-infused or bovine serum albumin-infused animals (controls) was greater in 3-day cultures compared with 1-day cultures, whereas the production of IL-6 and PGE2 was less. CONCLUSIONS: These results indicate that TNF infusion caused cells in the bone marrow to be tolerant to lipopolysaccharide stimulation or that TNF infusion programmed the cells to become tolerant to lipopolysaccharide stimulation on differentiation and/or maturation. The results also indicate that bone marrow cells may be regulated by TNF (probably indirectly) at different phases of maturation and/or differentiation with respect to the production of different mediators. Although TNF is considered to be an inflammatory cytokine, at low concentrations it may be an important down-regulator of the inflammatory response.

The gut as a source of inflammatory cytokines after stimulation with endotoxin.

OBJECTIVE: To find out if endotoxin (LPS) can mediate the production of inflammatory cytokines by enterocytes. DESIGN: Laboratory experiment. SETTING: Teaching hospital and burns unit, USA. MATERIAL: Caco-2 cells (HTB38, human adenocarcinoma, and colon). MAIN OUTCOME MEASURES: Concentrations of tumour necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6) and prostaglandin E2 (PGE2) in cell culture supernatants. RESULTS: LPS significantly increased the production of TNF from 8.9 to 26.4 units/ml in 24 h and this increase persisted at a lower level for 4 days with an increase from 2.3 to 9 units/ml at a cell concentration of 2 x 10(5) cells/ml. There was no increase in TNF production when the cells were cultured at 5 x 10(5) cells ml with LPS. At a concentration of 2 x 10(5) cells/ml, the cells produced small amounts of IL-6 in 24 h or 4 day cultures with or without LPS. At a concentration of 5 x 10(5) cells/ml, LPS significantly increased IL-6 production in 24 h from 142 to 433 units/ml and from 106 to 250 units/ml in 4 days. The amount of IL-6 produced by LPS-stimulated cells was greater at 1 day than at 4 days. There was no significant difference in PGE2 production by the cells under any of the incubation conditions. CONCLUSION: Enterocytes can produce TNF and IL-6, and endotoxin can increase the production of these cytokines by enterocytes. The gut therefore has the potential to become an important source of inflammatory cytokines.

Thermal injury induces the development of inflammatory macrophages from nonadherent bone marrow cells.

The normal course of hematopoiesis is controlled by growth factors and cytokines and, therefore, should be susceptible to alterations induced by systemic mediator release such as that seen following thermal injury. We hypothesized that a brief exposure of developing macrophages to the postthermal injury state would result in functionally altered progeny. We measured the production of inflammatory mediators by rat, bone-marrow macrophage precursors harvested 24 h following a 30% TBSA burn after subsequent maturation in a controlled, in vitro environment. Interleukin (IL)-6, tumor necrosis factor (TNF), and prostaglandin (PG) E2 levels in response to 24 h stimulation with lipopolysaccharide (LPS) were measured following 4 or 8 days of incubation with IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), or both. Flow cytometric analysis showed that bone marrow cells harvested from burn and sham animals cultured in GM-CSF developed principally into macrophages (His48+, R21A6A+, CD11b+. Unstimulated cells produced negligent levels of cytokines and PGE2. Stimulated burn-derived cells released greater amounts of IL-6 and TNF at 4 or 8 days of culture depending on the conditions. Elevated PGE2 release was noted in all GM-CSF containing cultures, with burn-derived cells showing a trend towards reduced prostaglandin release. Detection of mRNA for cytokines after LPS stimulation showed no change in IL-6 or TNF transcripts. A short exposure to the systemic effects of thermal injury preprogramed macrophage progenitor cells with the propensity to develop into inflammatory macrophages, secreting higher levels of TNF and IL-6. This shift towards proinflammatory functions in these cells suggests they could be a source of enhanced inflammatory mediator release at 4 or more days post thermal injury.

Production of cytokines and prostaglandin E2 by subpopulations of guinea pig enterocytes: effect of endotoxin and thermal injury.

BACKGROUND: There is increasing evidence that cells other than immune cells have the potential for producing immunomediators. This study determined whether distinct populations of enterocytes from unburned and burned animals responded differently to endotoxin regarding production of tumor necrosis factor, interleukin-1 and -6 and prostaglandin E2. METHODS: Three subpopulations of enterocytes, progressing from the villus tip towards the crypt, were obtained from washes of the small intestine. The cells were cultured in the presence of endotoxin, and the supernatants were assayed for the mediators. RESULTS: Thermal injury primed all three populations of enterocytes to produce larger amounts of tumor necrosis factor and interleukin-6 compared to cells from unburned animals. Enterocytes that were nearer the crypt produced the largest amounts of the cytokines. CONCLUSION: These observations may be important because, as gut integrity is compromised after thermal injury, enterocytes that may have previously been unexposed or less exposed to endotoxin can become a significant source of inflammatory cytokines.

The increased potential for the production of inflammatory cytokines by Kupffer cells and splenic macrophages eight days after thermal injury.

Burn patients often experience a devastating inflammatory response to infection within the first two weeks after thermal injury. The inflammatory cytokines IL-6, TNF and IL-1 have been implicated in this condition but most studies have focused on the abnormal levels of cytokines in the plasma. In this study the production of cytokines was compared for Kupffer cells versus splenic macrophages; endotoxin (LPS) stimulation versus no stimulation; and burn (post burn days 1, 3 and 8) versus no burn (control). Corresponding serum levels of IL-6 were also determined. Kupffer cells from normal or burned animals were shown to produce much higher amounts of the inflammatory cytokines than that produced by splenic macrophages. An exception to this was the equal production of TNF by LPS-stimulated hepatic and splenic cells. Both LPS-stimulated Kupffer cells and splenic macrophages produced larger amounts of the cytokines than that produced by the unstimulated cells. There was a significant effect of thermal injury on cytokine production by LPS-stimulated Kupffer cells at post burn day 8 and on TNF production by stimulated splenic macrophages also at post burn day eight. Although there was a statistically significant effect of thermal injury at post burn day 8 on IL-1 production by unstimulated splenic macrophages, the absolute amount of cytokine produced was very small. The results suggest that by post burn day 8 the cells may have become primed to respond to a stimulus such as endotoxin (LPS), a condition that could arise in a burn patient from sepsis. Strangely, the large spike in serum IL-6 level occurred at post burn day one and the level of the cytokine returned nearly to the control value on post burn days 3 and 8.

The mRNA expression and in vitro production of cytokines and other proteins by hepatocytes and Kupffer cells following thermal injury.

Inflammatory cytokines and acute-phase proteins are closely interrelated, and their levels of production by various cells are increased by thermal injury. It was hypothesized that burn-mediated increases in the production of TNF and IL-6 by Kupffer cells and the production of acute phase proteins by hepatocytes are paralleled by increases in the corresponding message RNA levels in these cells. The mRNA expression of the cytokines, IL-6 and TNF alpha, and acute phase proteins, alpha-1 acid glycoprotein (alpha-1 AGP), and albumin in liver tissue were determined in rats 24 h after thermal injury. Also IL-6 and TNF alpha from in vitro cultured Kupffer cells, and alpha-1 AGP and albumin from in vitro cultured hepatocytes, and the serum levels of these proteins, were determined. An increased expression of IL-6 mRNA in liver tissue from animals of the burned group was accompanied by an elevation of IL-6 released from cultured Kupffer cells and by increased serum levels of this cytokine. Thermal injury caused a decrease in TNF mRNA but no change in the production of this cytokine by Kupffer cells, and TNF could not be found in the serum. Also, an increase in alpha-1 AGP mRNA expression following thermal injury was consistent with the increase of alpha-1 AGP production by hepatocytes and with the elevated serum level of this acute phase protein. Thermal injury caused no change in albumin mRNA expression or in the in vitro production of this negative acute phase protein, however, the serum level of albumin increased. The results suggest that thermal injury may cause alterations in the cytokine and acute phase protein mRNA levels in liver, which may cause alterations in the cellular production and serum levels of the corresponding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of hepatocytes from normal or endotoxin treated animals on the production of tumour necrosis factor, interleukin-6, and prostaglandin-E2 by macrophages incubated in vitro with various fatty acids.

OBJECTIVE: To find out if the in vitro production of tumour necrosis factor (TNF), interleukin 6 (IL-6) and prostaglandin E2 (PGE2) by macrophages (P3888D1) were influenced by the addition to the culture media of hepatocytes from normal guinea pigs and from guinea pigs after infusion with endotoxin with and without fatty acids. DESIGN: Laboratory experiment. SETTING: University hospital, United States MATERIAL: 12 male Hartley guinea pigs. MAIN OUTCOME MEASURES: Concentrations of TNF-alpha, IL-6, and PGE2. RESULTS: Hepatocytes inhibited the production of TNF by macrophages. Those from animals pretreated with endotoxin caused a large increase in the production of PGE2 and affected the production of IL-6 only in the presence of fatty acids. Fatty acids differed from each other in their effects on production of TNF-alpha and PGE2, but had no effect on the production of IL-6. CONCLUSION: These results support the concept that hepatocytes and macrophages may interact in vivo, and this interaction may be influenced both by fatty acids that arise from lipid metabolism and from endotoxin present during sepsis and translocation.

The 1994 Lindberg Award. The production of tumor necrosis factor, interleukin-1, interleukin-6, and prostaglandin E2 by isolated enterocytes and gut macrophages: effect of lipopolysaccharide and thermal injury.

Increasing evidence shows that cells other than immune cells have the potential for producing cytokines and arachidonate metabolites. It was the purpose of this study to determine whether isolated enterocytes could produce tumor necrosis factor, interleukin-1, interleukin-6, and prostaglandin E2, to compare the production with that of isolated gut macrophages, and to determine whether a difference existed in the production of these mediators after thermal injury. Guinea pigs received a 30% total body surface area burn and were killed 24 hours after injury. Isolated enterocytes and related intestinal macrophages (5 x 10(5) cells/ml) were cultured for 24 hours in the presence and absence of endotoxin, and the supernatants were assayed for the mediators. An increase was seen in production of interleukin-6 by enterocytes and by macrophages after thermal injury. In general enterocytes and gut macrophages produced about the same amounts of the different mediators. In contrast to macrophages from other tissues, enterocytes did not produce more prostaglandin E2 after stimulation with lipopolysaccharide, and with one exception gut macrophages did not produce larger amounts of mediators after stimulation with lipopolysaccharide. Enterocytes may be a significant source of immunomediator production and could contribute to the inflammatory response.

Heterogeneity of Kupffer cells and splenic, alveolar, and peritoneal macrophages for the production of TNF, IL-1, and IL-6.

Kupffer cells and alveolar, splenic, and peritoneal macrophages from normal rats were incubated for various periods of time in the presence of LPS, and the culture supernatants were analyzed for IL-6, IL-1, and TNF. There was very little difference in the amounts of the cytokines produced by the macrophages when stimulated with 0.01-10 micrograms/ml of LPS. The shapes of the time course curves for the production of the cytokines by the different types of macrophages were generally similar, although only Kupffer cells continued to produce IL-6 throughout the entire incubation period and splenic macrophages showed a lag period in the production of IL-1. Kupffer cells produced more IL-6 than that produced by the other populations of macrophages, and alveolar macrophages produced more IL-1 compared to that produced by splenic cells. Kupffer cells and peritoneal macrophages produced more IL-6 in 24 h than in 6 h of culture, and splenic macrophages produced more IL-1 in 24 compared to 6 h of culture. Alveolar macrophages produced more TNF than that produced by the other populations of cells but only when integrated over the entire incubation period. These results confirm and extend the observed functional heterogeneity of macrophages obtained from different tissues of the same animal. This study and future studies will lead to a better understanding of the role of cytokines in the inflammatory response.

Production of tumor necrosis factor, interleukin-6 and prostaglandin E2 by LPS-stimulated rat bone marrow macrophages after thermal injury: effect of indomethacin.

The effect of thermal injury on the in vitro production of TNF, IL-6, and PGE2 by bone marrow-derived, LPS-stimulated rat macrophages was studied. Thermal injury caused a general hyperactivity in the production of the mediators by the cells. Indomethacin, a cyclooxygenase inhibitor of PGE2 synthesis, inhibited the production of IL-6 and PGE2 but had no effect on the production of TNF. These results suggest that the observed low concentration of PGE2 produced by the cells was insufficient to cause inhibition of TNF synthesis; thus, the effect of indomethacin would be undetectable. The results also suggest that indomethacin may act directly in inhibiting the production of IL-6 by the macrophages. The hyperactive effect of thermal injury on the production of inflammatory mediators by newly differentiated bone marrow derived macrophages can be important in the overall systemic response to the insult.

Time course of production of cytokines and prostaglandin E2 by macrophages isolated after thermal injury and bacterial translocation.

The relationship of translocation of bacteria from the gut of burned guinea pigs and the in vitro production of tumor necrosis factor (TNF), interleukin (IL)-1 and IL-6, and prostaglandin E2 (PGE2) by lipopolysaccharide (LPS)-stimulated mesenteric lymph node and splenic macrophages was investigated at two early times after thermal injury. Two hr postburn, there was a large number of translocated bacteria in the mesenteric lymph nodes and a large proportion was killed; at 24 hr postburn, there were fewer translocated bacteria, but a large proportion was viable. In some cases, there were very large differences compared to controls in the amounts of TNF, IL-6, and PGE2, but not of IL-1, produced by the macrophages at different times postburn and at different in vitro incubation times. The results suggest that the macrophages were primed by the burn or the translocated bacteria to produce in vitro different and sometimes large amounts of cytokines or PGE2 after further stimulation with LPS. Although there was no direct correlation between production of cytokines or PGE2 and time postburn, the early increased production of PGE2 by splenic macrophages could have depressed the animal's ability to kill translocated bacteria by 24 hr postburn, and could be one of the mechanisms of the cause of systemic infection after burn injury.

Effect of glutamine on phagocytosis and bacterial killing by normal and pediatric burn patient neutrophils.

Glutamine is essential for the function of lymphocytes and macrophages, where it serves, among other things, as a source of energy. Little information is available concerning the fuel that polymorphonuclear cells use for their metabolic and bactericidal functions. It was the purpose of this study to determine whether glutamine would enhance the in vitro bactericidal function of normal neutrophils and whether the amino acid would restore the observed impaired function in burn patients to or above the normal level. Twelve burn patients with total body surface area burns ranging from 32% to 87% were studied. At various postburn times, neutrophils were isolated and their ability to kill Staphylococcus aureus in the presence and absence of glutamine was determined and compared with that in normal subjects. Glutamine enhanced the bactericidal function of normal neutrophils. In every patient, at all but two postburn times, glutamine caused an improvement in the observed abnormal neutrophil bactericidal function and often restored it to or slightly above the normal level. Glutamine had no effect on the expression of C3b receptors (CR1 or CD35) or on phagocytosis by the cells. This study confirms the beneficial effects of glutamine in at least one arm of the immune system and adds evidence for the possible advantage of including this amino acid in the diets of burn and other trauma patients.

Production of cytokines and PGE2 and cytotoxicity of stimulated bone marrow macrophages after thermal injury and cytotoxicity of stimulated U-937 macrophages.

Bone marrow-derived macrophages from normal and burned rats were cultured for one and four days in the presence of LPS, PHA, or opsonized zymosan as activators, and the supernatants were assayed for the inflammatory mediators TNF, IL-6, and PGE2 and the cells assayed for cytotoxicity. The macrophages responded differently to the various stimuli regarding cytotoxicity and the production of mediators, perhaps implicating the complement receptor CR1 in TNF production and the LPS receptor CD14 or the PHA lectin receptor in IL-6 and PGE2 production and for cytotoxicity. The response of the cells also depended on culture time and postburn time; in addition, macrophages from burned and unburned animals responded differently, depending on postburn day and the type of stimulus. TNF production was generally higher for one-day compared to four-day cultures (i.e., TNF was disappearing in the cultures), but IL-6 and PGE2 production was greater in four-day cultures. The results of this study suggest that thermal injury can contribute to the development of inflammatory and cytotoxic macrophages from bone marrow progenitor cells.

A comparison of hepatic, splenic, peritoneal and alveolar macrophages with respect to PGE2, TXB2, production and ADCC function.

The in vitro production of PGE2 and TxA2, measured as TxB2, and the antibody-dependent cellular cytotoxicity (ADCC) as well as spontaneous cellular cytotoxicity (SCC) displayed by hepatic, peritoneal, splenic, and alveolar macrophages from 12 rats was determined and compared. Kupffer cells, the fixed macrophages of the liver, were the most active cells in the production of PGE2. Kupffer cells and peritoneal macrophages released about equal amounts of TxB2 which was much higher than that released by splenic macrophages. Kupffer cells displayed the strongest ADCC activity, and alveolar macrophages had the lowest value. These results confirm the previously reported heterogeneity among different sources of macrophages and emphasize the enhanced activity of Kupffer cells with respect to eicosanoid production and ADCC function.

The activation of bone marrow macrophages 24 hours after thermal injury.

We determined the effect of thermal injury on the in vitro production of the immunoactive substances tumor necrosis factor, interleukin 1, prostaglandin E2, and complement component C3 by lipopolysaccharide-stimulated guinea pig bone marrow macrophages and on the cytotoxicity of these cells. Macrophages from burned animals produced different amounts of these mediators compared with unburned animals at certain culture times, suggesting that thermal injury could program the bone marrow cells to respond differently from normal cells to in vitro stimulation with lipopolysaccharide. Also, the macrophages from burned animals displayed greater cytotoxicity towards L929 target cells. These results suggest that there is a complex interaction among cellular secretory products, especially after thermal injury, that may be important in host defense.

Adhesive effect of certain cytokines and other perturbants on human neutrophils.

Pretreatment of normal human neutrophils with certain cytokines and other mediators caused some of the cells to become adhesive and stick to the plastic (polypropylene) incubation tubes during pretreatment and during the assay for phagocytosis of C3b.IgG-coated microspheres. Often as much as 40% of the cells were adherent to the tubes after the reaction. This sticking of the neutrophils to the plastic tubes was confirmed by increase in cytometer sipping time and by lactic dehydrogenase assay of the suspended cells and of the cells stuck on the sides of the empty incubation tubes. Only those perturbants that caused an up-regulation of C3b receptors (CR1, CD35) and in most cases caused an enhancement of phagocytosis mediated the adhesiveness of the cells. Unless these stuck cells were detached by vigorous flushing with cold buffer containing EDTA, many of the cells were not admitted into the cytometer for determination of the effect of the perturbants on binding and phagocytic capacity of the neutrophils. This observation could have implications regarding the possibility of subpopulations of neutrophils and differences in function of adherent cells versus cells in suspension. In the cases studied there was no appreciable difference between the total binding and phagocytic capacities of the adherent and suspended cells.

Effects of combination of tumor necrosis factor alpha and chemotactic peptide, f-Met-Leu-Phe, on phagocytosis of opsonized microspheres by human neutrophils.

Pretreatment of normal, human neutrophils with 8 units/ml of TNF-alpha followed by treatment with 10(-8) M FMLP resulted in a synergistic effect of the combination of the two mediators on the enhancement of the phagocytic capacity of the cells. This enhancement of phagocytosis occurred without an additional increase in the upregulation of C3b receptors (CR1) beyond that caused by each mediator alone. Pretreatment of the cells with 8 units/ml of TNF-alpha followed by 10(-6) M FMLP resulted in an additive effect of the mediators on neutrophil phagocytosis, again without an additional up-regulation of CR1. This additive effect resulted in an increase in phagocytic capacity of the neutrophils greater than that obtained by treatment of the cells with 10(-6) M FMLP alone, which heretofore has resulted in the greatest enhancement of phagocytic capacity obtained by any pretreatment condition. These synergistic and additive effects of the combination of mediators could be of great importance in host defense against bacterial infections and have important implications regarding the mechanisms of receptor upregulation and phagocytosis.