Diacylglycerols induce both ion pumping in patch-clamped guard-cell protoplasts and opening of intact stomata

Stomatal guard cells in leaves regulate the apertures of microscopic pores through which photosynthetic gas exchange and water vapor loss occur. Environmental signals, including light, high humidity, and low CO2 concentrations, open stomata by increasing the volume of guard cells. Activation of a plasma membrane H+ pump initiates K+ and Cl- influx, accompanied by malate synthesis, resulting in osmotic water flow into the guard cells, a bowing apart of the guard-cell pair, and consequent stomatal opening. Physiological and electrophysiological techniques were employed to investigate the possibility that a second-messenger lipid, 1,2-diacylglycerol, is involved in the transduction of opening stimuli. The synthetic diacylglycerols 1,2-dihexanoylglycerol and 1,2-dioctanoylglycerol enhanced light-induced stomatal opening in Commelina communis and induced stomatal opening under darkness, whereas an isomer with no known second-messenger role, 1,3-dioctanoylglycerol, did not affect stomatal responses. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, the enzyme typically activated by 1,2-diacylglycerol in animal cells, inhibited light-stimulated stomatal opening and enhanced dark-induced stomatal closure. N-[(2-Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), which inhibits cyclic nucleotide-dependent protein kinases preferentially over lipid-dependent protein kinases such as protein kinase C, had little effect on stomatal apertures. Whole-cell patch clamping of guard-cell protoplasts of Vicia faba revealed that 1,2-dihexanoylglycerol and 1-oleoyl-2-acetylglycerol activated an ATP-dependent, voltage-independent current, suggesting activation of an electrogenic ion pump such as the H+ pump. Diacylglycerol or functionally similar lipids may act through protein phosphorylation to provide the intracellular signals that mediate H+-ATPase activation and stomatal opening in response to light or other opening stimuli.     

Diacylglycerols mimic phorbol diester induction of leukemic cell differentiation.

Activation of cellular protein kinase C appears to be involved in the mechanism by which phorbol diesters induce differentiation of human myeloid leukemia cells (HL-60). Protein kinase C is thought to be physiologically activated by diacylglycerol derived from receptor-mediated phosphatidylinositol hydrolysis. sn-1,2-diacylglycerols with short saturated acyl side chains (C4-C10) were synthesized and found to be potent activators of protein kinase C partially purified from HL-60 cells. These diacylglycerols were also competitive inhibitors of [3H]phorbol dibutyrate binding to the soluble phorbol diester receptor. The most potent diacylglycerol, sn-1,2-dioctanoylglycerol, displaced greater than 90% of [3H]phorbol dibutyrate from the phorbol diester receptor of intact HL-60 cells. Because of probable cellular metabolism of sn-1,2-dioctanoylglycerol, hourly doses were required to maintain persistent occupancy of the phorbol diester binding site. Treatment of HL-60 cells with either phorbol 12-myristate 13-acetate or sn-1,2-dioctanoylglycerol produced identical phosphoprotein changes. Finally, sn-1,2-dioctanoylglycerol induced differentiation of the HL-60 cells into cells with morphologic characteristics of macrophages. Substitution of the hydroxyl group at position 3 with a hydrogen, chloro, or sulfhydryl moiety inactivated sn-1,2-dioctanoylglycerol. These data strengthen the hypothesis that protein kinase C activation plays a role in macrophage differentiation.     

Urokinase in conditioned medium from phorbol ester-pretreated endothelial cells promotes polymorphonuclear leukocyte migration.

Serum-free conditioned medium (CM) generated by human umbilical vein endothelial cell monolayers following pretreatment with 100 ng/ml of phorbol myristate acetate (PMA) promoted human polymorphonuclear leukocyte (PMNL) migration as assayed in blindwell chambers. Stimulation of PMNL migration in response to CM was dependent on the dose of PMA used to pretreat the endothelial cells as well as the duration of incubation time to generate CM. Phorbol esters have been previously shown to release plasminogen activators from vascular endothelial cells. In the present study, pretreatment of endothelial cells with PMA also increased plasminogen activator activity in CM at a time course similar to the generation of PMNL chemoattractant activity. Treatment of CM with a polyclonal antibody against human urokinase-type plasminogen activator (uPA) not only inhibited uPA activity, but also significantly reduced PMNL chemoattractant activity when compared with untreated CM. In contrast, treatment of CM with an antibody directed against tissue-type plasminogen activator (tPA) did not affect PMNL migratory activity. Furthermore, when CM was passed over an anti-uPA immunoaffinity column, plasminogen activator activity was reduced 90% and chemoattractant activities was reduced 68%. Both plasminogen activator and chemoattractant activities were reconstituted in the eluate from the anti-uPA column. These data demonstrate that uPA present in the CM from PMA-pretreated endothelial cells stimulates PMNL chemoattractant activity and suggests a possible role for endothelial cell-derived uPA in stimulating migration of peripheral blood leukocytes at an inflammatory locus.     

Characterization of the chemotactic defect in polymorphonuclear leukocytes exposed to influenza virus in vitro

The mechanism by which influenza virus interferes with polymorphonuclear leukocyte (PMN) chemotaxis was investigated. Incubation of human PMN with influenza A virus in vitro for 30 minutes significantly decreased PMN migration under agarose in response to N- formyl-methionyl-leucyl-phenylalanine (FMLP) or zymosan-activated serum. Virus-treated PMN tended to aggregate in the under-agarose assay. Aggregation was avoided by using a more dilute PMN suspension in filter assays. Virus treatment significantly decreased migration through 100-micron thick cellulose nitrate filters but had no effect on migration through 10-micron thick polycarbonate filters or on PMN bipolar shape change. Virus was not chemotactic in the polycarbonate filter assay and did not induce shape change in purified PMN. It was concluded that influenza virus did not interfere with the ability of PMN to recognize a chemoattractant, undergo shape change, and move a short distance but did limit the extent of migration. Inhibition could not be explained by chemotactic deactivation, since the virus was not chemotactic.     

Chemotactic activity of recombinant human granulocyte colony- stimulating factor

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) induced migration across polycarbonate filters of human polymorphonuclear leukocytes (PMN). rhG-CSF was active in inducing PMN migration at concentrations greater than or equal to 10 to 100 U/mL (7 to 70 ng/mL). rhG-CSF did not contain appreciable levels of endotoxin contamination as assessed by Limulus amebocyte assay, and Polymixin B did not affect the chemotactic activity of rhG-CSF. A monoclonal anti-G- CSF antibody blocked the induction of migration by G-CSF, thus establishing that the cytokine was responsible for the activity of the recombinant preparation. Checkerboard analysis was performed by seeding different concentrations of G-CSF above and/or below the filter and revealed that the migratory response to this cytokine was best observed in the presence of a positive concentration gradient between the lower and upper compartments of the chamber, thus indicating an actual chemotactic effect. When different migrating cells were examined, rhG- CSF was inactive on large granular lymphocytes and endothelial cells under conditions in which appropriate reference attractants were active. In contrast, rhG-CSF elicited a chemotactic response in monocytes inhibited by specific antibody. Thus, G-CSF is a chemotactic signal for phagocytes. This cytokine, when produced at inflammatory sites, may contribute to the recruitment of phagocytes from the blood compartment to amplify resistance against certain noxious agents.     

Blocking p21-activated kinase reduces lipopolysaccharide-induced acute lung injury by preventing polymorphonuclear leukocyte infiltration

RATIONALE: Excessive recruitment of polymorphonuclear leukocytes (PMNs) to the lung promotes acute lung injury (ALI). Chemokine receptors and adhesion molecules initiate leukocyte-endothelial interactions, but mediators of PMN migration through the alveolo-capillary membrane remain to be identified. p21-Activated kinase (PAK) is an effector of small GTPases and has been implicated in cell migration. OBJECTIVES: To test the role of PAK in ALI. METHODS: An inhibitory PAK peptide was used to determine the role of PAK in cytoskeletal actin polymerization, cell adhesion, and oxidative burst. PMN migration was investigated in vitro and in a murine model of lipopolysaccharide-induced lung injury. MEASUREMENTS AND MAIN RESULTS: PMN migration into lung interstitium and alveolar space was suppressed by an inhibitory PAK peptide. Neutrophils that had taken up the inhibitory PAK peptide were unable to enter the alveolar space. CXCL2/3, an important PMN chemoattractant in murine lung injury, induced PAK phosphorylation in PMNs. Blocking PAK function inhibited chemotaxis, chemokine-induced cytoskeletal actin polymerization, and adhesion-induced oxidative burst. CONCLUSIONS: We conclude that neutrophil PAK is a critical mediator of PMN migration and may be an attractive target in ALI.     

Effect of steroid treatment on the migration behaviour of neutrophils in patients with early rheumatoid arthritis

The influence of methylprednisolone on the migratory characteristics of neutrophil granulocytes was investigated in 10 patients with early rheumatoid arthritis (RA) and compared to 12 controls. The migration of neutrophils was measured with a whole-blood membrane filter assay with and without stimulation by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP). Total migration index (TMI), distribution characteristics (DC) and the product of TMI and DC (neutrophil migratory activity; NMA) served to characterize the migratory behaviour of neutrophils. The data demonstrated an increased polymorphonuclear leucocyte (PMN) migration in patients with early RA, indicating a bystander role of PMNs in inflammatory joint injury. Treatment with methylprednisolone reduced significantly the penetration depth (DC) of neutrophils, but did not influence the number of migrating cells (TMI). The unstimulated NMA was significantly reduced due to the marked DC reduction, whereas steroids did not influence the stimulated NMA of neutrophils. A significant reduction in PMN penetration depth was demonstrated only after a steroid therapy of at least 10 days, suggesting that a longer period of steroid therapy is necessary to provide effective inflammatory control. Received: 24 March 1997 / Accepted: 8 September 1997     

1,2-Diacylglycerol and phorbol ester inhibit agonist-induced formation of inositol phosphates in human platelets: possible implications for negative feedback regulation of inositol phospholipid hydrolysis.

The present study has demonstrated that pretreatment of human platelets with either phorbol ester or 1,2-diacylglycerol inhibits agonist-induced formation of inositol phosphates; this inhibition can be correlated with a decrease in the release of ATP and 5-hydroxytryptamine by thrombin. The mechanism of this action is not known, but a role for protein kinase C is suggested, as both phorbol ester and 1,2-diacylglycerol have in common the ability to activate this enzyme. These results have important implications as a possible negative feedback control over agonist-induced hydrolysis of inositol phospholipids.     

Molecular and functional analysis of a lymphocyte chemoattractant factor: association of biologic function with CD4 expression.

Lymphocyte chemoattractant factor (LCF) is a lymphocyte cell product that stimulates a migratory response in CD4+ lymphocytes, monocytes, and eosinophils. In concert with its chemoattractant activity, LCF induces human T-lymphocyte expression of interleukin 2 receptor. Here we describe the molecular cloning of cDNA encoding human LCF. It is a novel interleukin with no significant homology to any previously described cytokine families. There is an absolute requirement for both autoaggregation of LCF monomers and for membrane-expressed CD4 molecules for LCF-induced migration in lymphocytes.     

1,2-diacylglycerol content in thoracic aorta of spontaneously hypertensive rats

Phosphoinositide metabolism participates in the control of cell calcium homeostasis. Because a notable neutral lipid (1,2-diacylglycerol) is generated from phosphoinositide hydrolysis and is assumed to be a secondary messenger, we determined 1,2-diacylglycerol content and its fatty acid profiles in the thoracic aorta of spontaneously hypertensive rats (SHR) and compared it with those of normotensive Wistar-Kyoto (WKY) rats. After the aorta was exposed to 10(-5) M norepinephrine as a stimulant, 1,2-diacylglycerol content in SHR was significantly higher by 33% than in WKY rats at 4 weeks of age, whereas there was no difference in 1,2-diacylglycerol content between the two strains at 20 weeks of age. Before norepinephrine stimulation, there was no significant difference in 1,2-diacylglycerol level between the two strains at 4 weeks of age. Analysis on a gas chromatograph showed that 1,2-diacylglycerol was composed of similar molecular species of fatty acids in aortas obtained from SHR and WKY rats. On the other hand, the cholesterol content of aortas was higher in SHR than in WKY rats at 20 weeks of age, whereas the difference at 4 weeks was not significant. Phosphatidylcholine, phosphatidylethanolamine, and triglyceride showed no significant difference between the two strains. It is concluded that norepinephrine-induced 1,2-diacylglycerol production increases in the thoracic aorta of SHR before the development of hypertension.     

Activation of human neutrophils by monoclonal antibody PMN7C3: cell movement and adhesion can be triggered independently from the respiratory burst

Anti-neutrophil monoclonal antibody PMN7C3 (IgG3) recognizes glycoproteins bearing the oligosaccharide lacto-N-fucopentaose III, including the C3bi receptor, LFA-1, and p150,95 on the plasma membrane and a group of granule-associated glycoproteins. We have previously shown that binding of this antibody to polymorphonuclear leukocytes (PMNs) stimulates a transient rise in cytosolic free calcium concentration but does not trigger the neutrophil respiratory burst. We now demonstrate that binding of PMN7C3 (and five other monoclonal antibodies recognizing the same antigen) to human neutrophils activates several other cellular responses. Addition of PMN7C3 to monolayers of neutrophils induces a rapid change in cell shape followed by pseudopod formation and increased migration. With incubation at 37 degrees C, the neutrophils aggregate in clusters (leukoagglutination). Quantitation of cell movement in a multiwell chemotaxis assembly or by migration of PMNs under agarose revealed that PMN7C3 is both chemotactic and chemokinetic. Pretreatment with the antibody inhibits subsequent chemotactic response to other stimuli. Monoclonal antibodies binding to other neutrophil antigens do not mimic these effects. These data suggest that cell movement and adhesion can be triggered independently from the respiratory burst. PMN7C3 may be a useful probe with which to study the events that link receptor-ligand binding to cellular response.     

Activation of resting human T cells requires prolonged stimulation of protein kinase C.

Purified resting human T cells can be induced to express the alpha subunit of the interleukin 2 receptor and to proliferate by treatment with 12-O-tetradecanoylphorbol-13-acetate plus ionomycin but not with 1,2-dioctanoylglycerol plus ionomycin. Determination of the translocation of protein kinase C showed that 12-O-tetradecanoylphorbol-13-acetate plus ionomycin caused a prolonged membrane association of the enzyme for more than 4 hr, whereas 1,2-dioctanoylglycerol plus ionomycin induced a transient membrane association, which was maximal at 20 min. Delivery of multiple additions of 1,2-dioctanoylglycerol plus ionomycin to the T cells resulted in progressively increased expression of the alpha subunit of the interleukin 2 receptor and proliferation commensurate with the number of multiple additions delivered, suggesting that prolonged protein kinase C activity is required for T-cell activation.     

Neutrophil response following intratracheal instillation of collagen peptides into rat lungs

Inflammatory lung diseases are associated with destruction of interstitial collagen and release of degraded collagen fragments into the lower respiratory tract. To determine whether degraded collagen might be one factor mediating the cellular influx, we measured polymorphonuclear leukocytes (PMN) in bronchoalveolar lavage fluid following intratracheal instillation of collagen peptides. The responses to collagenous peptides derived from collagen digested with bacterial collagenase and to the collagenous-like polytripeptide (proline-proline-glycine) were examined. Both types of collagen peptides were chemotactic for human PMN in vitro. Two days following instillation of 5 mg collagenous peptides, we observed a threefold increase in the percentage of PMN in lavage fluid with a maximum (fivefold) response on day 6. Since other chemoattractants produce a response within hours when instilled intratracheally, we postulated that the late neutrophil influx produced with collagen may be the result of production of a neutrophil chemoattractant by alveolar macrophages. Alveolar macrophages treated with collagenous or collagenous-like peptides released PMN chemotactic factors, and the time course of release of chemotactic activity by alveolar macrophages in vitro correlated with the in vivo finding of a 2-6-day delay in PMN accumulation in the lungs. These observations are consistent with the idea that collagen peptides may stimulate alveolar macrophages to produce chemotactic factors for neutrophils. This mechanism may play a role in the accumulation of phagocytic cells in the lung following injury.     

Early pulmonary granulocyte recruitment in response to Streptococcus pneumoniae

Although polymorphonuclear leukocytes (PMN) are a conspicuous histologic feature of clinical and experimental pneumococcal pneumonia, neither the mechanism nor the magnitude of recruitment of these cells to the lung following lesser pneumococcal challenge is known. We have, therefore, investigated the early process of recruitment of PMN to alveolar spaces after pulmonary inoculation of Streptococcus pneumoniae in doses less than those causing pneumonia. We injected Balb/c mice with water and varying inoculums of pneumococci via an endobronchial catheter. Bronchoalveolar lavage (BAL) was performed on the inoculated lung at 0, 2, or 4 h after injection. Cellular response was measured and chemotactic activity was assayed on BAL supernatants at each time interval using the migration of human PMN through 3-micron filters in modified Boyden chambers by the leading front techniques. The BAL of normal and control animals (inoculum of sterile water only used for the control animals) yielded 5.03 +/- 1.51 X 10(2) and 0.17 +/- 0.04 X 10(5) PMN, respectively. The PMN recruitment at 4 h as a function of pneumococcal inoculum was described by the following equation: log PMN = 0.751 log Pn + 1.119 (r2 = 0.82, p less than 0.001). The PMN were, therefore, recruited in a dose-dependent manner. That recruitment may be caused by chemotactic substance(s) was suggested by the significant correlation between the PMN response and the distance of in vitro migration: log PMN = 0.057 micron + 0.52 (r = 0.77, p less than 0.005). We have defined quantitatively the recruitment of PMN to the lung after pneumococcal challenge.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro modulation of porcine Leydig cell steroidogenesis by phorbol-12-myristate-13-acetate and 1,2-dioctanoylglycerol

Serum-free primary cultures of neonatal (1-day-old) porcine Leydig cells were used to study the effects of phorbol-12-myristate-13-acetate and 1,2-dioctanoylglycerol on testosterone and pregnenolone production. Phorbol-12-myristate-13-acetate alone from 0.001-10 mumol/l stimulated testosterone and pregnenolone production, whereas 1,2-dioctanoylglycerol alone had no effect on steroid production, relative to control. Phorbol-12-myristate-13-acetate and 1,2-dioctanoylglycerol each inhibited pLH-stimulated testosterone and pregnenolone production. To further clarify the influence of these protein kinase C activators on steroidogenesis, cultured Leydig cells were treated with either phorbol-12-myristate-13-acetate or 1,2-dioctanoylglycerol plus forskolin (an adenylate cyclase activator). Both phorbol-12-myristate-13-acetate and 1,2-dioctanoylglycerol inhibited forskolin-stimulated testosterone production. Phorbol-12-myristate-13-acetate had no effect on forskolin-stimulated pregnenolone production and only the highest concentration of 1,2-dioctanoylglycerol (100 mumol/l) inhibited forskolin-stimulated production of pregnenolone. These data demonstrate that porcine Leydig cell steroidogenesis can be modulated by interactions of the protein kinase C and protein kinase A second messenger systems.     

Hydrolysis of phosphatidylinositol by human endometrium: modulating effects of steroids on arachidonic acid and 1,2-diacylglycerol release

Phospholipase C and 1,2-diacylglycerol lipase activities were demonstrated in human endometrium using 1-stearoyl-2-[1-14C]arachidonyl phosphatidylinositol as substrate. Phosphatidylinositol is hydrolysed by phospholipase C to inositol phosphates and to 1,2-diacylglycerol which is then further metabolized by 1,2-diacylglycerol lipase to release free arachidonic acid. In the present study the radiolabelled products formed (1,2-diacylglycerol and arachidonic acid) were measured following chloroform/methanol extraction and thin-layer chromatography. Phospholipase C activity was calcium dependent and optimal at pH 5.0-5.5 and 7.5; 1,2-diacylglycerol lipase activity was also calcium dependent, with an optimum pH of 5.5. A significant increase in 1,2-diacylglycerol production was stimulated by steroid sulphates. Pregnenolone sulphate, oestrone sulphate, testosterone sulphate and dehydroepiandrosterone sulphate stimulated 4, 3.2-, 1.8- and 2.6-fold increases in release respectively. Oestradiol sulphate stimulated a 25% increase in diacylglycerol release which was not significantly different from the control value. Progesterone stimulated a fourfold increase but other free steroids had no effect. Arachidonic acid release was increased in the presence of oestradiol sulphate, oestrone and oestradiol but reduced by oestrone sulphate, dehydroepiandrosterone sulphate, progesterone, dehydroepiandrosterone and, to a lesser extent, by pregnenolone sulphate and testosterone sulphate. 5-Androstene-3 beta,17 beta-diol had no effect on the liberation of either product. This study demonstrates a potential route for the liberation of arachidonic acid from phosphatidylinositol in human endometrium. The opposing effects of steroids on phospholipase C and 1,2-diacylglycerol lipase activity could be important in regulating the release of arachidonic acid by this pathway.     

A secreted Salmonella protein induces a proinflammatory response in epithelial cells, which promotes neutrophil migration

In response to Salmonella typhimurium, the intestinal epithelium generates an intense inflammatory response consisting largely of polymorphonuclear leukocytes (neutrophils, PMN) migrating toward and ultimately across the epithelial monolayer into the intestinal lumen. It has been shown that bacterial-epithelial cell interactions elicit the production of inflammatory regulators that promote transepithelial PMN migration. Although S. typhimurium can enter intestinal epithelial cells, bacterial internalization is not required for the signaling mechanisms that induce PMN movement. Here, we sought to determine which S. typhimurium factors and intestinal epithelial signaling pathways elicit the production of PMN chemoattractants by enterocytes. Our results suggest that S. typhimurium activates a protein kinase C-dependent signal transduction pathway that orchestrates transepithelial PMN movement. We show that the type III effector protein, SipA, is not only necessary but is sufficient to induce this proinflammatory response in epithelial cells. Our results force us to reconsider the long-held view that Salmonella effector proteins must be directly delivered into host cells from bacterial cells.     

Specificity and mechanism of protein kinase C activation by sn-1,2-diacylglycerols.

The specificity of protein kinase C activation by sn-1,2-diacylglycerols and analogues was investigated by using a Triton X-100 mixed micellar assay [Hannun, Y. A., Loomis, C. R. & Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043]. Analogues containing acyl or alkyl chains eight carbons in length were synthesized because sn-1,2-dioctanoylglycerol is an effective cell-permeant activator of protein kinase C. These analogues were tested as activators and antagonists of rat brain protein kinase C to determine the exact structural features important for activity. The analogues established that activation of protein kinase C by diacylglycerols is highly specific. Several analogues established that both carbonyl moieties of the oxygen esters are required for maximal activity and that the 3-hydroxyl moiety is also required. None of the analogues were antagonists. These data, combined with previous investigations, permitted formulation of a model of protein kinase C activation. A three-point attachment of sn-1,2-diacylglycerol to the surface-bound protein kinase C-phosphatidylserine-Ca2+ complex is envisioned to cause activation. Direct ligation of diacylglycerol to Ca2+ is proposed to be an essential step in the mechanism of activation of protein kinase C.     

Translocation of the FGR protein-tyrosine kinase as a consequence of neutrophil activation.

Recent studies of the FGR protooncogene have shown that expression of its mRNA is limited to mature peripheral blood granulocytes, monocytes, and tissue macrophages. In the present study, we have investigated p55c-fgr expression in normal human neutrophils [polymorphonuclear leukocytes (PMN)] and have found enzymatically active p55c-fgr to be abundant in lysates of PMN and murine fibroblasts transfected with a FGR expression plasmid but not control cells. Fractionation studies revealed that neutrophil p55c-fgr was present in plasma membrane-enriched fractions as well as fractions containing secondary and tertiary granules. Little change in the distribution of p55c-fgr or FGR kinase activity was observed under conditions favoring tertiary granule release. In contrast, when secondary granule secretion was induced with the chemoattractant peptide, formyl-Met-Leu-Phe, a marked decrease in p55c-fgr and FGR kinase was observed in fractions depleted of secondary granules. Concomitantly, the relative concentration of p55c-fgr and its enzymatic activity were increased in fractions containing plasma membrane. From these findings we conclude that p55c-fgr is associated with functional secretory granules and is redistributed within normal neutrophils in response to their activation.     

Effects of serine protease inhibitors on accumulation of polymorphonuclear leukocytes in the lung induced by acute pancreatitis in rats

The administration of a high-dose of a serine protease inhibitor is recommended in patients complicated by multiple organ failure (MOF), including adult respiratory distress syndrome (ARDS), induced by acute pancreatitis. The accumulation of polymorphonuclear leukocytes (PMN) in affected organs is considered to be one of the causative factors of MOF. Adhesion to endothelial cells (EC), via adhesion molecules, and the transendothelial migration of PMN is closely associated with the accumulation of PMN. We examined the effects of two serine protease inhibitors, ulinastatin (UT) and gabexate mesilate (GM), on EC-PMN adhesion and transendothelial migration in human umbilical vein EC and⁵¹Cr-labeled PMN in vitro. EC-PMN adhesion, and the expression of intercellular adhesion molecule-1 (ICAM-1) and endothelial cell adhesion molecule-1 (ELAM-1) on EC induced by IL-1β and TNFα, were reduced by the pretreatment of EC with these inhibitors. The transendothelial migration of PMN stimulated by IL-8 was also inhibited by pretreating PMN with UT or GM. We also examined whether these inhibitors reduced PMN accumulation in the lung in rats with acute pancreatitis induced by a closed duodenal loop. The myeloperoxidase activity in and histological findings of the lung suggested that UT and GM reduced PMN accumulation. In conclusion, serine protease inhibitors may inhibit PMN accumulation in ARDS due to acute pancreatitis.