NLRC4 biology in immunity and inflammation.

Inflammasomes are cytosolic multiprotein complexes that sense microbial infections or host cell damage, triggering cytokine production and a proinflammatory form of cell death, called pyroptosis. Whereas pyroptosis and cytokine production may often promote host resistance to infections, uncontrolled inflammasome activation leads to autoinflammatory diseases in humans. Among the multiple inflammasomes described, the neuronal apoptosis inhibitory protein/nucleotide-binding domain leucine-rich repeat-containing protein family caspase activation and recruitment domain-containing protein 4 (NLRC4) inflammasome emerged as a critical component for the restriction of bacterial infections. Accordingly, our understanding of this inflammasome advanced remarkably over the last 10 yr, expanding our knowledge about ligand-receptor interaction; cryo-EM structure; and downstream effectors and substrates, such as gasdermin-D, caspase-1, caspase-8, and caspase-7. In this review, we discuss recent advances on the biology of the NLRC4 inflammasome, in terms of structure and activation mechanisms, importance in bacterial and nonbacterial diseases, and the identification of NLRC4 gain-of-function mutations leading to NLRC4-associated autoinflammatory diseases in humans.

Gasdermin-D and Caspase-7 are the key Caspase-1/8 substrates downstream of the NAIP5/NLRC4 inflammasome required for restriction of Legionella pneumophila.

Inflammasomes are cytosolic multi-protein complexes that detect infection or cellular damage and activate the Caspase-1 (CASP1) protease. The NAIP5/NLRC4 inflammasome detects bacterial flagellin and is essential for resistance to the flagellated intracellular bacterium Legionella pneumophila. The effectors required downstream of NAIP5/NLRC4 to restrict bacterial replication remain unclear. Upon NAIP5/NLRC4 activation, CASP1 cleaves and activates the pore-forming protein Gasdermin-D (GSDMD) and the effector caspase-7 (CASP7). However, Casp1-/- (and Casp1/11-/-) mice are only partially susceptible to L. pneumophila and do not phenocopy Nlrc4-/-mice, because NAIP5/NLRC4 also activates CASP8 for restriction of L. pneumophila infection. Here we show that CASP8 promotes the activation of CASP7 and that Casp7/1/11-/- and Casp8/1/11-/- mice recapitulate the full susceptibility of Nlrc4-/- mice. Gsdmd-/- mice exhibit only mild susceptibility to L. pneumophila, but Gsdmd-/-Casp7-/- mice are as susceptible as the Nlrc4-/- mice. These results demonstrate that GSDMD and CASP7 are the key substrates downstream of NAIP5/NLRC4/CASP1/8 required for resistance to L. pneumophila.

Flagellin/NLRC4 Pathway Rescues NLRP3-Inflammasome Defect in Dendritic Cells From HIV-Infected Patients: Perspective for New Adjuvant in Immunocompromised Individuals.

Introduction: NLRP3 inflammasome plays a key role in dendritic cells (DC) activation in response to vaccine adjuvants, however we previously showed that it is not properly activated in DC from HIV-infected patients (HIV-DC), explaining, at least in part, the poor response to immunization of these patients. Taking in account that several cytoplasmic receptors are able to activate inflammasome, and that bacterial components are considered as a novel and efficient adjuvant, we postulated that bacterial flagellin (FLG), a natural ligand of NAIP/NLRC4 inflammasome, could rescue the activation of the complex in HIV-DC. Objective: Demonstrate that FLG is able to activate monocyte-derived dendritic cells from HIV-infected individuals better than LPS, and to what extent the entity of inflammasome activation differs between DC from HIV-infected patients and healthy donors. Methods: Monocyte-derived dendritic cells from HIV-infected patients (HIV-DC) and healthy donors (HD-DC) were stimulated with FLG, and inflammasome as well as DC activation (phenotypic profile, cytokine production, autologous lymphocytes activation) were compared. Chemical and genetic inhibitors were used to depict the relative contribution of NLRC4 and NLRP3 in HIV/HD-DC response to FLG. Results: FLG properly activates HD-DC and HIV-DC. FLG induces higher inflammasome activation than LPS in HIV-DC. FLG acts through NLRC4 and NLRP3 in HD-DC, but at a lesser extent in HIV-DC due to intrinsic NLRP3 defect. Conclusions: FLG by-passes NLRP3 defect in HIV-DC, through the activation of NAIP/NLRC4 inflammasome, indicating possible future use of the bacterial component as an efficient adjuvant in immunocompromised individuals.

Interleukin-18 diagnostically distinguishes and pathogenically promotes human and murine macrophage activation syndrome.

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperferritinemic systemic inflammatory disorders. Although profound cytotoxic impairment causes familial HLH (fHLH), the mechanisms driving non-fHLH and MAS are largely unknown. MAS occurs in patients with suspected rheumatic disease, but the mechanistic basis for its distinction is unclear. Recently, a syndrome of recurrent MAS with infantile enterocolitis caused by NLRC4 inflammasome hyperactivity highlighted the potential importance of interleukin-18 (IL-18). We tested this association in hyperferritinemic and autoinflammatory patients and found a dramatic correlation of MAS risk with chronic (sometimes lifelong) elevation of mature IL-18, particularly with IL-18 unbound by IL-18 binding protein, or free IL-18. In a mouse engineered to carry a disease-causing germ line NLRC4T337S mutation, we observed inflammasome-dependent, chronic IL-18 elevation. Surprisingly, this NLRC4T337S-induced systemic IL-18 elevation derived entirely from intestinal epithelia. NLRC4T337S intestines were histologically normal but showed increased epithelial turnover and upregulation of interferon-γ-induced genes. Assessing cellular and tissue expression, classical inflammasome components such as Il1b, Nlrp3, and Mefv predominated in neutrophils, whereas Nlrc4 and Il18 were distinctly epithelial. Demonstrating the importance of free IL-18, Il18 transgenic mice exhibited free IL-18 elevation and more severe experimental MAS. NLRC4T337S mice, whose free IL-18 levels were normal, did not. Thus, we describe a unique connection between MAS risk and chronic IL-18, identify epithelial inflammasome hyperactivity as a potential source, and demonstrate the pathogenicity of free IL-18. These data suggest an IL-18-driven pathway, complementary to the cytotoxic impairment of fHLH, with potential as a distinguishing biomarker and therapeutic target in MAS.

Inflammasome-dependent Mechanisms Involved in Sensing and Restriction of Bacterial Replication.

Inflammasomes are multiprotein platforms assembled in the cytosol in response to pathogens and cell stress. Inflammasomes are recognized by their important role on defenses against bacterial infections and have been also implicated in a range of human inflammatory disorders. Intracellular sensors such as NLRP1, NLRP3, NLRC4, AIM2 and Pyrin induce assembly of inflammasomes, while caspase-11 induces the non-canonical pathway for activation of the NLRP3 inflammasome. The formation of the inflammasome leads to caspase-1 activation that triggers pyroptosis and activation of interleukin-1ß (IL-1ß) and IL-18. Pyroptotic cell death and cytokines production are involved in restriction of bacterial replication by limiting the replication niche of intracellular bacteria and by inducing inflammatory responses. In this review we focus on the mechanisms mediated by inflammasome activation that leads to inflammatory responses and restriction of bacterial infection.

Immunization with a nontoxic naturally occurring Clostridium perfringens alpha toxin induces neutralizing antibodies in rabbits.

Clostridium perfringens alpha toxin, encoded by plc gene, has been implicated in gas gangrene, a life threatening infection. Vaccination is considered one of the best solutions against Clostridium infections. Although studies have identified many low quality clostridial vaccines, the use of recombinant proteins has been considered a promising alternative. Previously, a naturally occurring alpha toxin isoform (αAV1b) was identified with a mutation at residue 11 (His/Tyr), which can affect its enzymatic activity. The aim of the present study was to evaluate whether the mutation in the αAV1b isoform could result in an inactive toxin and was able to induce protection against the native alpha toxin. We used recombinant protein techniques to determine whether this mutation in αAV1b could result in an inactive toxin compared to the active isoform, αZ23. Rabbits were immunized with the recombinant toxins (αAV1b and αZ23) and with native alpha toxin. αAV1b showed no enzymatic and hemolytic activities. ELISA titration assays showed a high titer of both anti-recombinant toxin (anti-rec-αAV1b and anti-rec-αZ23) antibodies against the native alpha toxin. The alpha antitoxin titer detected in the rabbits' serum pool was 24.0 IU/mL for both recombinant toxins. These results demonstrate that the inactive naturally mutated αAV1b is able to induce an immune response, and suggest it can be considered as a target for the development of a commercial vaccine against C. perfringens alpha toxin.

Synthesis of a model trisaccharide for studying the interplay between the anti α-Gal antibody and the trans-sialidase reactions in Trypanosoma cruzi.

Trypanosoma cruzi, the etiologic agent of Chagas disease, is covered by a dense glycocalix mainly composed by glycoproteins called mucins which are also the acceptors of sialic acid in a reaction catalyzed by a trans-sialidase (TcTS). Sialylation of trypomastigote mucins protects the parasite from lysis by the anti α-Galp antibodies from serum. The TcTS is essential for the infection process since T. cruzi is unable to biosynthesize sialic acid. The enzyme specifically transfers it from a terminal ß-d-Galp unit in the host glycoconjugate to terminal ß-d-Galp units in the parasite mucins to construct the d-NeuNAc(α2→3)ß-d-Galp motif. On the other hand, although galactose is the most abundant sugar in mucins of both, the infective trypomastigotes and the insect stage epimastigotes, α-d-Galp is only present in the infective stage whereas ß-d-Galf is characteristic of the epimastigote stage of the less virulent strains. Neither α-d-Galp nor d-Galf is acceptor of sialic acid. In the mucins, some of the oligosaccharides are branched with terminal ß-d-Galp units to be able to accept sialic acid in the TcTS reaction. Based on previous reports showing that anti α-Galp antibodies only partially colocalize with sialic acid, we have undertaken the synthesis of the trisaccharide α-d-Galp(1→3)-[ß-d-Galp(1→6)]-d-Galp, the smallest structure containing both, the antigenic d-Galp(α1→3)-d-Galp unit and the sialic acid-acceptor ß-d-Galp unit. The trisaccharide was obtained as the 6-aminohexyl glycoside to facilitate further conjugation for biochemical studies. The synthetic approach involved the α-galactosylation at O-4 of a suitable precursor of the reducing end, followed by ß-galactosylation at O-6 of the same precursor and introduction of the 6-aminohexyl aglycone. The fully deprotected trisaccharide was successfully sialylated by TcTS using either 3'-sialyllactose or fetuin as donors. The product, 6-aminohexyl α-d-NeuNAc(2→3)-ß-d-Galp(1→6)-[α-d-Galp(1→3)]-ß-d-Galp, was purified and characterized.

Sublingual flagellin protects against acute pneumococcal pneumonia in a TLR5-dependent and NLRC4-independent fashion.

AIM: To evaluate efficacy of sublingual flagellin to treat acute pneumonia. MATERIALS & METHODS: Mice were treated sublingually with flagellin and challenged intranasally with a lethal dose of pneumococcus. Flagellins lacking TLR5 or NLRC4 activation domains were used to assess their contribution to protection. RESULTS: Sublingual flagellin protected mice in a TLR5-dependent, NLRC4-independent fashion. Neutrophils were required for protection. Flagellin-stimulated lung epithelial cells recapitulated the lung's transcriptional profile suggesting they could be targeted by flagellin in vivo. CONCLUSION: Ligation of TLR5, a pathogen recognition receptor not naturally engaged by pneumococcus, protects mice from invasive pneumonia when administered via sublingual route. This can be a highly cost-effective alternative therapy against pneumonia.

Immunogenicity of a Trivalent Recombinant Vaccine Against Clostridium perfringens Alpha, Beta, and Epsilon Toxins in Farm Ruminants.

Clostridium perfringens is an anaerobic bacterium that produces several toxins. Of these, the alpha, beta, and epsilon toxins are responsible for causing the most severe C. perfringens-related diseases in farm animals. The best way to control these diseases is through vaccination. However, commercially available vaccines are based on inactivated toxins and have many production drawbacks, which can be overcome through the use of recombinant antigens. In this study, we produced recombinant alpha, beta, and epsilon toxins in Escherichia coli to formulate a trivalent vaccine. Its effectiveness was evaluated through a potency test in rabbits, in which the vaccine generated 9.6, 24.4, and 25.0 IU/mL of neutralizing antibodies against the respective toxins. Following this, cattle, sheep, and goats received the same formulation, generating, respectively, 5.19 ± 0.48, 4.34 ± 0.43, and 4.70 ± 0.58 IU/mL against alpha toxin, 13.71 ± 1.17 IU/mL (for all three species) against beta toxin, and 12.74 ± 1.70, 7.66 ± 1.69, and 8.91 ± 2.14 IU/mL against epsilon toxin. These levels were above the minimum recommended by international protocols. As such, our vaccine was effective in generating protective antibodies and, thus, may represent an interesting alternative for the prevention of C. perfringens-related intoxications in farm animals.

Caspase-1 but Not Caspase-11 Is Required for NLRC4-Mediated Pyroptosis and Restriction of Infection by Flagellated Legionella Species in Mouse Macrophages and In Vivo.

Gram-negative bacteria from the Legionella genus are intracellular pathogens that cause a severe form of pneumonia called Legionnaires' disease. The bacteria replicate intracellularly in macrophages, and the restriction of bacterial replication by these cells is critical for host resistance. The activation of the NAIP5/NLRC4 inflammasome, which is readily triggered in response to bacterial flagellin, is essential for the restriction of bacterial replication in murine macrophages. Once activated, this inflammasome induces pore formation and pyroptosis and facilitates the restriction of bacterial replication in macrophages. Because investigations related to the NLRC4-mediated restriction of Legionella replication were performed using mice double deficient for caspase-1 and caspase-11, we assessed the participation of caspase-1 and caspase-11 in the functions of the NLRC4 inflammasome and the restriction of Legionella replication in macrophages and in vivo. By using several species of Legionella and mice singly deficient for caspase-1 or caspase-11, we demonstrated that caspase-1 but not caspase-11 was required for pore formation, pyroptosis, and restriction of Legionella replication in macrophages and in vivo. By generating F1 mice in a mixed 129 × C57BL/6 background deficient (129 × Casp-11(-/-) ) or sufficient (129 × C57BL/6) for caspase-11 expression, we found that caspase-11 was dispensable for the restriction of Legionella pneumophila replication in macrophages and in vivo. Thus, although caspase-11 participates in flagellin-independent noncanonical activation of the NLRP3 inflammasome, it is dispensable for the activities of the NLRC4 inflammasome. In contrast, functional caspase-1 is necessary and sufficient to trigger flagellin/NLRC4-mediated restriction of Legionella spp. infection in macrophages and in vivo.

Neonatal treatment with monosodium glutamate lastingly facilitates spreading depression in the rat cortex.

AIMS: Monosodium glutamate (MSG) is a neuroexcitatory amino acid used in human food to enhance flavor. MSG can affect the morphological and electrophysiological organization of the brain. This effect is more severe during brain development. Here, we investigated the electrophysiological and morphological effects of MSG in the developing rat brain by characterizing changes in the excitability-related phenomenon of cortical spreading depression (CSD) and microglial reaction. MAIN METHODS: From postnatal days 1-14, Wistar rat pups received 2 or 4 g/kg MSG (groups MSG-2 and MSG-4, respectively; n=9 in each group), saline (n=10) or no treatment (naïve group; n=5) every other day. At 45-60 days, CSD was recorded on two cortical points for 4h. The CSD parameters velocity, and amplitude and duration of the negative potential change were calculated. Fixative-perfused brain sections were immunolabeled with anti-IBA-1 antibodies to identify and quantify cortical microglia. KEY FINDINGS: MSG-4 rats presented significantly higher velocities (4.59 ± 0.34 mm/min) than the controls (saline, 3.84 ± 0.20mm/min; naïve, 3.71 ± 0.8mm/min) and MSG-2 group (3.75 ± 0.10mm/min). The amplitude (8.8 ± 2.2 to 11.2 ± 1.9 mV) and duration (58.2 ± 7.1 to 73.6 ± 6.0s) of the negative slow potential shift was similar in all groups. MSG-treatment dose-dependently increased the microglial immunolabeling. SIGNIFICANCE: The results demonstrate a novel, dose-dependent action of MSG in the developing brain, characterized by acceleration of CSD and significant microglial reaction in the cerebral cortex. The CSD effect indicates that MSG can influence cortical excitability, during brain development, as evaluated by CSD acceleration. Data suggest caution when consuming MSG, especially in developing organisms.

Distribution of the parvalbumin, calbindin-D28K and calretinin immunoreactivity in globus pallidus of the Brazilian short-tailed opossum (Monodelphis domestica).

This study describes the topography, borders and divisions of the globus pallidus in the Brazilian short-tailed opossum (Monodelphis domestica) and distribution of the three calcium binding proteins, parvalbumin (PV), calbindin D-28k (CB) and calretinin (CR) in that nucleus. The globus pallidus of the opossum consists of medial and lateral parts that are visible with Nissl or Timm's staining and also in PV and CR immunostained sections. Neurons of the globus pallidus expressing these proteins were classified into three types on the basis of size and shape of their soma and dendritic tree. Type 1 neurons had medium-sized fusiform soma with dendrites sprouting from the opposite poles. Neurons of the type 2 had medium-to-large, multipolar soma with scarce, thin dendrites. Cell bodies of type 3 neurons were small and either ovoid or round. Immunostaining showed that the most numerous were neurons expressing PV that belonged to all three types. Density of the PV-immunopositive fibers and puncta correlated with the density of the PV-labeled neurons. Labeling for CB resulted mainly in the light staining of neuropil in both parts of the nucleus, while the CB-expressing cells (mainly of the type 2) were scarce and placed only along the border of the globus pallidus and putamen. Staining for calretinin resulted in labeling almost exclusively the immunoreactive puncta and fibers that were distributed with medium-to-high density throughout the nucleus. Close to the border of globus pallidus with the putamen these fibers (probably dendrites) were long, thin and varicous, while more medially bundles of thick, short and smooth fibers predominated. Single CR-ir neurons (all of the type 3) were scattered through the globus pallidus. Colocalization of two calcium binding proteins in one neuron was. never observed. The CB-ir puncta (probably terminals of axons projecting to the nucleus) frequently formed basket-like structures around the PV-ir neurons. Therefore, the globus pallidus in the opossum, much as that in the rat, consists of a heterogeneous population of neurons, probably playing diversified functions.

Cloning and partial characterization of a Boophilus microplus (Acari: Ixodidae) calreticulin.

We report the cloning, sequence characterization and expression analysis of a calreticulin (CRT) coding cDNA of Boophilus microplus. CRT is a calcium-binding protein involved in multiple cell functions and possibly implicated in parasites host immune system evasion. The CRT cDNA sequence and its molecular characterization are described. Sequence similarity and phylogenetic analyses indicate a close relationship to other arthropod CRT sequences. The CRT cDNA was also expressed in a procariotic system and the recombinant protein (rBmCRT) was used to raise antibodies in a rabbit. Expression analyses of the corresponding gene in different developmental stages and tissues were performed by RT-PCR and Western-blot, which indicated a ubiquitous expression of the B. microplus calreticulin gene and demonstrated its presence in saliva. Sera of tick-infested bovines suggested that this protein may not be able to induce an IgG-based humoral response in its natural host.

Prevalence of superantigen-secreting bacteria in patients with Kawasaki disease.

OBJECTIVES: To assess the prevalence of superantigen secreting bacteria in children with acute Kawasaki disease (KD) relative to control patients. STUDY DESIGN: Bacterial cultures were obtained in a blinded fashion from the throat, rectum, and groin of 45 patients with untreated acute KD and 37 febrile control patients from 6 centers in the United States. Cultures were processed for the presence of superantigen-producing bacteria at a central laboratory. RESULTS: Staphylococci or streptococci that produced superantigens (TSST-1, SEB, SEC, SPEB, SPEC) were isolated from 25 of 45 patients with KD (56%) as compared with 13 of 37 (35%) control patients (P =.078). Because SEB- and SEC-producing Staphylococcus aureus have not been associated with KD and because they do not induce a Vbeta2+ T-lymphocyte response, we analyzed the difference between groups relative to superantigens TSST-1 or SPEB/SPEC production. TSST-1 secreting S aureus or SPEB/SPEC producing group A streptococci were isolated from 20 of 45 (44%) patients with KD compared with 7 of 37 (19%) control patients (P =.019). CONCLUSIONS: The overall isolation rates of superantigen (TSST-1, SPEB, SPEC, SEB, SEC) producing bacteria between patients with KD and febrile control patients were not statistically significant. However, future studies should further examine the potential role of Vbeta2-stimulatory superantigens (TSST-1 and SPEB/SPEC) in KD.

Development of an immunoenzymatic assay for the detection of human antibodies against Trypanosoma cruzi calreticulin, an immunodominant antigen.

We have developed an indirect immunoenzymatic assay (ELISA) for the detection of human antibodies against calreticulin (formerly known as Tc45), a dimorphic Trypanosoma cruzi antigen, described in our laboratory. PVC microtitration plates were sensitized with the monoclonal anti-calreticulin antibody (MoAb) and reacted with calreticulin present in a partially purified preparation. The presence of anti-T. cruzi calreticulin IgG in sera from infected individuals was tested. The data generated with this assay were validated by correlation, in a regression analysis, with those obtained by an indirect immunoradiometric assay (IRMA). From the 12 seropositive sera (as defined by a commercial test), eight came out positive and four negative in both assays. The 12 human sera were also analyzed in direct immunometric assays (ELISA and IRMA), where the solid phase was sensitized with a whole parasite extract. The direct ELISA and IRMA correlated positively (P<0.01). Further validation of this ELISA was achieved with an indirect immunofluorescense assay. The high degree of significance obtained when the indirect IRMA and ELISA systems were compared, indicated that the relatively small sample number used (12) was statistically satisfactory for the purposes of this investigation. Thus, the IRMA can be replaced by the ELISA, with advantages mainly derived from the cumbersome manipulation of radioactive wastes. The MoAb used as an antigen capture agent in the ELISA proposed here, recognizes a homologous protein in Trypanosoma rangeli, suggesting that individuals infected with this parasite might have crossreactive antibodies. However, the system retains its diagnostic interest, given the facts that the MoAb does not recognize a homologous protein in Leishmania mexicana, Leishmania donovani, or Crithidia fasciculata.

Trypanosoma cruzi calreticulin is a lectin that binds monoglucosylated oligosaccharides but not protein moieties of glycoproteins.

Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse-chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin-cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-beta-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin-cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages.

Antinociceptive effect of the calcium-binding protein MRP-14 and the role played by neutrophils on the control of inflammatory pain.

Macrophages secrete a variety of chemical mediators that play a central role in the pathophysiology of inflammatory pain. Therefore, the activation or deactivation of these cells in an inflammatory focus could modulate the intensity of the algogenic response. Based on these premises and on our previous demonstration that the calcium-binding protein MRP-14, highly expressed in neutrophils, deactivates activated macrophages in vitro, we decided to investigate the role of MRP-14 and of neutrophils in the control of inflammatory pain in mice. Our results show that this protein is endowed with antinociceptive activity. When tested in the writhing model it was able to inhibit pain response but did not change the behavior of the animals in the hot plate test. This observation indicates that MRP-14 down-regulates inflammatory but not central pain. Using a model of acute neutrophilic peritonitis induced by glycogen, a close correlation between neutrophil migration and antinociception was detected. Surgical adrenalectomy demonstrated that the antinociceptive response induced by glycogen was not due to endogenous liberation of glucocorticoids. The treatment of animals either with a monoclonal antibody anti-MRP-14 or a monoclonal antibody that depletes the animals of neutrophils reverts the antinociceptive response observed in the glycogen-induced peritonitis. These data define the calcium-binding protein MRP-14 as a novel mediator for the control of inflammatory pain and consequently discloses an anti-inflammatory role for the neutrophil.