Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4.

Toll-like receptor 4 (TLR4), the principal signaling receptor for lipopolysaccharide (LPS) in mammals, requires the binding of MD-2 to its extracellular domain for maximal responsiveness. MD-2 contains a leader sequence but lacks a transmembrane domain, and we asked whether it is secreted into the medium as an active protein. As a source of secreted MD-2 (sMD-2), we used culture supernatants from cells stably transduced with epitope-tagged human MD-2. We show that sMD-2 exists as a heterogeneous collection of large disulfide-linked oligomers formed from stable dimeric subunits and that concentrations of sMD-2 as low as 50 pM enhance the responsiveness of TLR4 reporter cells to LPS. An MD-2-like activity is also released by monocyte-derived dendritic cells from normal donors. When coexpressed, TLR4 indiscriminately associates in the endoplasmic reticulum/cis Golgi with different-sized oligomers of MD-2, and excess MD-2 is secreted into the medium. We conclude that normal and transfected cells secrete a soluble form of MD-2 that binds with high affinity to TLR4 and that could play a role in regulating responses to LPS and other pathogen-derived substances in vivo.

Lipopolysaccharide tolerance and ethanol modulate hepatic nitric oxide production in a gender-dependent manner.

This study was directed at the role of tolerance to endotoxin (lipopolysaccharide, LPS) and ethanol (EtOH) intoxication in modulating hepatic nitric oxide (NO) production, and the demonstration of gender differences. Previous studies demonstrated that tolerance to either LPS or EtOH was associated with reduced hepatic production of superoxide anions. We now tested the hypothesis that the reduced hepatic production of superoxide anions during tolerance to LPS and the altered response to EtOH are accompanied by increased sensitivity of hepatic NO release to stimulation. Age-matched male and female Sprague-Dawley rats were made tolerant to LPS by an i.v. injection of LPS (0.5 or 0.45 mg/kg) 2 days prior to an in vivo EtOH infusion for 3 h (LPS-EtOH group). Control groups were saline-pretreated, saline-infused; saline-pretreated, EtOH-infused; and LPS-pretreated, saline-infused. At the end of the infusion, isolated hepatocytes, Kupffer, and sinusoidal endothelial cells were cultured for 20 h for subsequent measurement of basal (spontaneous) and in vitro-stimulated nitrite release. LPS-tolerance resulted in significantly enhanced stimulated NO production by hepatocytes and Kupffer cells in both male and female rats. EtOH abolished this priming effect in hepatocytes from male, but not from female rats. The priming effect was markedly diminished by EtOH in Kupffer cells of female rats only. LPS-tolerance increased NO production by stimulated endothelial cells of males, and decreased NO production by cells of females. EtOH infusion did not influence NO production by endothelial cells from male rats and it reversed the LPS-tolerance-induced inhibition in females. These data demonstrate that modulation by LPS-tolerance of hepatic NO release in EtOH-treated rats leads to enhanced stimulated NO production, while hepatic superoxide anion release was previously shown to be reduced within the same time frame. Since NO is able to scavenge superoxide, the LPS-tolerance-induced alterations in the EtOH effects on NO production may have a potential significance in modulating - in a time-dependent manner - oxidative injury associated with LPS and acute EtOH intake.

Serine/threonine phosphorylation in cellular signaling for alveolar macrophage phagocytic response to endotoxin.

Protein serine/threonine (ser/thr) phosphorylation is an early signaling event in macrophage activation. We investigated the changes in stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) activity and effects of phosphatase inhibition on alveolar macrophage (AM) function in rats challenged with intratracheal endotoxin. Animals were sacrificed 90 min post intratracheal lipopolysaccharide (LPS, 100 microg/rat) challenge. AMs were incubated with or without phosphatase inhibitors at 37 degrees C for 30 min. Phagocytosis, CD18 expression, SAPK/JNK and phosphatase activities of AMs were determined. LPS challenge activated SAPK/JNK activity and enhanced phagocytosis of AMs without altering phosphatase activity in these cells. Inhibition of phosphatase 1 and 2A activity with okadaic acid and calyculin A exerted a bi-phasic effect on AM phagocytic function. Okadaic acid at a concentration of 1 microM increased the mean channel fluorescence intensity (MCF) and the percentage of cells engaged in phagocytosis (percent phagocytosis) in AMs from saline-treated rats. This inhibitor at concentrations of 0.5 and 1 microM enhanced both the MCF and percent phagocytosis of AMs from LPS-challenged rats. Calyculin A at a concentration of 10 nM increased the MCF phagocytosis of AMs from LPS-challenged rats. At higher concentrations (20 and 30 nM), calyculin A showed a suppression on both the MCF and percent phagocytosis of AMs in both saline and LPS groups. AM CD18 expression was not altered following LPS challenge. Phosphatase inhibitors at doses that enhanced AM phagocytosis showed either no effect (okadaic acid) or inhibition (calyculin A) of AM CD18 expression. These results suggest that ser/thr phosphorylation and dephosphorylation participate in mediating the phagocytic response of AMs to LPS.

Gender differences in some host defense mechanisms.

Sexual dimorphism exists in the immune response. Both humoral and cell-mediated immunity are more active in females than in males, and steroid gonadal hormones may play an important role in regulating this response. We have documented gender differences in several aspects of neutrophil and macrophage functions elicited by lipopolysaccharide (LPS) (endotoxin) treatment and/or acute ethanol intoxication. In LPS-treated female rats, circulating neutrophils and alveolar macrophages are resistant to the deleterious effects of surgery and anesthesia on phagocytosis observed in male rats. The generation of cytokine-induced neutrophil chemoattractant (CINC) by hepatocytes and Kupffer cells of LPS-treated rats, as well as TNF-alpha secretion by Kupffer cells and alveolar macrophages of acutely ethanol intoxicated rats are also gender dependent. The effects of alcohol on the immune response are expressed differently in males and females. In LPS plus ethanol-treated rats gender differences were noted in terms of adhesion molecule (CD11b/c) expression on circulating neutrophils, and cytoskeletal reorganization in blood-recruited neutrophils and Kupffer cells. Nitric oxide (NO) plays an important role in inflammatory processes. We found gender differences in NO production by alveolar macrophages of LPS-treated rats; this difference was abrogated by ethanol treatment. LPS tolerance and ethanol treatment modulate hepatic NO production in rats in a cell- and gender-dependent fashion, which may exert a protective influence against oxidative injury in the female liver.

Acute ethanol intoxication suppresses the pulmonary inflammatory response in rats challenged with intrapulmonary endotoxin.

The effects of acute ethanol intoxication on the functional activities of circulating and lung-recruited polymorphonuclear leukocytes (PMNs) and alveolar macrophages (AMs) were determined in rats challenged with intratracheal endotoxin to elucidate the mechanisms underlying the defects of pulmonary host defenses caused by acute ethanol intoxication. Acute ethanol Intoxication was induced by an intraperitoneal injection of 20% ethanol at a dose of 5.5 g of ethanol/kg. The control animals were injected with an equal amount of saline. Thirty min after intraperitoneal injection, rats were challenged with intratracheal endotoxin (300 micrograms/kg in 0.5 ml of saline) or saline. The rats were killed 3 h after intratracheal injection. CD11b/c expression on PMNs and phagocytosis and hydrogen peroxide generation of PMNs and AMs were determined by flow cytometry. Cytokine-Induced neutrophil chemoattractant (CINC) level in bronchoalveolar lavage fluid was measured with a specific ELISA. Intratracheal endotoxin caused a significant PMN recruitment into the lung in control animals. Acute ethanol intoxication completely suppressed the endotoxin-induced pulmonary recruitment of PMNs. Pulmonary-recruited PMNs exhibited a significant upregulation (8-fold) of CD11b/c expression when compared with circulating PMNs. This upregulation of CD11b/c expression was abolished by ethanol intoxication. Ethanol intoxication suppressed hydrogen peroxide generation by AMs and lung-recruited PMNs, and the phagocytosis of circulating PMNs. In contrast, acute ethanol intoxication did not affect pulmonary CINC production. These data indicate that the antiinflammatory effects of alcohol seem to be primarily based on the effects of ethanol on the PMNs themselves and not on the generation of certain chemotactic stimuli. In addition to the impairment of PMN recruitment, the suppression of AM and PMN activities also contributes to the mechanisms underlying ethanol-induced defects of pulmonary host defenses.

Acute ethanol administration modulates leukocyte actin polymerization in endotoxic rats.

The effects of acute ethanol intoxication with or without endotoxemia were studied on chemoattractant-initiated actin polymerization in circulating and liver-recruited neutrophils [polymorphonuclear leukocytes (PMNs)] and Kupffer cells of age-matched male and female rats. In female rats, F-actin content in response to f-Met-Leu-Phe in circulating PMNs was significantly upregulated by ethanol+endotoxin (ET) treatment relative to saline+ET-treated animals. Male rats did not show this upregulated response. In liver-recruited PMNs, the F-actin response did not change significantly due to ethanol + ET treatment in cells of either gender. The actin polymerization response in Kupffer cells was smaller than in liver sequestered PMNs and also exhibited a gender difference. In Kupffer cells of female rats, only ethanol+ET treatment elicited an F-actin response to f-Met-Leu-Phe, whereas in cells of male rats similar increases in F-actin content were observed in both saline+ET and ethanol+ET groups. We conclude that ethanol-enhanced actin polymerization in rats is associated with gender differences. This may be one of the underlying mechanisms resulting in upregulated leukocyte activation in acutely ethanol-intoxicated endotoxic animals.

Ceramide inhibits nitric oxide production in alveolar macrophages of endotoxin and ethanol plus endotoxin-treated rats.

The effect of ceramide on nitric oxide (NO) formation was studied in rat alveolar macrophages (AMs). Rats were infused with ethanol (EtOH) for 3 h, or the EtOH infusion was combined with i.v. injection of endotoxin (ET) 90 min into the infusion. Controls were infused with saline. Alveolar macrophages were obtained by bronchoalveolar lavage and were cultured for 20 h in the presence and absence of ET, interferon-gamma (IFN), C6 ceramide (N-hexanoylsphingosine), and C2 dihydroceramide. Nitric oxide formation was assessed by measurement of nitrites in the medium. C6 ceramide significantly suppressed NO formation in response to in vitro addition of ET, but not IFN. C2 dihydroceramide caused no inhibition. The ceramide effect appears to be not only stimulus specific, but also specific to AMs, since NO formation by Kupffer cells and liver infiltrated neutrophils was not affected. The results suggest involvement of the sphingomyelin cycle in ET-stimulated NO formation in rat AMs.

Modulation of the lung host response by granulocyte colony-stimulating factor in rats challenged with intrapulmonary endotoxin.

The effects of granulocyte colony-stimulating factor (G-CSF) on the functional activities of circulating and lung-recruited neutrophils (PMNs) and alveolar macrophages (AMs) were studied in rats to further elucidate the mechanisms underlying G-CSF-enhanced pulmonary host defense. Animals received G-CSF or vehicle twice a day for 2 days, followed by an intratracheal challenge with endotoxin or saline. G-CSF up-regulated CD11b/c expression and mean channel fluorescence intensity of phagocytosis in circulating PMNs. G-CSF also enhanced phagocytic activities, reflected by both the percentage of phagocytosis and mean channel fluorescence intensity in lung-recruited PMNs and AMs in intratracheal endotoxin-challenged rats. The endotoxin-induced increase in pulmonary production of tumor necrosis factor-alpha and cytokine-induced neutrophil chemoattractant was not affected by G-CSF pretreatment. These data demonstrate that G-CSF-enhanced pulmonary recruitment of PMNs is primarily based on the effects of G-CSF on the PMNs themselves, rather than the generation of certain chemotactic stimuli, i.e., cytokine-induced neutrophil chemoattractant and tumor necrosis factor-alpha. The enhanced phagocytic activities of lung-recruited PMNs and AMs also augment pulmonary host defenses in G-CSF-pretreated animals.

Gender differences in nitric oxide production by alveolar macrophages in ethanol plus lipopolysaccharide-treated rats.

Alveolar macrophages (AMs) constitute an important first line of host defense against infection in the lung, and NO is an essential component of the microbicidal activity of cytokine-activated macrophages. Previously we studied the respiratory burst, protein kinase C activity, and NO generation in tumor necrosis factor (TNF) and lipopolysaccharide (LPS)-treated AMs and gender differences in phagocytosis in ethanol (EtOH)-intoxicated rats. Now we have investigated NO production by AMs in EtOH plus LPS-treated male and female rats. Rats were infused iv with EtOH for 3 h to a blood level of approximately 180 mg/dl. At 90 min of infusion, Escherichia coli LPS (750 microg/kg) was injected i.v. Controls received saline (SAL) + LPS. AMs were isolated by bronchoalveolar lavage and cultured for 20 h in the absence and presence of LPS, interferon-y (IFN), and LPS + IFN. Nitrite was determined in the medium and was taken as an index of NO production. EtOH alone resulted in no significant differences compared with SAL infusion. LPS treatment caused a decrease in basal and an increase in LPS and IFN-stimulated generation of NO in males and females. EtOH + LPS treatment vs EtOH showed no significant differences. There are gender differences in both spontaneous and in vitro stimulated NO production by AMs. AMs of female rats treated with SAL + LPS released significantly more NO spontaneously than AMs of equally treated male rats. Cells of SAL + LPS-treated male rats activated in vitro by LPS and IFN-gamma produced significantly greater amounts of NO than AMs of female rats. These differences in activated induction of NO production were abrogated by ethanol treatment.

The marine toxin okadaic acid reduces O2- generation and tyrosine phosphorylation in LPS-primed rat neutrophils.

Contrasting effects of okadaic acid (OKA) on neutrophil (PMN) superoxide anion (O2-) generation have been reported. In this study, we examined the effect of OKA on phorbol myristate acetate (PMA)-stimulated O2- generation in rat PMNs primed with LPS in vivo (LPS-PMN) and saline-treated rat PMNs (SAL-PMN). The following results were observed: (1) OKA, but neither genistein nor vanadate, markedly reduced O2- generation in a dose and time-dependent manner; (2) genistein, a tyrosine kinase inhibitor, as well as OKA, reduced tyrosine phosphorylation; (3) sodium orthovanadate, a tyrosine phosphatase inhibitor, potently enhanced tyrosine phosphorylation. Our studies suggest that OKA might reduce tyrosine phosphorylation by affecting the activity of tyrosine phosphatases regulated by serine-threonine phosphorylation.

Effects of female hormones (17beta-estradiol and progesterone) on nitric oxide production by alveolar macrophages in rats.

The effect of female sex hormones on nitric oxide (NO) production was studied in alveolar macrophages (AMs). Male rats were treated with endotoxin (LPS) intratracheally or saline as control. AMs were obtained by bronchoalveolar lavage 90 min later and were cultured in the presence or in the absence of LPS and 17beta-estradiol or progesterone (10(-9) to 10(-4) M). NO production was assessed by measurement of nitrites in the medium. In some experiments, NO production by AMs was measured in intratracheally LPS-treated orchidectomized rats or in female control and ovariectomized rats. Both spontaneous and stimulated NO production were higher in AMs from female than from male rats, but without statistical significance. However, ovariectomy induced significant inhibition in spontaneous production of NO by AMs. In orchidectomized rats, the NO response by AMs to LPS stimulation relative to spontaneous NO production was significantly downregulated. Female sex hormones in physiological concentrations seem to be necessary for spontaneous NO production in female rats. Pharmacological doses of estradiol inhibited in vitro LPS-stimulated NO production in AMs of both saline- and LPS-treated rats, and basal NO production only in LPS-treated male rats. Progesterone at 10(-4) M inhibited basal and in vitro LPS-stimulated NO generation by AMs of both saline- and LPS-treated male rats. In LPS-treated female rats in vitro LPS-stimulated NO production was not affected by estradiol treatment. In ovariectomized LPS-treated female rats progesterone at 10(-5) M significantly inhibited NO production by in vitro-stimulated AMs. Thus female sex hormones may contribute to the gender-related differences in the immune response.

Protein tyrosine kinase activity and the influence of gender in phagocytosis and tumor necrosis factor secretion in alveolar macrophages and lung-recruited neutrophils.

The role of tyrosine phosphorylation in endotoxin-induced phagocytic and tumor necrosis factor secretory responses was studied in rat alveolar macrophages and lung-recruited neutrophils. Exploration of sexual dimorphism in some aspects of these functions was also a specific aim. Male and female rats were injected intratracheally with endotoxin or saline. Two and a half hours later the animals were subjected to bronchoalveolar lavage, and alveolar macrophages and lung-recruited neutrophils were isolated. Circulating neutrophils of endotoxin-treated rats were also isolated at this time. Phagocytosis and CD11b/c and CD18 expression were measured by flow cytometry; tumor necrosis factor was measured with a cytotoxicity assay. Using the protein tyrosine kinase inhibitor AG126 and phosphotyrosine immunoblotting, we demonstrated that tyrosine phosphorylation is an important signaling pathway in the activation of these cells by endotoxin and that it is coupled to phagocytosis and tumor necrosis factor secretion, but not to beta 2 integrin expression. Conditioned medium of alveolar macrophages of endotoxin-injected rats upregulates phagocytosis by blood neutrophils of naive rats and this upregulating activity is tyrosine phosphorylation dependent. The substrates for tyrosine phosphorylation are different in alveolar macrophages and lung neutrophils, as are their sensitivities to AG126. Significant gender differences exist in the modulation of phagocytosis by inhibition of tyrosine phosphorylation and in tumor necrosis factor secretion by endotoxin-stimulated alveolar macrophages.

Gender differences in neutrophil function and cytokine-induced neutrophil chemoattractant generation in endotoxic rats.

Several lines of evidence indicate sexual dimorphism in the immune response. We explored gender differences in phagocytosis by neutrophils (PMNs), CD11b/c expression, generation of cytokine-induced neutrophil chemoattractant (CINC) and the influence of developmental stages on some of these parameters. Phagocytosis by PMNs of reproductive female rats was not suppressed by anesthesia and surgery as it was in age-matched males. The phagocytic response to an endotoxir (ET) challenge was also higher in PMNs of reproductive females than in males or in prereproductive or postreproductive females. CINC generation in reproductive females was lower than in age-matched males. Phagocytosis in saline-treated postreproductive females was reduced compared to reproductive and prereproductive females, but was not different from adult males. CD11b/c expression was greater in PMNs of saline treated postreproductive females, than in reproductive or prereproductive animals, but an ET challenge upregulated CD11b/c expression to the same level in all three groups. No gender difference was observed in this parameter. These data indicate that in terms of phagocytosis PMNs of reproductive female rats are more resistant to the effects of anesthesia and surgery and respond to an ET challenge more vigorously than cells of age-matched males. CINC generation in adult rats is also gender dependent. The developmental stages in females modulate phagocytosis and beta 2-integrin expression in PMNs.

Gender differences in phagocytic responses in the blood and liver, and the generation of cytokine-induced neutrophil chemoattractant in the liver of acutely ethanol-intoxicated rats.

Phagocytosis by circulating and liver-recruited polymorphonuclear leukocytes (PMNs) and Kupffer cells was studied in acutely ethanol-intoxicated, age-matched male and female rats. Acute ethanol intoxication in female rats is associated with a more effective phagocytic PMN response in the circulating blood, but with lower phagocytic activities by liver-recruited PMNs and Kupffer cells than in their male counterparts. Endotoxin [lipopolysaccharide (LPS)] treatment (consisting of a 90-min intravenous infusion of a nonlethal dose) of acutely ethanol-intoxicated male and female rats results in enhanced phagocytic responses in liver-sequestered PMNs and Kupffer cells, but not in circulating PMNs. However, the LPS challenge elicits a lesser phagocytic response in liver PMNs and Kupffer cells of female rats than in males. Significant gender differences exist in the extent of hepatic PMN infiltration in ethanol plus LPS-treated rats, which is paralleled by very similar differences in CD11b/c adhesion molecule expression in circulating PMNs and cytokine-induced neutrophil chemoattractant generation by hepatocytes and Kupffer cells. Taken together, these data indicate a smaller phagocytic response to fight infection in the liver of acutely alcohol-intoxicated female rats, but also a mechanism to afford some protection against neutrophil-associated tissue injury.

Attenuation of hepatic neutrophil sequestration by anti-CINC antibody in endotoxic rats.

Cytokine-induced neutrophil chemoattractant (CINC) is a member of the chemokine alpha sub-family. It is induced in rats by tumor necrosis factor-alpha (TNF-alpha), interleukin-1, and lipopolysaccharide and is implicated in neutrophil infiltration in response to inflammatory stimuli. We tested the hypothesis that pretreatment with anti-CINC antibody or by cobra venom factor attenuates hepatic neutrophil accumulation induced by a 90 min infusion of Escherichia coli endotoxin. Changes in the expression of CD11b/c and CD18 and in plasma TNF-alpha levels were also investigated. Cultured hepatocytes and Kupffer cells of endotoxic rats produced significantly more CINC than those of saline-infused controls. CINC generation by Kupffer cells was much lower than generation by hepatocytes. Pretreatment with anti-CINC antibody or cobra venom factor significantly reduced hepatic neutrophil sequestration, but did not affect the up-regulation of CD11b/c and CD18 expression on liver-sequestered neutrophils or plasma TNF-alpha levels. We conclude that CINC-mediated hepatic neutrophil accumulation may not be necessarily associated with up-regulation of neutrophil adhesion molecules or elevated circulating TNF-alpha levels. Attenuation of hepatic neutrophil sequestration by anti-CINC antibody is likely based on blocking of the chemotactic activity of CINC and thus diminishing the chemotactic gradient established in the liver.

Generation of a CD11b/c upregulating and chemotactic factor by hepatocytes of endotoxic rats--nonidentity with interleukin-8.

To further clarify the mechanism of polymorphonuclear leukocyte (PMN) recruitment into the liver associated with short term endotoxin infusion (1), we investigated the effect of a noval factor generated by hepatocytes of such endotoxic rats on the expression of PMN adhesion molecules CD11b/c and chemotactic activity. Conditioned medium of hepatocytes from endotoxin-infused rats shows a fast induction and dose-dependent activity for upregulating CD11b/c expression in and chemotactic activity for blood PMN of naive rats. Supernatants of naive control rats cultured in the presence of endotoxin and Kupffer cells and liver PMNs of endotoxic rats also produce activation, but to a much lesser extent. The upregulating activity can be reduced significantly by heat inactivation at 100 degrees C for 10 min and by pronase hydrolysis at 37 degrees C for 60 min. Generation of the activity does not depend on cyclooxygenase products or phospholipase A2 activity, and it does not seem to be associated with the complement pathway. The activity is associated with molecular masses of 9-12 and 27-32 kDa and cannot be reduced by antiserum to rat interleukin-8 in serial dilutions ranging from 1:50 to 1:25,600. The results show that hepatocytes from acutely endotoxin infused rats generate a small molecular weight protein factor (or factors) that is capable of upregulating PMN 11b/c expression and chemotactic activity and is seemingly different from rat interleukin-8. Thus, hepatocytes in endotoxemia may play an important role in modulating neutrophil function and contributing to the mechanism of neutrophil sequestration into the liver.

Prohormone convertases PC2 and PC3 in rat neutrophils and macrophages. Parallel changes with proenkephalin-derived peptides induced by LPS in vivo.

Prohormone- or proneuropeptide-converting enzymes PC2 and PC3 have been observed exclusively in nervous and endocrine tissues. In this work the presence of these enzymes in cells of the immune system was demonstrated. PC2 was detected in peripheral and liver-infiltrating polymorphonuclear leukocytes (PMN) but not in alveolar macrophages (AM) or spleen mononuclear cells (SMC). PC2 proteins corresponded to 75, 71 and 56 kDa forms. PC3 appeared in AM and SMC but not in PMN, and a 66 kDa protein was the only PC3 form detected. Proenkephalin-derived peptides (PENKp) were observed in PMN and AM, showing peptides of 35, 28, 21, 18 and 14 kDa in the former cells and a doublet of 35 and 32 kDa in the latter. PC2 proteins and PENKp decreased in liver PMN and peripheral PMN 90 min after intravenous (i.v.) infusion of LPS, suggesting an increased release. However, in vitro assays showed that the chemotactic peptide FMLP but not LPS increased the basal secretion of PC2 proteins and PENKp in PMN. These results indicate that PC2 proteins are released from PMN, together with PENKp, and suggest that LPS in vivo may act through an indirect mechanism. Low levels of PC3 and PENK were detected in the AM of rats treated for 90 min with SAL or LPS. However, a significant increase of PC3 and PENKp appeared 30 h after LPS infusion. These results show for the first time that PC2 and PC3 are differentially expressed in PMN and AM, respectively, which were paralleled by the presence of different post-translational products of PENK. In addition, the in vivo effect of LPS on PC2, PC3 and PENKp levels in PMN and AM resembles the effect of LPS on prohormone levels in endocrine tissues, suggesting that similar mechanisms may control the turnover of PENK in endocrine and in these immune cells.

Increased surface expression of CD11b/c and CD18 appears to be dissociated from anti-CD11b/c monoclonal antibody stimulated O2- anion generation in in vivo Escherichia coli lipopolysaccharide and tumor necrosis factor-alpha-treated rat neutrophils.

The purpose of this investigation was to determine the effect of anti-rat CD11b/c monoclonal antibody (MAb) on in vitro superoxide anion (O2-) generation in in vivo Escherichia coli lipopolysaccharide (LPS) and tumor necrosis-alpha (TNF)-treated rat polymorphonuclear leukocytes (PMN). After a continuous infusion of a nonlethal dose of E. coli LPS (.5 mg/kg) or TNF (6.0 x 10(5) units) into rats, PMN were recovered by centrifugal elutriation and discontinuous density gradient centrifugation from the liver (LPS-treated) and whole blood (LPS- and TNF-treated) and compared for CD11b/c, CD11a, and CD18 upregulation and their capacity for basal and agonist-stimulated O2- production. Immunofluorescence flow cytometry studies of rat whole blood PMN demonstrated that, upon LPS infusion, there was a time-dependent upregulation of CD11b/c and CD18, but not of CD11a. Similarly, TNF infusion upregulated CD11b/c although to a lesser degree. Stimulation of LPS- and TNF-treated PMN with phorbol 12-myristate 13-acetate (PMA), opsonized zymosan (OPZ), and anti-rat CD11b/c MAb triggered O2- generation. Although total O2- generated by OPZ and anti-rat CD11b/c MAb was less than that generated by PMA stimulation, the in vivo LPS- and TNF-induced beta 2 integrin upregulation did not result in a statistically significant enhancement of O2- generation with respect to normal saline-treated PMN. Our results do not appear to support the hypothesis that enhanced expression of CD11b/c or CD18 might be associated with enhanced in vitro anti-CD11b/c MAb-triggered O2- generation in LPS- and TNF-treated PMN in vivo.