High-mobility group box 1 protein is an inflammatory mediator in necrotizing enterocolitis: protective effect of the macrophage deactivator semapimod

High-mobility group box 1 (HMGB1) is a late mediator of endotoxemia known to stimulate the production of proinflammatory cytokines that are putative mediators of intestinal inflammation associated with necrotizing enterocolitis (NEC). We hypothesized that HMGB1 is also involved in the pathogenesis of NEC. We examined the expression of HMGB1 and the effect of the novel drug semapimod on intestinal inflammation in an experimental model of NEC in neonatal rats. Newborn rats were subjected to hypoxia and fed a conventional formula by gavage (FFH) or were breast fed (BF). Rats were killed on day 4, and the distal ileum was harvested for morphological studies and Western blot analysis. FFH newborn rats but not BF controls developed intestinal inflammation similar to the histological changes observed in human NEC. We found that the expression of HMGB1 and its receptor for advanced glycation end products (RAGE) as well as that of other apoptosis/inflammation-related proteins (Bad, Bax, inducible nitric oxide synthase, and cyclooxygenase 2) was upregulated in the ileal mucosa of FFH newborn rats compared with BF animals. Administration of the drug semapimod inhibited the upregulation of those proteins and partially protected the animals against the FFH-induced intestinal injury. Elevated levels of HMGB1 were also found in ileal samples from infants undergoing intestinal resection for acute NEC. Our results implicate HMGB1 and RAGE as important mediators of enterocyte cell death and hypoxia-induced injury in NEC and support the hypothesis that inhibitors such as semapimod might play a therapeutic role in chronic intestinal inflammation characterized by this animal model.     

Nitric oxide inhibits enterocyte migration through activation of RhoA-GTPase in a SHP-2-dependent manner

Diseases of intestinal inflammation like necrotizing enterocolitis (NEC) are associated with impaired epithelial barrier integrity and the sustained release of intestinal nitric oxide (NO). NO modifies the cytoskeletal regulator RhoA-GTPase, suggesting that NO could affect barrier healing by inhibiting intestinal restitution. We now hypothesize that NO inhibits enterocyte migration through RhoA-GTPase and sought to determine the pathways involved. The induction of NEC was associated with increased enterocyte NO release and impaired migration of bromodeoxyuridine-labeled enterocytes from terminal ileal crypts to villus tips. In IEC-6 enterocytes, NO significantly inhibited enterocyte migration and activated RhoA-GTPase while increasing the formation of stress fibers. In parallel, exposure of IEC-6 cells to NO increased the phosphorylation of focal adhesion kinase (pFAK) and caused a striking increase in cell-matrix adhesiveness, suggesting a mechanism by which NO could impair enterocyte migration. NEC was associated with increased expression of pFAK in the terminal ileal mucosa of wild-type mice and a corresponding increase in disease severity compared with inducible NO synthase knockout mice, confirming the dependence of NO for FAK phosphorylation in vivo and its role in the pathogenesis of NEC. Strikingly, inhibition of the protein tyrosine phosphatase SHP-2 in IEC-6 cells prevented the activation of RhoA by NO, restored focal adhesions, and reversed the inhibitory effects of NO on enterocyte migration. These data indicate that NO impairs mucosal healing by inhibiting enterocyte migration through activation of RhoA in a SHP-2-dependent manner and support a possible role for SHP-2 as a therapeutic target in diseases of intestinal inflammation like NEC.     

Recruitment of dendritic cells is responsible for intestinal epithelial damage in the pathogenesis of necrotizing enterocolitis by Cronobacter sakazakii

Cronobacter sakazakii is a Gram-negative pathogen associated with the cases of necrotizing enterocolitis (NEC) that result from formula contamination. In a mouse model of NEC, we demonstrate that C. sakazakii infection results in epithelial damage by recruiting greater numbers of dendritic cells (DCs) than macrophages and neutrophils in the gut and suppresses DC maturation, which requires outer membrane protein A (OmpA) expression in C. sakazakii. Pretreatment of intestinal epithelial cell monolayers with supernatant from OmpA(+) C. sakazakii/DC culture markedly enhanced membrane permeability and enterocyte apoptosis, whereas OmpA(-) C. sakazakii/DC culture supernatant had no effect. Analysis of OmpA(+) C. sakazakii/DC coculture supernatant revealed significantly greater TGF-β production compared with the levels produced by OmpA(-) C. sakazakii infection. TGF-β levels were elevated in the intestinal tissue of mice infected with OmpA(+) C. sakazakii. Cocultures of CaCo-2 cells and DCs in a "double-layer" model followed by infection with OmpA(+) C. sakazakii significantly enhanced monolayer leakage by increasing TGF-β production. Elevated levels of inducible NO synthase (iNOS) were also observed in the double-layer infection model, and abrogation of iNOS expression prevented the C. sakazakii-induced CaCo-2 cell monolayer permeability despite the presence of DCs or OmpA(+) C. sakazakii/DC supernatant. Blocking TGF-β activity using a neutralizing Ab suppressed iNOS production and prevented apoptosis and monolayer leakage. Depletion of DCs in newborn mice protected against C. sakazakii-induced NEC, whereas adoptive transfer of DCs rendered the animals susceptible to infection. Therefore, C. sakazakii interaction with DCs in intestine enhances the destruction of the intestinal epithelium and the onset of NEC due to increased TGF-β production.     

A critical role for TLR4 in the pathogenesis of necrotizing enterocolitis by modulating intestinal injury and repair

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in preterm infants and is characterized by translocation of LPS across the inflamed intestine. We hypothesized that the LPS receptor (TLR4) plays a critical role in NEC development, and we sought to determine the mechanisms involved. We now demonstrate that NEC in mice and humans is associated with increased expression of TLR4 in the intestinal mucosa and that physiological stressors associated with NEC development, namely, exposure to LPS and hypoxia, sensitize the murine intestinal epithelium to LPS through up-regulation of TLR4. In support of a critical role for TLR4 in NEC development, TLR4-mutant C3H/HeJ mice were protected from the development of NEC compared with wild-type C3H/HeOUJ littermates. TLR4 activation in vitro led to increased enterocyte apoptosis and reduced enterocyte migration and proliferation, suggesting a role for TLR4 in intestinal repair. In support of this possibility, increased NEC severity in C3H/HeOUJ mice resulted from increased enterocyte apoptosis and reduced enterocyte restitution and proliferation after mucosal injury compared with mutant mice. TLR4 signaling also led to increased serine phosphorylation of intestinal focal adhesion kinase (FAK). Remarkably, TLR4 coimmunoprecipitated with FAK, and small interfering RNA-mediated FAK inhibition restored enterocyte migration after TLR4 activation, demonstrating that the FAK-TLR4 association regulates intestinal healing. These findings demonstrate a critical role for TLR4 in the development of NEC through effects on enterocyte injury and repair, identify a novel TLR4-FAK association in regulating enterocyte migration, and suggest TLR4/FAK as a therapeutic target in this disease.     

Intestinal and hepatic expression of BNIP3 in necrotizing enterocolitis: regulation by nitric oxide and peroxynitrite

Necrotizing enterocolitis (NEC) is characterized by the upregulation of proinflammatory proteins, nitrosative stress, and increased enterocyte apoptosis. We examined the expression and regulation of the Bcl-2/adenovirus EIB 19-kDa-interacting protein 3 (BNIP3), a pro-apoptotic gene regulated by nitric oxide (NO) in hepatocytes, in NEC. Newborn rats subjected to hypoxia and fed a conventional formula by gavage (FFH) developed NEC and demonstrated elevated expression of BNIP3 mRNA and protein in mucosal scrapings of the ileal samples and in the liver. In contrast, control rats [breast-fed (BF) without hypoxia] did not develop NEC or elevated BNIP3 expression in these tissues. BNIP3 expression paralleled the histological manifestation of NEC. Supplementation of the formula with L-Nomega-(1-iminoethyl)lysine, an inducible NO synthase inhibitor, reduced BNIP3 expression in FFH animals to the levels found in BF animals. Both hypoxia and peroxynitrite upregulated BNIP3 protein expression in human intestinal cells. Finally, ileal samples obtained from infants undergoing surgical resection for acute NEC demonstrated higher levels of BNIP3 protein. Because hypoxia and formation of reactive nitrogen species may promote gut barrier failure, we propose that upregulation of the cell death-related protein BNIP3 is one possible mechanism associated with enterocyte cell death observed in the intestine with NEC.     

NF-kappaB-mediated expression of iNOS promotes epithelial defense against infection by Cryptosporidium parvum in neonatal piglets

Cryptosporidium sp. parasitizes intestinal epithelium, resulting in enterocyte loss, villous atrophy, and malabsorptive diarrhea. We have shown that mucosal expression of inducible nitric oxide (NO) synthase (iNOS) is increased in infected piglets and that inhibition of iNOS in vitro has no short-term effect on barrier function. NO exerts inhibitory effects on a variety of pathogens; nevertheless, the specific sites of iNOS expression, pathways of iNOS induction, and mechanism of NO action in cryptosporidiosis remain unclear. Using an in vivo model of Cryptosporidium parvum infection, we have examined the location, mechanism of induction, specificity, and consequence of iNOS expression in neonatal piglets. In acute C. parvum infection, iNOS expression predominated in the villous epithelium, was NF-kappaB dependent, and was not restricted to infected enterocytes. Ongoing treatment of infected piglets with a selective iNOS inhibitor resulted in significant increases in villous epithelial parasitism and oocyst excretion but was not detrimental to maintenance of mucosal barrier function. Intensified parasitism could not be attributed to attenuated fluid loss or changes in epithelial proliferation or replacement rate, inasmuch as iNOS inhibition did not alter severity of diarrhea, piglet hydration, Cl- secretion, or kinetics of bromodeoxyuridine-labeled enterocytes. These findings suggest that induction of iNOS represents a nonspecific response of the epithelium that mediates enterocyte defense against C. parvum infection. iNOS did not contribute to the pathogenic sequelae of C. parvum infection.     

Nitrosative stress in an animal model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a disease of newborns characterized by gut barrier failure. We reasoned that upregulation of inducible nitric oxide synthase (iNOS) may result in nitrosative stress and accumulation of nitroso species in the intestine. Newborn rats were either breast-fed (BF), or formula-fed and additionally subjected to hypoxia (FFH). At Day 4 after birth, the distal ilea were harvested and processed for Western blot analysis and measurement of NO-related metabolites. While BF neonates showed normal morphology, FFH neonates developed signs of NEC by Day 4. These pathological changes correlated with upregulation of iNOS and increases in tissue nitrite, nitrosothiol, and nitrosamine concentrations. Enhanced nitroso levels were most prominent in the mucosal layers of the ileum and iNOS inhibition resulted in a significant decrease in both nitroso species and incidence of NEC. In contrast, increased nitrite levels were distributed evenly throughout the ileum and remained unchanged following iNOS inhibition. Similarly, specimens from NEC patients had higher intestinal levels of NO-related metabolites compared to non-NEC controls. This is the first report of tissue levels of nitroso species in the gut of an animal model of NEC and of human specimens. The results suggest that local nitrosative stress contributes to the pathology associated with NEC. Unexpectedly, the NO breakdown product nitrite, previously considered biologically inert, was found to be present throughout the ileal wall, suggesting that cellular NO metabolism is altered significantly in NEC. Whether nitrite plays a protective or deleterious role remains to be investigated.     

Curcumin attenuates ovalbumin-induced airway inflammation by regulating nitric oxide

Curcumin has been strongly implicated as an anti-inflammatory agent, but the precise mechanisms of its action are largely unknown. In this study, we show that curcumin contributes to anti-inflammatory activity in the murine asthma model and lung epithelial cell A549 through suppression of nitric oxide (NO). To address this problem, curcumin was injected into the peritoneum of ovalbumin (OVA)-sensitized mice before the last allergen challenge. OVA challenge resulted in activation of the production of inducible nitric oxide (iNOS) in lung tissue, inflammatory cytokines, recruitment of eosinophils to lung airways, and airway hyper-responsiveness to inhaled methacholine. These effects of ovalbumin challenge were all inhibited by pretreatment of mice with curcumin. Furthermore, supplementation with curcumin in the A549 human airway epithelial cells decreased iNOS and NO production induced by IFN-γ. These findings show that curcumin may be useful as an adjuvant therapy for airway inflammation through suppression of iNOS and NO.     

The effect of alpha-melanocyte stimulating hormone on endotoxin-induced intestinal injury.

We investigated the effect of alpha-melanocyte stimulating hormone (alpha-MSH) on endotoxin-induced intestinal inflammation and the role of nitric oxide and prostaglandins in this response. alpha-MSH treatment (25 microg/rat, intraperitoneally (i.p.); twice daily) reduced the severity of the lesions macroscopically and microscopically. This protective effect was found to be confined mainly to the distal ileum. These lesions were reversed by pretreatment with the non-selective COX inhibitor indomethacin (10 mg/kg, subcutaneously (s.c.)) but not by the selective COX-2 inhibitor nimesulide (3 mg/kg, s.c.), the NO donor sodium nitroprusside (4 mg/kg, i.v.) or the iNOS inhibitor dexamethasone (3 mg./kg, i.p.) at macroscopic level and reversed by Indo or Dex at microscopic level. Increased peroxidase activity -index of tissue neutrophil infiltration- in the distal ileum of LPS-treated rats was decreased by alpha-MSH and this effect was reversed by pretreatment with Indo. In conclusion, the neuropeptide alpha-MSH has a beneficial effect on endotoxin-induced distal intestinal lesions by a mechanism which probably involves nitric oxide and COX-1 derived prostaglandins.     

Pomegranate seed oil reduces intestinal damage in a rat model of necrotizing enterocolitis

Pomegranate seed oil (PSO), which is the major source of conjugated linolenic acids such as punicic acid (PuA), exhibits strong anti-inflammatory properties. Necrotizing enterocolitis (NEC) is a devastating disease associated with severe and excessive intestinal inflammation. The aim of this study was to evaluate the effects of orally administered PSO on the development of NEC, intestinal epithelial proliferation, and cytokine regulation in a rat model of NEC. Premature rats were divided into three groups: dam fed (DF), formula-fed rats (FF), or rats fed with formula supplemented with 1.5% of PSO (FF + PSO). All groups were exposed to asphyxia/cold stress to induce NEC. Intestinal injury, epithelial cell proliferation, cytokine production, and trefoil factor 3 (Tff3) production were evaluated in the terminal ileum. Oral administration of PSO (FF+PSO) decreased the incidence of NEC from 61 to 26%. Feeding formula with PSO improved enterocyte proliferation in the site of injury. Increased levels of proinflammatory IL-6, IL-8, IL-12, IL-23, and TNF-α in the ileum of FF rats were normalized in PSO-treated animals. Tff3 production in the FF rats was reduced compared with DF but not further affected by the PSO. In conclusion, administration of PSO protects against NEC in the neonatal rat model. This protective effect is associated with an improvement of intestinal epithelial homeostasis and a strong anti-inflammatory effect of PSO on the developing intestinal mucosa.     

Activated macrophages inhibit enterocyte gap junctions via the release of nitric oxide

Enterocytes exist in close association with tissue macrophages, whose activation during inflammatory processes leads to the release of nitric oxide (NO). Repair from mucosal injury requires the migration of enterocytes into the mucosal defect, a process that requires connexin43 (Cx43)-mediated gap junction communication between adjacent enterocytes. Enterocyte migration is inhibited during inflammatory conditions including necrotizing enterocolitis, in part, through impaired gap junction communication. We now hypothesize that activated macrophages inhibit gap junctions of adjacent enterocytes and seek to determine whether NO release from macrophages was involved. Using a coculture system of enterocytes and macrophages, we now demonstrate that "activation" of macrophages with lipopolysaccharide and interferon reduces the phosphorylation of Cx43 in adjacent enterocytes, an event known to inhibit gap junction communication. The effects of macrophages on enterocyte gap junctions could be reversed by treatment of macrophages with the inducible nitric oxide synthase (iNOS) inhibitor l-Lysine omega-acetamidine hydrochloride (l-NIL) and by incubation with macrophages from iNOS(-/-) mice, implicating NO in the process. Activated macrophages also caused a NO-dependent redistribution of connexin43 in adjacent enterocytes from the cell surface to an intracellular location, further suggesting NO release may inhibit gap junction function. Treatment of enterocytes with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) markedly inhibited gap junction communication as determined using single cell microinjection of the gap junction tracer Lucifer yellow. Strikingly, activated macrophages inhibited enterocyte migration into a scraped wound, which was reversed by l-NIL pretreatment. These results implicate enterocyte gap junctions as a target of the NO-mediated effects of macrophages during intestinal inflammation, particularly where enterocyte migration is impaired.     

Alpha2-adrenergic regulation of NO production alters postoperative intestinal smooth muscle dysfunction in rodents

Alpha2-adrenergic receptor activation plays an important role in the development of postoperative ileus. Alpha2-adrenergic receptors also regulate nitric oxide (NO) production by the mononuclear phagocyte system. We have previously shown that intestinal manipulation leads to a significant increase in NO production by infiltrating monocytes within the intestinal muscularis. The purpose of this study was to investigate whether alpha2-adrenergic blockade with yohimbine would alter postsurgical intestinal smooth muscle dysfunction and NO production by infiltrating monocytes and macrophages within the intestinal muscularis. Rats underwent small bowel intestinal manipulation with or without yohimbine. In vivo gastrointestinal transit and in vitro jejunal circular muscle contractility was measured 24 h postoperatively. RT-PCR was used to detect inducible NO synthase (iNOS) expression. NO levels in tissue culture supernatants were measured. Immunohistochemistry was used to localize alpha2-adrenergic receptor expression in the intestinal muscularis. Yohimbine significantly decreased manipulation-induced delay in gastrointestinal transit and reversed the postoperative decrease in intestinal muscle contractility. Intestinal manipulation resulted in significant iNOS mRNA induction in the intestinal muscularis, which was markedly attenuated after yohimbine treatment. Yohimbine also significantly decreased the postoperative increase in NO released into intestinal muscularis tissue culture supernatant. Immunohistochemistry identified alpha2-adrenergic receptors on monocytes recruited postoperatively into the intestinal muscularis. This study demonstrates that alpha2-adrenergic receptor stimulation of the inflamed postoperative intestinal muscularis plays a significant role in aggravating postoperative ileus through an enhanced induction of iNOS mRNA and increased release of NO from manipulated intestinal muscularis.     

The therapeutic effect of recombinant human trefoil factor 3 on hypoxia-induced necrotizing enterocolitis in immature rat

Trefoil peptides are a new class of regulatory peptides involved in mucosal protection and repair in the gastrointestinal tract. Among them, trefoil factor 3 (TFF3) (intestinal trefoil factor) is known to be cytoprotective in the gut. The aim of this study was to determine the effect of recombinant human trefoil factor 3 (rhTFF3) on hypoxia-induced necrotizing enterocolitis (NEC) in immature rats. In the present study, thirty-two 1-day-old Wistar rat pups were randomly divided into four groups. Group 1 served as nonhypoxic controls. Group 2 rats were subjected to hypoxia reoxygenation (H/O) and then were returned to their mothers. Groups 3 and 4 rats were subjected to H/O, were returned to their mothers, and were treated with rhTFF3 intraperitoneally (0.5 mg) and subcutaneously (0.2 mg), respectively, for the next 3 days. All animals were killed on day 4, and intestine specimens were obtained to determine the histological changes, tissue level of interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), malondialdehyde (MDA), prostaglandin E2 (PGE2), tromboxane B2 (TXB2), and nitric oxide (NO). In addition, the effects of rhTFF3 on abundance of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) were also investigated. In neonatal NEC (group 2), necrosis of villi and crypts and, in some cases, transmural necrosis was observed under light microscopy. Tissue level of interleukin-8, tumor necrosis factor-alpha, malondialdehyde, prostaglandin E2, tromboxane B2, and nitric oxide were significantly higher than group 1. In addition, abundance of inducible nitric oxide synthase and cyclooxygenase 2 was markedly increased. In groups 3 and 4, only very slight intestinal injury was observed. The tissue level of interleukin-8, tumor necrosis factor-alpha, malondialdehyde, prostaglandin E2, tromboxane B2, and nitric oxide were significantly decreased in comparison to the group 2. Meanwhile, the abundance of inducible nitric oxide synthase and cyclooxygenase 2 was also marked decreased in comparison to group 2. The current study suggests a therapeutic role of TFF3 in an experimental model of NEC. Our findings may open a new insight into the treatment of NEC in newborns.     

Chronic blockade of NO synthase paradoxically increases islet NO production and modulates islet hormone release

Islet production of nitric oxide (NO) and CO in relation to islet hormone secretion was investigated in mice given the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) in their drinking water. In these mice, the total islet NO production was paradoxically increased, reflecting induction of inducible NOS (iNOS) in background of reduced activity and immunoreactivity of constitutive NOS (cNOS). Unexpectedly, normal mice fasted for 24 h also displayed iNOS activity, which was further increased in L-NAME-drinking mice. Glucose-stimulated insulin secretion in vitro and in vivo was increased in fasted but unaffected in fed mice after L-NAME drinking. Glucagon secretion was increased in vitro. Control islets incubated with different NOS inhibitors at 20 mM glucose displayed increased insulin release and decreased cNOS activity. These NOS inhibitors potentiated glucose-stimulated insulin release also from islets of L-NAME-drinking mice. In contrast, glucagon release was suppressed. In islets from L-NAME-drinking mice, cyclic nucleotides were upregulated, and forskolin-stimulated hormone release, CO production, and heme oxygenase (HO)-2 expression increased. In conclusion, chronic NOS blockade evoked iNOS-derived NO production in pancreatic islets and elicited compensatory mechanisms against the inhibitory action of NO on glucose-stimulated insulin release by inducing upregulation of the islet cAMP and HO-CO systems.     

Hypoxia-induced regulation of nitric oxide synthase in cardiac endothelial cells and myocytes and the role of the PI3-K/PKB pathway

The roles of endothelial nitric oxide synthase (eNOS), and its putative association with protein kinase B (PKB), and inducible nitric oxide synthase (iNOS) are not well characterized in hypoxic cardiac cells and there is a lack of studies that measure nitric oxide (NO) directly. Objective To measure NO production in cardiomyocytes and cardiac microvascular endothelial cells (CMECs) under baseline and hypoxic conditions and to evaluate the expression, regulation and activation of eNOS, iNOS and PKB. The effect of PI3-K/PKB inhibition on NO production and eNOS expression/activation was also investigated. Methods Adult rat cardiomyocytes and rat CMECs were made hypoxic by cell pelleting and low PO₂ incubation. Intracellular NO was measured by FACS analysis of DAF-2/DA fluorescence, and eNOS, iNOS and PKB were evaluated by Western blotting or flow cytometry. Upstream PKB inhibition was achieved with wortmannin. Results (1) NO levels increased in both cell types after exposure to hypoxia. (2) In hypoxic CMECs, eNOS was upregulated and activated, no iNOS expression was observed and PKB was activated. (3) In myocytes, hypoxia did not affect eNOS expression, but increased its activation. Activated PKB also increased during hypoxia. FACS analysis showed increased iNOS in hypoxic myocytes. (4) Wortmannin resulted in decreased hypoxia-induced NO production and reduced activated eNOS levels. Conclusions Cardiomyocytes and CMECs show increased NO production during hypoxia. eNOS seems to be the main NOS isoform involved as source of the increased NO generation, although there may be a role for iNOS and other non-eNOS sources of NO in the hypoxic myocytes. Hypoxia-induced PKB and eNOS activation occurred simultaneously in both cell types, and the PI3-K/PKB pathway was associated with hypoxia-induced NO production via eNOS activation.     

Inhaled NO reduces leukocyte-endothelial cell interactions and myocardial dysfunction in endotoxemic rats

Inhaled nitric oxide (NO) has been shown to have some protective effect in the peripheral distal inflamed vasculature. The objective of the study was to determine whether inhaled NO would reduce endotoxin-induced leukocyte activation and myocardial contractile dysfunction. Rats were treated with either saline or endotoxin (10 mg/kg iv) and then allowed to breathe (4 h) either air or air plus NO (10 ppm). In endotoxemic rats, mesenteric venular endothelium leukocyte firm adhesion increased compared with control rats (1.15 +/- 0.32 vs. 4.08 +/- 0.96 leukocytes/100 microm; P < 0.05). Inhaled NO significantly attenuated endotoxin-induced venular endothelium leukocyte adhesion (4.08 +/- 0.96 vs. 1.86 +/- 0.76 leukocytes/100 microm; P < 0.05) and FITC-conjugated anti-intercellular adhesion molecule-1 fluorescence intensity. Endotoxin-induced myocardial dysfunction and leukocyte content increases were reduced in inhaled NO-treated rats. These observations suggest that inhaled NO reduces the degree of cardiovascular dysfunction and inflammation in endotoxemic rats.