Opioid-Induced Hyperalgesia and Tolerance Are Driven by HCN Ion Channels.

Prolonged exposure to opioids causes an enhanced sensitivity to painful stimuli (opioid-induced hyperalgesia, OIH) and a need for increased opioid doses to maintain analgesia (opioid-induced tolerance, OIT), but the mechanisms underlying both processes remain obscure. We found that pharmacological block or genetic deletion of HCN2 ion channels in primary nociceptive neurons of male mice completely abolished OIH but had no effect on OIT. Conversely, pharmacological inhibition of central HCN channels alleviated OIT but had no effect on OIH. Expression of C-FOS, a marker of neuronal activity, was increased in second-order neurons of the dorsal spinal cord by induction of OIH, and the increase was prevented by peripheral block or genetic deletion of HCN2, but block of OIT by spinal block of HCN channels had no impact on C-FOS expression in dorsal horn neurons. Collectively, these observations show that OIH is driven by HCN2 ion channels in peripheral nociceptors, while OIT is driven by a member of the HCN family located in the CNS. Induction of OIH increased cAMP in nociceptive neurons, and a consequent shift in the activation curve of HCN2 caused an increase in nociceptor firing. The shift in HCN2 was caused by expression of a constitutively active µ-opioid receptor (MOR) and was reversed by MOR antagonists. We identified the opioid-induced MOR as a six-transmembrane splice variant, and we show that it increases cAMP by coupling constitutively to Gs HCN2 ion channels therefore drive OIH, and likely OIT, and may be a novel therapeutic target for the treatment of addiction.

HCN2 Ion Channels Drive Pain in Rodent Models of Migraine.

Migraine is believed to be initiated by neuronal activity in the CNS, that triggers excitation of nociceptive trigeminal ganglion (TG) nerve fibers innervating the meninges and thus causes a unilateral throbbing headache. Drugs that precipitate or potentiate migraine are known to elevate intracellular levels of the cyclic nucleotides cAMP or cGMP, while anti-migraine treatments couple to signaling pathways that reduce cAMP or cGMP, suggesting an involvement of these cyclic nucleotides in migraine. Members of the HCN ion channel family are activated by direct binding of cAMP or cGMP, suggesting in turn that a member of this family may be a critical trigger of migraine. Here, we show that pharmacological block or targeted genetic deletion of HCN2 abolishes migraine-like pain in three rodent migraine models (in both sexes). Induction of migraine-like pain in these models triggered expression of the protein C-FOS, a marker of neuronal activity, in neurons of the trigeminocervical complex (TCC), where TG neurons terminate, and C-FOS expression was reversed by peripheral HCN2 inhibition. HCN2 block in vivo inhibited both evoked and spontaneous neuronal activity in nociceptive TG neurons. The NO donor glyceryl trinitrate (GTN) caused an increase in cGMP in the TG in vivo Exposing isolated TG neurons to GTN caused a rightward shift in the voltage dependence of HCN currents and thus increased neuronal excitability. This work identifies HCN2 as a novel target for the development of migraine treatments.SIGNIFICANCE STATEMENT Migraine is believed to be initiated by localized excitability of neurons within the CNS, but the most disturbing symptom, the characteristic throbbing migraine headache pain, is widely agreed to be caused by activity in afferent pain-sensitive (nociceptive) nerve fibers of the trigeminal nerve. Using a variety of preclinical models of migraine, we identify the HCN2 ion channel as the molecular source of trigeminal hyperexcitability in migraine and we show that pharmacological or genetic inhibition of HCN2 can relieve migraine-like pain symptoms. The work highlights the HCN2 ion channel as a potential pharmacological target for the development of novel analgesics effective in migraine.

Medication adherence in systemic lupus erythematosus during Brazilian COVID-19 pandemic.

BACKGROUND: Patients with chronic diseases are potential candidates for inadequate follow-up of drug therapy, tending to incur damage to the intended results. This deserves greater attention in the pandemic period, as they are in the considered risk group. METHODS: We aim to assess Treatment Adherence Measure and analyze associations with characteristics related to the patient, treatment, disease, health professionals and service, and sociodemographic issues in patients with Systemic Lupus Erythematosus (SLE). W conducted a cross-sectional study with a sample of 116 participants, whose data were collected through individual interviews and review of medical records, during the first months of the COVID-19 pandemic in Brazil. Adherence was measured using the Treatment Adherence Measure, and associations were evidenced through described and inferential statistics. RESULTS: The percentage of adherent patients was 55.2%. An association was found between MTA (Medication Treatment Adherence) and physical exercise practice (p = 0.032), and difficulties with treatment (p = 0.002). Conclusion: Participants who did not practice physical exercise were 3.71 times more likely to not adhere to the treatment. Individuals who identified difficulties in the treatment were 3.43 times more likely to not adhere to the treatment; we believe that the pandemic may have influenced this result. More targeted studies are needed to measure the impact on MTA in these patients.

Angiotensin type 2 receptor antagonism as a new target to manage gout.

BACKGROUND: There is a growing search for therapeutic targets in the treatment of gout. The present study aimed to evaluate the analgesic and anti-inflammatory potential of angiotensin type 2 receptor (AT2R) antagonism in an acute gout attack mouse model. METHODS: Male wild-type (WT) C57BL/6 mice either with the AT2R antagonist, PD123319 (10 pmol/joint), or with vehicle injections, or AT2R KO mice, received intra-articular (IA) injection of monosodium urate (MSU) crystals (100 µg/joint), that induce the acute gout attack, and were tested for mechanical allodynia, thermal hyperalgesia, spontaneous nociception and ankle edema development at several times after the injections. To test an involvement of AT2R in joint pain, mice received an IA administration of angiotensin II (0.05-5 nmol/joint) with or without PD123319, and were also evaluated for pain and edema development. Ankle joint tissue samples from mice undergoing the above treatments were assessed for myeloperoxidase activity, IL-1ß release, mRNA expression analyses and nitrite/nitrate levels, 4 h after injections. RESULTS: AT2R antagonism has robust antinociceptive effects on mechanical allodynia (44% reduction) and spontaneous nociception (56%), as well as anti-inflammatory effects preventing edema formation (45%), reducing myeloperoxidase activity (54%) and IL-1ß levels (32%). Additionally, Agtr2tm1a mutant mice have largely reduced painful signs of gout. Angiotensin II administration causes pain and inflammation, which was prevented by AT2R antagonism, as observed in mechanical allodynia 4 h (100%), spontaneous nociception (46%), cold nociceptive response (54%), edema formation (83%), myeloperoxidase activity (48%), and IL-1ß levels (89%). PD123319 treatment also reduces NO concentrations (74%) and AT2R mRNA levels in comparison with MSU untreated mice. CONCLUSION: Our findings show that AT2R activation contributes to acute pain in experimental mouse models of gout. Therefore, the antagonism of AT2R may be a potential therapeutic option to manage gout arthritis.

The role of Acanthamoeba spp. in biofilm communities: a systematic review.

Acanthamoeba spp. have always caused disease in immunosuppressed patients, but since 1986, they have become a worldwide public health issue by causing infection in healthy contact lens wearers. Amoebae of the Acanthamoeba genus are broadly distributed in nature, living either freely or as parasites, and are frequently associated with biofilms throughout the environment. These biofilms provide the parasite with protection against external aggression, thus favoring its increased pathogeny. This review aims to assess observational studies on the association between Acanthamoeba spp. and biofilms, opening potential lines of research on this severe ocular infection. A systematic literature search was conducted in May 2020 in the following databases: PubMed Central®/Medline, LILACS, The Cochrane Library, and EMBASE®. The studies were selected following the inclusion and exclusion criteria specifically defined for this review. Electronic research recovered 353 publications in the literature. However, none of the studies met the inclusion criterion of biofilm-producing Acanthamoeba spp., inferring that the parasite does not produce biofilms. Nonetheless, 78 studies were classified as potentially included regarding any association of Acanthamoeba spp. and biofilms. These studies were allocated across six different locations (hospital, aquatic, ophthalmic and dental environments, biofilms produced by bacteria, and other places). Acanthamoeba species use biofilms produced by other microorganisms for their benefit, in addition to them providing protection to and facilitating the dissemination of pathogens residing in them.

Effects of anesthesiologists awareness on the control of cuff pressure in patients submitted to cardiac surgery.

INTRODUCTION: Sedatives applied to cardiac surgery patients can act on the respiratory tract, creating a demand for a tracheal prosthesis in the artificial route, whose distal part (cuff) has a recommended pressure of 20 to 25 mm Hg. The professional's lack of knowledge about procedures and adequate pressure can pose risks to patients' health. OBJECTIVE: To analyze the effect of anesthesiologists awareness on the control of cuff pressure. METHODOLOGY: A prospective cohort study. At the beginning of the research, cuff pressures were consecutively measured immediately after the patient's admission to the intensive care unit (ICU). After this period, anesthesiologists were trained by the responsible researchers for 1 month. In the final 2.5 months of the research, cuff pressure was again measured immediately after the patient's admission to the ICU. RESULTS: A total of 70 patients were evaluated, 37 of whom were pre-awareness and 33 were post-awareness. Male sex was the most prevalent with 46 (66%) patients and the mean age was 58 ± 10 years. There was a reduction from 76 ± 14 to 28 ± 9 in cuff pressure (P < .01). CONCLUSION: The training of anesthesiologists who assist cardiac surgery patients allowed a reduction in cuff pressure abnormalities.

Low expression of CD39 on regulatory T cells as a biomarker for resistance to methotrexate therapy in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by joint destruction and severe morbidity. Methotrexate (MTX) is the standard first-line therapy of RA. However, about 40% of RA patients are unresponsive to MTX treatment. Regulatory T cells (Tregs, CD4(+)CD25(+)FoxP3(+)) are thought to play an important role in attenuating RA. To investigate the role of Tregs in MTX resistance, we recruited 122 RA patients (53 responsive, R-MTX; 69 unresponsive, UR-MTX) and 33 healthy controls. Three months after MTX treatment, R-MTX but not UR-MTX showed higher frequency of peripheral blood CD39(+)CD4(+)CD25(+)FoxP3(+) Tregs than the healthy controls. Tregs produce adenosine (ADO) through ATP degradation by sequential actions of two cell surface ectonucleotidases: CD39 and CD73. Tregs from UR-MTX expressed a lower density of CD39, produced less ADO, and had reduced suppressive activity than Tregs from R-MTX. In a prospective study, before MTX treatment, UR-MTX expressed a lower density of CD39 on Tregs than those of R-MTX or control (P < 0.01). In a murine model of arthritis, CD39 blockade reversed the antiarthritic effects of MTX treatment. Our results demonstrate that MTX unresponsiveness in RA is associated with low expression of CD39 on Tregs and the decreased suppressive activity of these cells through reduced ADO production. Our findings thus provide hitherto unrecognized mechanism of immune regulation in RA and on mode of action of MTX. Furthermore, our data suggest that low expression of CD39 on Tregs could be a noninvasive biomarker for identifying MTX-resistant RA patients.

Spinal cord oligodendrocyte-derived alarmin IL-33 mediates neuropathic pain.

Neuropathic pain from injury to the peripheral and CNS represents a major health care issue. We have investigated the role of IL-33/IL-33 receptor (ST2) signaling in experimental models of neuropathic pain in mice. Chronic constriction injury (CCI) of the sciatic nerve induced IL-33 production in the spinal cord. IL-33/citrine reporter mice revealed that oligodendrocytes are the main cells expressing IL-33 within the spinal cord together with a minor expression by neurons, microglia. and astrocytes. CCI-induced mechanical hyperalgesia was reduced in IL-33R (ST2)(-/ -) mice compared with wild-type (WT) mice. Intrathecal treatment of WT mice with soluble IL-33 receptor (IL-33 decoy receptor) markedly reduced CCI-induced hyperalgesia. Consistent with these observations, intrathecal injection of IL-33 enhanced CCI hyperalgesia and induced hyperalgesia in naive mice. IL-33-mediated hyperalgesia during CCI was dependent on a reciprocal relationship with TNF-α and IL-1ß. IL-33-induced hyperalgesia was markedly attenuated by inhibitors of PI3K, mammalian target of rapamycin, MAPKs (p38, ERK, and JNK), NF-κB, and also by the inhibitors of glial cells (microglia and astrocytes). Furthermore, targeting these signaling pathways and cells inhibited IL-33-induced TNF-α and IL-1ß production in the spinal cord. Our study, therefore, reveals an important role of oligodendrocyte-derived IL-33 in neuropathic pain.

Targeting the minor pocket of C5aR for the rational design of an oral allosteric inhibitor for inflammatory and neuropathic pain relief.

Chronic pain resulting from inflammatory and neuropathic disorders causes considerable economic and social burden. Pharmacological therapies currently available for certain types of pain are only partially effective and may cause severe adverse side effects. The C5a anaphylatoxin acting on its cognate G protein-coupled receptor (GPCR), C5aR, is a potent pronociceptive mediator in several models of inflammatory and neuropathic pain. Although there has long been interest in the identification of C5aR inhibitors, their development has been complicated, as for many peptidomimetic drugs, mostly by poor drug-like properties. Herein, we report the de novo design of a potent and selective C5aR noncompetitive allosteric inhibitor, DF2593A, guided by the hypothesis that an allosteric site, the "minor pocket," previously characterized in CXC chemokine receptors-1 and -2, is functionally conserved in the GPCR class. In vitro, DF2593A potently inhibited C5a-induced migration of human and rodent neutrophils. In vivo, oral administration of DF2593A effectively reduced mechanical hyperalgesia in several models of acute and chronic inflammatory and neuropathic pain, without any apparent side effects. Mechanical hyperalgesia after spared nerve injury was also reduced in C5aR(-/-) mice compared with WT mice. Furthermore, treatment of C5aR(-/-) mice with DF2593A did not produce any further antinociceptive effect compared with C5aR(-/-) mice treated with vehicle. The successful medicinal chemistry strategy confirms that a conserved minor pocket is amenable for the rational design of selective inhibitors and the pharmacological results support that the allosteric blockade of the C5aR represents a highly promising therapeutic approach to control chronic inflammatory and neuropathic pain.

Intrusive luxation of an immature permanent incisor: a 10-year follow-up.

Intrusive luxation in the permanent dentition is rare and considered the most severe form of dental trauma. Intrusion is characterized by the axial displacement of the tooth into the alveolar bone. It normally affects pulpal vitality and the periodontal ligament. The most common forms of treatment are waiting for spontaneous reeruption, repositioning with orthodontic procedures, or immediate surgical repositioning. A well-planned approach to the treatment of an intrusive luxation is necessary to obtain a successful result. This case report describes treatment of an 8-year-old boy who suffered intrusive luxation of his permanent maxillary right central incisor while root formation was incomplete. The patient was followed clinically and radiographically for 10 years.

Therapeutic Treatment of Arthritic Mice with 15-Deoxy Δ12,14-Prostaglandin J2 (15d-PGJ2) Ameliorates Disease through the Suppression of Th17 Cells and the Induction of CD4+CD25-FOXP3+ Cells.

The prostaglandin, 15-deoxy Δ12,14-prostaglandin J2 (15d-PGJ2), is a lipid mediator that plays an important role in the control of chronic inflammatory disease. However, the role of prostanoid in rheumatoid arthritis (RA) is not well determined. We demonstrated the therapeutic effect of 15d-PGJ2 in an experimental model of arthritis. Daily administration of 15d-PGJ2 attenuated the severity of CIA, reducing the clinical score, pain, and edema. 15d-PGJ2 treatment was associated with a marked reduction in joint levels of proinflammatory cytokines. Although the mRNA expression of ROR-γt was profoundly reduced, FOXP3 was enhanced in draining lymph node cells from 15d-PGJ2-treated arthritic mice. The specific and polyclonal CD4+ Th17 cell responses were limited during the addition of prostaglandin to cell culture. Moreover, in vitro 15d-PGJ2 increased the expression of FOXP3, GITR, and CTLA-4 in the CD4+CD25- population, suggesting the induction of Tregs on conventional T cells. Prostanoid addition to CD4+CD25- cells selectively suppressed Th17 differentiation and promoted the enhancement of FOXP3 under polarization conditions. Thus, 15d-PGJ2 ameliorated symptoms of collagen-induced arthritis by regulating Th17 differentiation, concomitant with the induction of Tregs, and, consequently, protected mice from diseases aggravation. Altogether, these results indicate that 15d-PGJ2 may represent a potential therapeutic strategy in RA.

Bioadhesive Surfactant Systems for Methotrexate Skin Delivery.

Methotrexate (MTX) is an immunosuppressive drug for systemic use in the treatment of skin diseases, however, MTX presents a number of side effects, such as hepatotoxicity. To overcome this limitation, this study developed skin MTX delivery surfactant systems, such as a microemulsion (ME) and a liquid crystalline system (LCS), consisting of a glycol copolymer-based silicone fluid (SFGC) as oil phase, polyether functional siloxane (PFS) as surfactant, and carbomer homopolymer type A (C971) dispersion at 0.5% (wt/wt) as aqueous phase. Polarized light microscopy and small-angle X-ray scattering evidenced the presence of hexagonal and lamellar LCSs, and also a ME. Texture profile and in vitro bioadhesion assays showed that these formulations are suitable for topical application, showing interesting hardness, adhesiveness and compressibility values. Rheology analysis confirmed the Newtonian behaviour of the ME, whereas lamellar and hexagonal LCSs behave as pseudoplastic and dilatant non-Newtonian fluids, respectively. In vitro release profiles indicated that MTX could be released in a controlled manner from all the systems, and the Weibull model showed the highest adjusted coefficient of determination. Finally, the formulations were not cytotoxic to the immortalized human keratinocyte line HaCaT. Therefore, these bioadhesive surfactant systems established with PFS and C971 have great potential as skin delivery systems.

Gastrin-releasing peptide receptor (GRPR) mediates chemotaxis in neutrophils.

Neutrophil migration to inflamed sites is crucial for both the initiation of inflammation and resolution of infection, yet these cells are involved in perpetuation of different chronic inflammatory diseases. Gastrin-releasing peptide (GRP) is a neuropeptide that acts through G protein coupled receptors (GPCRs) involved in signal transmission in both central and peripheral nervous systems. Its receptor, gastrin-releasing peptide receptor (GRPR), is expressed by various cell types, and it is overexpressed in cancer cells. RC-3095 is a selective GRPR antagonist, recently found to have antiinflammatory properties in arthritis and sepsis models. Here we demonstrate that i.p. injection of GRP attracts neutrophils in 4 h, and attraction is blocked by RC-3095. Macrophage depletion or neutralization of TNF abrogates GRP-induced neutrophil recruitment to the peritoneum. In vitro, GRP-induced neutrophil migration was dependent on PLC-ß2, PI3K, ERK, p38 and independent of Gαi protein, and neutrophil migration toward synovial fluid of arthritis patients was inhibited by treatment with RC-3095. We propose that GRPR is an alternative chemotactic receptor that may play a role in the pathogenesis of inflammatory disorders.

Joint NOD2/RIPK2 signaling regulates IL-17 axis and contributes to the development of experimental arthritis.

Intracellular pattern recognition receptors such as the nucleotide-binding oligomerization domain (NOD)-like receptors family members are key for innate immune recognition of microbial infection and may play important roles in the development of inflammatory diseases, including rheumatic diseases. In this study, we evaluated the role of NOD1 and NOD2 on development of experimental arthritis. Ag-induced arthritis was generated in wild-type, NOD1(-/-), NOD2(-/-), or receptor-interacting serine-threonine kinase 2(-/-) (RIPK2(-/-)) immunized mice challenged intra-articularly with methylated BSA. Nociception was determined by electronic Von Frey test. Neutrophil recruitment and histopathological analysis of proteoglycan lost was evaluated in inflamed joints. Joint levels of inflammatory cytokine/chemokine were measured by ELISA. Cytokine (IL-6 and IL-23) and NOD2 expressions were determined in mice synovial tissue by RT-PCR. The NOD2(-/-) and RIPK2(-/-), but not NOD1(-/-), mice are protected from Ag-induced arthritis, which was characterized by a reduction in neutrophil recruitment, nociception, and cartilage degradation. NOD2/RIPK2 signaling impairment was associated with a reduction in proinflammatory cytokines and chemokines (TNF, IL-1ß, and CXCL1/KC). IL-17 and IL-17 triggering cytokines (IL-6 and IL-23) were also reduced in the joint, but there is no difference in the percentage of CD4(+) IL-17(+) cells in the lymph node between arthritic wild-type and NOD2(-/-) mice. Altogether, these findings point to a pivotal role of the NOD2/RIPK2 signaling in the onset of experimental arthritis by triggering an IL-17-dependent joint immune response. Therefore, we could propose that NOD2 signaling is a target for the development of new therapies for the control of rheumatoid arthritis.

Description of four new HLA-B alleles in Brazilian bone marrow donors.

Four new HLA-B alleles were identified in Brazilian individuals using next generation sequencing.

Two novel HLA-DRB3 alleles identified in Brazilian bone marrow donors: HLA-DRB3*01:122 and -DRB3*01:123.

We identified two new HLA-DRB3 alleles in Brazilian individuals using next generation sequencing.

Adhesion of Acanthamoeba on Scleral Contact Lenses According to Lens Shape.

Purpose: To investigate the adhesion of Acanthamoeba to scleral contact lens (ScCL) surface according to lens shape. Methods: Two strains of A. polyphaga (CDC:V062 and ATCC 30461) and one clinical Acanthamoeba isolate, were inoculated onto five contact lens (CL): one first-generation silicone hydrogel (SHCL; lotrafilcon B; adhesion control) containing plasma surface treatment; two ScCL (fluorosilicone acrylate) one containing surface treatment composed of plasma and the other containing plasma with Hydra-PEG, and two CL designed with a flat shape having the same material and surface treatments of the ScCL. Trophozoites that adhered to the lens's surfaces were counted by inverted optical light microscopy. Possible alterations of the lens surface that could predispose amoeba adhesion and Acanthamoeba attached to these lens surfaces were evaluated by scanning electron microscopy (SEM). Results: All strains revealed greater adhesion to the ScCL when compared with the flat lenses (P < 0.001). The clinical isolate and the ATCC 30461 had a higher adhesion (P < 0.001) when compared with the CDC:V062. A rough texture was observed on the surface of the lenses that have been examined by SEM. Also, SEM revealed that the isolates had a rounded appearance on the surface of the ScCL in contrast with an elongated appearance on the surface of the silicone hydrogel. Conclusions: The findings revealed that the curved shape of the ScCL favors amoeba adhesion.

Interdisciplinary treatment of a class III patient with congenitally absent maxillary lateral incisors.

UNLABELLED: The purpose of this paper is to present a case report of an adult Class III patient presenting bilateral congenitally missing maxillary lateral incisors that compromised occlusal function as well as smile and facial esthetics. After the interdisciplinary diagnosis, spaces for prosthetic substitution were opened with the help of miniscrews. The orthodontic therapy achieved a better dental relationship, and the final interdisciplinary treatment results represented a significant improvement in function and both dental and facial esthetics. CLINICAL SIGNIFICANCE: The interdisciplinary treatment of orthodontics and restorative dentistry is very important because the two complement each other in search of the best for the patient. This case demonstrates very well that where orthodontics provided the best tooth position prior to implant placement and restorations.

Risk factors for work disability in Brazilian patients with systemic lupus erythematosus.

BACKGROUND: Systemic Lupus Erythematosus (SLE) predominantly affects young females who are in their most productive years of life. SLE can cause organ damage and affects daily functioning and quality of life, causing work disability (WD). METHODS: We developed a longitudinal study with 110 SLE patients, whose data were collected through individual standardized interview and review of medical records. We aimed to determine the prevalence of WD and its possible associated risk factors (sociodemographic, lifestyle habits, quality of life, clinical characteristic, cumulative organ damage and disease activity). To identify variables associated with work disability, two different multivariate regression models using a stepwise backward method were performed. RESULTS: The percentage of WD due to SLE was 76.3%. An association was found between WD and lack of physical exercise (p=0.017) and high physical work demand (p=0.037). Clinical characteristics were not significant predictors of work dysfunction. CONCLUSION: 76.3% of our sample developed WD after SLE diagnosis. Participants who did not practice physical exercise and those who had a high-demand physical work were, respectively, 3.78 and 4.80 times more likely to have WD. Although we were not able to analyze the influence of COVID-19 in WD development, COVID-19 pandemic could have exacerbated the inequalities among people with chronic health conditions, especially in a low-income population, which could have influenced our results. Additional researches to evaluate risk factors for WD in low-income SLE patients and on strategies for reducing its impact are needed.

Endothelin-1 induces neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2 in mice.

Endothelin mediates neutrophil recruitment during innate inflammation. Herein we address whether endothelin-1 (ET-1) is involved in neutrophil recruitment in adaptive inflammation in mice, and its mechanisms. Pharmacological treatments were used to determine the role of endothelin in neutrophil recruitment to the peritoneal cavity of mice challenged with antigen (ovalbumin) or ET-1. Levels of ET-1, tumour necrosis factor α (TNFα), and CXC chemokine ligand 1 (CXCL1) were determined by enzyme-linked immunosorbent assay. Neutrophil migration and flow cytometry analyses were performed 4 h after the intraperitoneal stimulus. ET-1 induced dose-dependent neutrophil recruitment to the peritoneal cavity. Treatment with the non-selective ET(A)/ET(B) receptor antagonist bosentan, and selective ET(A) or ET(B) receptor antagonists BQ-123 or BQ-788, respectively, inhibited ET-1- and ovalbumin-induced neutrophil migration to the peritoneal cavity. In agreement with the above, the antigen challenge significantly increased levels of ET-1 in peritoneal exudates. The ET-1- and ovalbumin-induced neutrophil recruitment were reduced in TNFR1 deficient mice, and by treatments targeting CXCL1 or CXC chemokine receptor 2 (CXCR2); further, treatment with bosentan, BQ-123, or BQ-788 inhibited ET-1- and antigen-induced production of TNFα and CXCL1. Furthermore, ET-1 and ovalbumin challenge induced an increase in the number of cells expressing the Gr1(+) markers in the granulocyte gate, CD11c(+) markers in the monocyte gate, and CD4(+) and CD45(+) (B220) markers in the lymphocyte gate in an ET(A)- and ET(B)-dependent manner, as determined by flow cytometry analysis, suggesting that ET-1 might be involved in the recruitment of neutrophils and other cells in adaptive inflammation. Therefore, the present study demonstrates that ET-1 is an important mediator for neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2-dependent mechanism.