Targeting peptidylarginine deiminase reduces neutrophil extracellular trap formation and tissue injury in severe acute pancreatitis

Recent evidence suggests that neutrophil extracellular traps (NETs) play an important role in the development of acute pancreatitis (AP). Herein, we examined the role of peptidylarginine deiminase (PAD), which has been shown to regulate NET formation, in severe AP. AP was induced by retrograde of taurocholate infusion into pancreatic duct in C57BL/6 mice. PAD was pharmacologically inhibited using Cl-amidine, a pan-PAD inhibitor. Pancreata were collected, and histones, citrullinated histone 3, chemokines, myeloperoxidase, and NETs were quantified. Chemokines, matrix metalloproteinase-9 (MMP-9), interleukin-6 (IL-6), and DNA-histone complexes were determined in plasma samples. Infusion of taurocholate induced formation of NETs in pancreatic tissues of mice. Pretreatment with Cl-amidine markedly reduced the NET formation in the inflamed pancreas. Moreover, inhibition of PAD decreased the levels of blood amylase as well as edema, acinar cell necrosis, hemorrhage, and neutrophil infiltration in the pancreas of animals with AP. Administration of Cl-amidine attenuated the myeloperoxidase levels in the pancreas and lung of mice exposed to taurocholate. In addition, Cl-amidine decreased pancreatic levels of CXC chemokines, plasma levels of IL-6, and MMP-9 in mice with severe AP. This study shows that Cl-amidine is a potent inhibitor of NET formation in severe AP. Also, our results suggest that PAD regulates pathological inflammation and tissue damage in the inflamed pancreas. Thus, targeting PAD might be a useful strategy to treat patients with severe AP.     

In vivo evidence for the role of GM-CSF as a mediator in acute pancreatitis-associated lung injury

Severe pancreatitis is frequently associated with acute lung injury (ALI) and the respiratory distress syndrome. The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in mediating the ALI associated with secretagogue-induced experimental pancreatitis was evaluated with GM-CSF knockout mice (GM-CSF -/-). Pancreatitis was induced by hourly (12x) intraperitoneal injection of a supramaximally stimulating dose of the cholecystokinin analog caerulein. The resulting pancreatitis was similar in GM-CSF-sufficient (GM-CSF +/+) control animals and GM-CSF -/- mice. Lung injury, quantitated by measuring lung myeloperoxidase activity (an indicator of neutrophil sequestration), alveolar-capillary permeability, and alveolar membrane thickness was less severe in GM-CSF -/- than in GM-CSF +/+ mice. In GM-CSF +/+ mice, pancreas, lung and serum GM-CSF levels increase during pancreatitis. Lung levels of macrophage inflammatory protein (MIP)-2 are also increased during pancreatitis, but, in this case, the rise is less profound in GM-CSF -/- mice than in GM-CSF +/+ controls. Administration of anti-MIP-2 antibodies was found to reduce the severity of pancreatitis-associated ALI. Our findings indicate that GM-CSF plays a critical role in coupling pancreatitis to ALI and suggest that GM-CSF may act indirectly by regulating the release of other proinflammatory factors including MIP-2.     

Treatment with bindarit, a blocker of MCP-1 synthesis, protects mice against acute pancreatitis

Chemokines are believed to play a key role in the pathogenesis of acute pancreatitis. We have earlier shown that pancreatic acinar cells produce the chemokine monocyte chemotactic protein (MCP)-1 in response to caerulein hyperstimulation, demonstrating that acinar-derived MCP-1 is an early mediator of inflammation in acute pancreatitis. Blocking chemokine production or action is a major target for pharmacological intervention in a variety of inflammatory diseases, such as acute pancreatitis. 2-Methyl-2-[[1-(phenylmethyl)-1H-indazol-3yl]methoxy]propanoic acid (bindarit) has been shown to preferentially inhibit MCP-1 production in vitro in monocytes and in vivo without affecting the production of the cytokines IL-1, IL-6, or the chemokines IL-8, protein macrophage inflammatory-1alpha, and RANTES. The present study aimed to define the role of MCP-1 in acute pancreatitis with the use of bindarit. In a model of acute pancreatitis induced by caerulein hyperstimulation, prophylactic as well as therapeutic treatment with bindarit significantly reduced MCP-1 levels in the pancreas. Also, this treatment significantly protected mice against acute pancreatitis as evident by attenuated hyperamylasemia neutrophil sequestration in the pancreas (pancreatic MPO activity), and pancreatic acinar cell injury/necrosis on histological examination of pancreas sections.     

Protective roles of redox-active protein thioredoxin-1 for severe acute pancreatitis

Severe acute pancreatitis is a disease with high mortality, and infiltration of inflammatory cells and reactive oxygen species have a crucial role in the pathophysiology of this disease. Thioredoxin-1 (TRX-1) is an endogenous redox-active multifunctional protein with antioxidant and anti-inflammatory effects. TRX-1 is induced in various inflammatory conditions and shows cytoprotective effects. The aim of the present study was to clarify the protective roles of TRX-1 in the host defense mechanism against severe acute pancreatitis. Experimental acute pancreatitis was induced by intraperitoneal administration of cerulein, a CCK analog, and aggravated by lipopolysaccharide injection in transgenic mice overexpressing human TRX-1 (hTRX-1) and control C57BL/6 mice. Transgenic overexpression of hTRX-1 strikingly attenuated the severity of experimental acute pancreatitis. TRX-1 overexpression suppressed neutrophil infiltration as determined by myeloperoxidase activity, oxidative stress as determined by malondialdehyde concentration, and cytoplasmic degradation of inhibitor of kappaB-alpha, thereby suppressing proinflammatory cytokines, tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6; a neutrophil chemoattractant, keratinocyte-derived chemokine; and inducible nitric oxide synthase in the pancreas. Administration of recombinant hTRX-1 also suppressed neutrophil infiltration, reduced the inflammation of the pancreas and the lung, and improved the mortality rate. The present study suggests that TRX-1 has potent antioxidant and anti-inflammatory actions in experimental acute pancreatitis and might be a new therapeutic strategy to improve the prognosis of severe acute pancreatitis.     

Reduced chemotactic peptide binding in juvenile periodontitis: a model for neutrophil function

Chemotactic peptide binding sites from peripheral blood neutrophils of Localized Juvenile Periodontitis patients and normal controls were quantitated using tritiated N-formylmethionylleucyl-phenylalanine and a rapid filtration assay. It was found that there is a significant reduction in the number of binding sites per cell on neutrophils from the patient group whereas binding affinity remained the same as control values. A direct correlation between $\text{in}\text{vitro}$ neutrophil chemotactic response and density of binding sites was found. Since these patients present little clinical illness, localized juvenile periodontitis can be a valuable model for the study of human neutrophil function.     

Local disruption of the celiac ganglion inhibits substance P release and ameliorates caerulein-induced pancreatitis in rats

Primary sensory neurons of the C and Adelta subtypes express the vanilloid capsaicin receptor TRPV1 and contain proinflammatory peptides such as substance P (SP) that mediate neurogenic inflammation. Pancreatic injury stimulates these neurons causing the release of SP in the pancreas resulting in pancreatic edema and neutrophil infiltration that contributes to pancreatitis. Axons of primary sensory neurons innervating the pancreas course through the celiac ganglion. We hypothesized that disruption of the celiac ganglion by surgical excision or inhibition of C and Adelta fibers through blockade of TRPV1 would reduce the severity of experimental pancreatitis by inhibiting neurogenic inflammation. Resiniferatoxin (RTX) is a specific TRPV1 agonist that, in high doses, selectively destroys C and Adelta fibers. Sprague-Dawley rats underwent surgical ganglionectomy or application of 10 microg RTX (vs. vehicle alone) to the celiac ganglion. One week later, pancreatitis was induced by six hourly intraperitoneal injections of caerulein (50 microg/kg). The severity of pancreatitis was assessed by serum amylase, pancreatic edema, and pancreatic myeloperoxidase (MPO) activity. SP receptor (neurokinin-1 receptor, NK-1R) internalization in acinar cells, used as an index of endogenous SP release, was assessed by immunocytochemical quantification of NK-1R endocytosis. Caerulein administration caused significant increases in pancreatic edema, serum amylase, MPO activity, and NK-1R internalization. RTX treatment and ganglionectomy significantly reduced pancreatic edema by 46% (P < 0.001) and NK-1R internalization by 80% and 51% (P < 0.001 and P < 0.05, respectively). RTX administration also significantly reduced MPO activity by 47% (P < 0.05). Neither treatment affected serum amylase, consistent with a direct effect of caerulein. These results demonstrate that disruption of or local application of RTX to the celiac ganglion inhibits SP release in the pancreas and reduces the severity of acute secretagogue-induced pancreatitis. It is possible that selectively disrupting TRPV1-bearing neurons could be used to reduce pancreatitis severity.     

Leukocyte chemoattractant peptides from the serpin heparin cofactor II

Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits the coagulant proteinase alpha-thrombin. We have recently demonstrated that proteolysis of HC by catalytic amounts of polymorphonuclear leukocyte proteinases (elastase or cathepsin G) generates leukocyte chemotaxins (Hoffman, M., Pratt, C. W., Brown, R. L., and Church, F. C. (1989) Blood 73, 1682-1685). One of four peptides produced when HC is degraded by neutrophil elastase has chemotactic activity for both monocytes and neutrophils with maximal migration comparable to formyl-Met-Leu-Phe, the "gold standard" bacterially derived chemotaxin. The amino-terminal sequence of this HC peptide is Asp-Phe-His-Lys-Glu-Asn-Thr-Val-... and the peptide corresponds to Asp-39 to Ile-66 of HC. A variety of synthetic peptides derived from this sequence were evaluated for leukocyte migration activity, and a dodecapeptide from Asp-49 to Tyr-60 (Asp-Trp-Ile-Pro-Glu-Gly-Glu-Glu-Asp-Asp-Asp-Tyr) was identified as the active site for leukocyte chemotactic action. The 12-mer synthetic peptide possesses significant neutrophil chemotactic action at 1 nM (60% of the maximal activity of formyl-Met-Leu-Phe), while a peptide with the reverse sequence has essentially no chemotactic activity. Cross-desensitization experiments also show that pretreatment of neutrophils with a 19-mer peptide (Asn-48 to Ile-66) greatly reduces subsequent chemotaxis to HC-neutrophil elastase proteolysis reaction products. When injected intraperitoneally in mice, the HC-neutrophil elastase digest elicits neutrophil migration. Our results demonstrate that not only does HC function as a thrombin inhibitor, but that limited proteolysis of HC near the amino terminus yields biologically active peptide(s) which might participate in inflammation and in wound healing and tissue repair processes.     

Complement factor C5a exerts an anti-inflammatory effect in acute pancreatitis and associated lung injury

Complement factor C5a acting via C5a receptors (C5aR) is recognized as an anaphylotoxin and chemoattractant that exerts proinflammatory effects in many pathological states. The effects of C5a and C5aR in acute pancreatitis and in pancreatitis-associated lung injury were evaluated using genetically altered mice that either lack C5aR or do not express C5. Pancreatitis was induced by administration of 12 hourly injections of cerulein (50 microg/kg ip). The severity of pancreatitis was determined by measuring serum amylase, neutrophil sequestration in the pancreas, and acinar cell necrosis. The severity of lung injury was evaluated by measuring neutrophil sequestration in the lung and pulmonary microvascular permeability. In both strains of genetically altered mice, the severity of pancreatitis and pancreatitis-associated lung injury was greater than that noted in the comparison wild-type strains of C5aR- and C5-sufficient animals. This exacerbation of injury in the absence of C5a function indicates that, in pancreatitis, C5a exerts an anti-inflammatory effect. Potentially, C5a and its receptor are capable of both promoting and reducing the extent of acute inflammation.     

Development of a new mouse model of acute pancreatitis induced by administration of L-arginine

The pathogenesis of acute pancreatitis is not fully understood. Experimental animal models that mimic human disease are essential to better understand the pathophysiology of the disease and to evaluate potential therapeutic agents. Given that the mouse genome is known completely and that a large number of strains with various genetic deletions are available, it is advantageous to have multiple reliable mouse models of acute pancreatitis. Presently, there is only one predominant model of acute pancreatitis in mice, in which hyperstimulatory doses of cholecystokinin or its analog caerulein are administered. Therefore, the aim of this study was to develop another mouse model of acute pancreatitis. In this study, C57BL/6 mice were injected intraperitoneally with L-arginine in two doses of 4 g/kg each, 1 h apart. Serum amylase, myeloperoxidase, and histopathology were examined at varying time points after injection to assess injury to the pancreas and lung. We found that injection of L-arginine was followed by significant increases in plasma amylase and pancreatic myeloperoxidase accompanied by marked histopathological changes. The injury to the pancreas was slow to develop and peaked at 72 h. Subsequent to peak injury, the damaged areas contained collagen fibers as assessed by increased Sirius red staining. In contrast, D-arginine or other amino acids did not cause injury to the pancreas. In addition, acute inflammation in the pancreas was associated with lung injury. Our results indicate that administration of L-arginine to mice results in severe acute pancreatitis. This model should help in elucidating the pathophysiology of pancreatitis.     

Chemotaxis of neutrophil leukocytes towards substratum-bound protein attractants

Human blood neutrophil leukocytes were shown by the checkerboard filter assay to be capable of chemotactic migration in response to gradients of filter-bound chemotactic proteins in the absence of protein in free solution. Chemotactic proteins (casein, denatured serum albumin) and chemokinetic proteins (native serum albumin) bind substantially to substrata such as filters and glass, whereas low molecular weight chemotactic factors such as formyl-methionyl-phenylalanine bind poorly. Cells do not locomote towards the latter factors in the absence of protein but can, when migrating on substratum-bound albumin, respond chemotactically to gradients of fluid-phase formyl peptide.     

Differentiated HL-60 cells are a valid model system for the analysis of human neutrophil migration and chemotaxis

We have carried out a detailed comparison of the motile properties of differentiated HL-60 cells and human peripheral blood neutrophils. We compared the effects of chemotactic stimuli and of inhibitors of signalling proteins on morphology, chemokinesis and chemotaxis of neutrophils and differentiated HL-60 cells using videomicroscopy and a filter assay for chemotaxis. We also assessed expression of signalling and cytoskeletal proteins using Western blotting.Chemotactic peptide induced a front-tail polarity in HL-60 cells comparable to that of neutrophils. Chemokinetic and chemotactic responses to chemotactic peptide were also very similar for both cell types, concerning mean speed of migration, the fraction of migrated cells and the concentration of stimulus optimal for activation. The cytokine interleukin-8 was in contrast clearly less effective in activating motile responses of differentiated HL-60 cells as compared to neutrophils.An important functional role of Rho-activated kinases and phosphatidylinositol 3-kinase in motile responses of HL-60 cells, consistent with their upregulation during differentiation, could be confirmed using inhibitors with specificity for the corresponding enzymes. The only difference observed here between HL-60 cells and neutrophils concerned the differential effects of a protein kinase C inhibitor.In summary, the results presented here show that differentiated HL-60 cells, stimulated with chemotactic peptide, are a valid model system to study molecular mechanisms of neutrophil emigration.     

Ameliorative Effects of Curcumin on Fibrinogen‐Like Protein‐2 Gene Expression,Some Oxido‐Inflammatory and Apoptotic Markers in a Rat Model of l‐Arginine‐Induced Acute Pancreatitis

The aim of the study was to investigate the ameliorative effects of curcumin on fibrinogen like protein‐2 (fgl‐2), some oxido‐inflammatory and apoptotic markers in rat‐induced acute pancreatitis (AP). Seventy‐five albino rats were divided into control group, l ‐arginine (l ‐Arg)‐induced AP group, curcumin pre‐treated group before AP induction, curcumin post‐treated group after AP induction, and curcumin injected group only. AP group showed severe necrotizing pancreatitis confirmed by histopathological changes and elevations in serum amylase and lipase activities, levels of epithelial neutrophil‐activating peptide 78, tissue content of protein carbonyls, levels of tumor necrosis factor α, and caspase‐3 as well as myeloperoxidase activity. Significant elevation in pancreatic fgl‐2 mRNA expression was detected in AP group. Improvement of all parameters was detected with increase of caspase‐3 in both curcumin‐treated groups that confirmed curcumin ameliorative effects against AP through induction of apoptosis and inhibition of micro‐thrombosis, inflammation, and oxidative stress.     

Fc gamma RIIIb allele-sensitive release of alpha-defensins: anti-neutrophil cytoplasmic antibody-induced release of chemotaxins

Antineutrophil cytoplasmic Abs (ANCA) can activate neutrophils in an FcgammaR-dependent manner, but the link between this ANCA-induced effect and mononuclear cell activation with the characteristic granuloma formation of Wegener's granulomatosis is unclear. Human alpha-defensins, small cationic antimicrobial peptides, are found in neutrophils and have chemotactic activity for T cells, dendritic cells, and monocytes. In this study, we quantitated the release of alpha-defensins (human neutrophil peptides 1-3) from human neutrophils after targeted FcgammaR cross-linking (XL). Homotypic XL of FcgammaRIIa, FcgammaRIIIb, or heterotypic XL of both receptors resulted in significant release of alpha-defensins, an effect also induced by both human polyclonal and murine monoclonal cytoplasmic staining ANCA (anti-proteinase 3). This release of alpha-defensins, as well as of other granule constituents (ANCA targets anti-proteinase 3 and myeloperoxidase and elastase), was significantly greater in donors homozygous for the NA1 allele of FcgammaRIIIb than in donors homozygous for NA2. Interestingly, the ANCA-induced release was completely inhibited by the IgG Fc-binding peptide TG19320, which blocks the IgG-Fc region from binding to FcgammaR. Based on their chemotactic properties, alpha-defensins and their release by ANCA may contribute to modulation of the acquired immune response and to granuloma formation. The greater activity of the FcgammaRIIIB-NA1 genotype may also explain the greater severity of disease and its flare-ups in patients with this allele.     

Small angle neutron scattering of the mitochondrial ADP/ATP carrier protein in detergent

We examined the potential role of fibronectin in chemotactic factor stimulation of neutrophil adherence to plastic. Monoclonal antibody to human fibronectin significantly reduced chemotactic peptide stimulation of adherence but did not reduce adherence stimulated by phorbol myristate acetate or aggregation stimulated by either agent. Stimulation of neutrophils by chemotactic peptide was also associated with loss of cell surface fibronectin detected by immunofluorescence or binding of radiolabeled collagen. These data suggest that chemotactic peptides stimulate neutrophils to release Fn and that Fn mediates the attachment of neutrophils to plastic surfaces.     

Chemotactic activity of oligopeptides containing an EWS motif on Tetrahymena pyriformis: the effect of amidation of the C-terminal residue

Chemotactic properties of 3-7-mer peptides containing an EWS motive and their peptide amides synthesized and characterized by us were investigated in Tetrahymena pyriformis GL model. Analysis of the peptide acids shows that SEWS possesses exceptionally strong (660%+/-21; 430%+/-18) chemoattractant ability at 10(-12) and 10(-11) m respectively. The shorter peptide (EWS) possesses chemorepellent activity, while longer peptides display neutral (WSEWS) or moderate chemoattractant (EWSEWS and GEWSEWS) chemotactic ability. Amidation of the C-terminus can significantly modify the character of peptides: it points to the conclusion that a free alpha-COOH group at this position is required for the high efficiency of SEWS, while in the shorter (EWS) and longer peptides (WSEWS and EWSEWS) amidation can result in chemoattractant ligands. Evaluation of the structure-function relationship of these compounds establishes the significance of Glu (E) with its high surface-exposed area and negatively-charged side chain. The high discriminative ability and good chemotactic responsiveness of Tetrahymena support the theory that a chemotactic signalling mechanism working in higher levels of phylogeny is a well conserved and inducible one even in protozoa.     

Oxidative inactivation of myeloperoxidase released from human neutrophils.

Within 1 min of stimulation of human neutrophils by the chemotactic peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) plus cytochalasin B, myeloperoxidase (together with other granule enzymes) was secreted and detected extracellularly. In contrast with the other granule constituents assayed (vitamin B12-binding protein and beta-glucuronidase), the activity of released myeloperoxidase rapidly decreased, so that, by 10 min after stimulation, only about 5% of the total cellular activity was detected. This inactivation was shown to be dependent on oxidant generation during the respiratory burst, since inactivation was not observed (a) after stimulation of anaerobic suspensions or (b) after release from neutrophils from a patient with chronic granulomatous disease; purified myeloperoxidase was rapidly inactivated after incubation with H2O2, presumably owing to the formation of an inactive enzyme-H2O2 complex. These results show that experiments designed to assess the role of myeloperoxidase in neutrophil functions which utilize assays based on peroxidase activity will grossly underestimate this enzyme if oxidant generation during the respiratory burst has also been activated.     

Serum complex of trypsin 2 and alpha 1 antitrypsin as diagnostic and prognostic marker of acute pancreatitis: clinical study in consecutive patients.

OBJECTIVE--To estimate the usefulness of serum concentrations of the complex of trypsin 2 and alpha 1 antitrypsin in diagnosing and assessing the severity of acute pancreatitis in comparison with serum C reactive protein, amylase, and trypsinogen 2 concentrations (reference markers). DESIGN--Markers were measured in consecutive patients admitted with acute abdominal pain that was either due to pancreatitis or to other disease unrelated to the pancreas (controls). SETTING--Department of surgery of a teaching hospital in Helsinki. SUBJECTS--110 patients with acute pancreatitis and 66 with acute abdominal diseases of extrapancreatic origin. On the basis of the clinical course, acute pancreatitis was classified as mild (82 patients) or severe (28 patients). MAIN OUTCOME MEASURES--Clinical diagnosis of acute pancreatitis and severity of the disease. RESULTS--At admission all patients with acute pancreatitis had clearly raised concentrations of trypsin 2-alpha 1 antitrypsin complex (32 micrograms/l), whereas only three of the controls had such values. Of the markers studied, trypsin 2-alpha 1 antitrypsin complex had the largest area under the receiver operating curve, both in differentiating acute pancreatitis from extrapancreatic disease and in differentiating mild from severe disease. CONCLUSIONS--Of the markers studied, trypsin 2-alpha 1 antitrypsin complex was the most accurate in differentiating between acute pancreatitis and extrapancreatic disease and in predicting a severe course for acute pancreatitis.     

Inhibition of cyclooxygenase-2 ameliorates the severity of pancreatitis and associated lung injury

Cyclooxygenase-2 (COX-2), a widely distributed enzyme, plays an important role in inflammation. We have studied the role of COX-2 in acute pancreatitis and pancreatitis-associated lung injury using both the pharmacological inhibition of COX-2 and genetic deletion of COX-2. Pancreatitis was induced in mice by 12 hourly injections of cerulein. The severity of pancreatitis was assessed by measuring serum amylase, pancreatic trypsin activity, intrapancreatic sequestration of neutrophils, and acinar cell necrosis. The severity of lung injury was evaluated by measuring lactate dehydrogenase levels in the bronchoalveolar lavage fluid and by quantitating neutrophil sequestration in the lung. In both the pharmacologically inhibited and genetically altered mice, the severity of pancreatitis and pancreatitis-associated lung injury was reduced compared with the noninhibited strains of COX-2-sufficient mice. This reduction in injury indicates that COX-2 plays an important proinflammatory role in pancreatitis and its associated lung injury. Our findings support the concept that COX-2 inhibitors may play a beneficial role in the prevention of acute pancreatitis or in the reduction of its severity.     

The role of Transient Receptor Potential Vanilloid 1 (TRPV1) channels in pancreatitis

Premature activation of digestive enzymes within the pancreas which leads to autodigestion of the gland is an early step in the pathogenesis of pancreatitis. Pancreatic injury is followed by other manifestations of inflammation including plasma extravasation, edema, and neutrophil infiltration which constitute the features of pancreatitis. Recent studies indicate that neural innervation of the pancreas may play an important role in the initiation and maintenance of the inflammatory response to injury. The pancreas is innervated by vagal, sympathetic and parasympathetic neurons, as well as sensory neurons. Activation of pancreatic primary sensory neurons causes the release of inflammatory neuropeptides both in the spinal cord to signal pain and in the pancreas itself where they produce plasma extravasation and neutrophil infiltration. Recent studies indicate that primary sensory neurons of the pancreas express transient receptor potential V1 (TRPV1) channels whose activation induces pancreatic inflammation. Moreover, blockade of these TRP channels significantly ameliorates experimental pancreatitis. This review describes our current understanding of the role of TRPV1 channels in pancreatitis and illustrates how this mechanism might be used to direct future treatments of pancreatic diseases.     

The beta-galactoside binding immunomodulatory lectin galectin-3 reverses the desensitized state induced in neutrophils by the chemotactic peptide f-Met-Leu-Phe: role of reactive oxygen species generated by the NADPH-oxidase and inactivation of the agonist

Neutrophils interacting with a chemoattractant gradually become nonresponsive to further stimulation by the same agonist, a process known as desensitization. Receptor desensitization is a highly regulated process that involves different mechanisms depending on which receptor-ligand pair that is studied. Galectin-3, a member of a large family of beta-galactoside-binding lectins, has been suggested to be a regulator of the inflammatory process, augmenting or directly triggering the neutrophil functional repertoire. We show here that the desensitized state of neutrophils interacting with the chemotactic peptide fMLF is broken by galectin-3 and that this is achieved through an oxygen radical-mediated inactivation of the chemoattractant. The effect was inhibited by the competitor lactose and required the affinity of galectin-3 for N-acetyllactosamine, a saccharide typically found on cell surface glycoproteins. The latter was shown using a galectin-3 mutant that lacked N-acetyllactosamine binding activity, and this protein was not active. The mechanism behind the inactivation of the chemoattractant was found to depend on the ability of galectin-3 to induce a neutrophil generation/secretion of reactive oxygen species which in combined action with myeloperoxidase inactivated the peptides.