Effect of rat CINC/gro, a member of the interleukin-8 family, on leukocytes in microcirculation of the rat mesentery.

Rat cytokine-induced neutrophil chemoattractant (CINC) is a member of the IL-8 family, and its human counterpart is gro/MGSA but not IL-8. We ascertained that chemically synthesized CINC was comparable to native CINC/gro with regard to chemotactic activity for rat neutrophils and studied the effect of synthesized CINC/gro on circulating leukocytes in microvascular vessels of rat mesentery. Exposure of rat mesentery to 10(-8)M authentic CINC/gro induced neutrophil adherence to and extravasation from postcapillary venules (PCVs) but not from capillaries or arterioles. CINC/gro concentrations as low as 10(-10) M were effective in causing neutrophil adherence. Neutrophils adhered to thin PCVs (mean diameter, approximately 25 microns) after exposure to CINC/gro for 15 min. The mean diameters of the PCV with adherence of neutrophils after exposure to CINC/gro for 30 and 60 min were 37 and 43 microns, respectively. The diameters of PCV with extravasation of neutrophils also increased in a time-dependent manner. The starting position of adherence of neutrophils was approximately 25-50 microns away from the upper junction of two vessels and remained virtually unchanged during exposure to CINC/gro for 60 min. However, the distance from the start to the end of neutrophil adherence increased in a time-dependent manner. The effect of CINC/gro on adherence and extravasation of leukocytes was neutrophil specific since other leukocytes such as lymphocytes and monocytes were not identified among the adherent and extravasated leukocytes.     

Level of neutrophil chemotactic factor CINC/gro, a member of the interleukin-8 family, associated with lipopolysaccharide-induced inflammation in rats.

Rat cytokine-induced neutrophil chemoattractant (CINC), which is a counterpart of human gro and belongs to the interleukin-8 family, has been quantified by a new sandwich enzyme-linked immunosorbent assay. Administration of lipopolysaccharide (LPS) into an air pouch performed by subcutaneous injection of air caused inflammation and severe neutrophil infiltration. After the LPS injection, changes in the concentration of CINC/gro, chemotactic activity, and the number of neutrophils in the air pouch exudate were determined. The chemotactic activity of neutrophils was augmented before practical neutrophil infiltration. More than half of the chemotactic activity was neutralized by the antisera. The time kinetics of the level of CINC/gro coincided with the changes in chemotactic activity. The maximal level of rat CINC/gro was 85 ng/ml, which is sufficient to cause neutrophil migration in vitro and in vivo as described previously. These data suggest that rat CINC/gro is a functional chemoattractant for neutrophils in LPS-induced inflammation in rats.     

Interleukin-1 stimulates rapid release of cytokine-induced neutrophil chemoattractant (CINC) in rat lungs

We found that intratracheal insufflation of interleukin-1 (IL-1) in rats rapidly increased lung lavage cytokine-induced neutrophil chemoattractant (CINC) concentrations, lung tissue myeloperoxidase (MPO) activity, and lung lavage neutrophil counts, and that CINC elevation preceded the migration of neutrophils into the lung. Further, we found that bolus CINC insufflation increased CINC concentrations in plasma, and we found that alveolar macrophages (AM) in lung tissue selections or AM recovered by lavage from rats given IL-1 intratracheally stained positively for CINC by immunohistochemistry. In addition, incubating rat AM with increasing doses of IL-1 in vitro progressively increased CINC concentrations in the culture medium. Our results suggest that the potent neutrophil chemoattractant CINC is rapidly produced and released by rat AM following challenge with IL-1 in vivo or in vitro, and support the hypothesis that CINC is an important mediator in the development of pulmonary inflammation in the rat.Parker B. Francis Fellow in Pulmonary Research.     

Ca2+]i changes and respiratory burst in human neutrophils and monocytes induced by NAP-1/interleukin-8, NAP-2, and gro/MGSA

The stimulatory effects of neutrophil-activating peptide 1 (NAP-1), also termed interleukin 8 (IL-8), neutrophil-activating peptide 2 (NAP-2), and melanoma growth-stimulatory activity (gro/MGSA) on human neutrophils and monocytes were compared on the basis of two responses that can be assessed in real time, the changes in cytosolic free calcium and the respiratory burst. All three peptides induced a rapid and transient rise of cytosolic-free calcium and the respiratory burst in neutrophils. Both responses were also obtained in monocytes on stimulation with NAP-1/IL-8 and gro/MGSA, but not with NAP-2, which appeared to be more selective for neutrophils. Pretreatment with concanavalin A (ConA) enhanced several fold the rate and duration of the respiratory burst of neutrophils stimulated with all three peptides and of monocytes stimulated with NAP-1/IL-8 and gro/MGSA, but not with NAP-2. Sequential stimulation showed mutual cross desensitization by NAP-2 and gro/MGSA in neutrophils. In addition, desensitization of neutrophils toward NAP-2 and gro/MGSA, and of monocytes toward gro/MGSA, was obtained by prestimulation with NAP-1/IL-8. Prestimulation with either NAP-2 or gro/MGSA, however, did not desensitize the cells for NAP-1/IL-8. These results suggest that under conditions where multiple stimulatory agents are produced, neutrophil-activating peptides may contribute to the formation of substantial amounts of oxygen-derived radicals. In addition, the study shows that NAP-1/IL-8 and gro/MGSA, but not NAP-2, have some stimulatory effects on monocytes as well.     

Stimulatory effect of the herbal medicine Keishi-bukuryo-gan on a cytokine-induced neutrophil chemoattractant, in rat ovarian cell culture

PROBLEM AND METHOD OF STUDY: We investigated the effects of Keishi-bukuryo-gan, a Japanese herbal medicine, and its crude ingredients in relation to the production of cytokine-induced neutrophil chemoattractant (CINC/gro), interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), which are known to stimulate the secretion of CINC/gro in the ovulatory process, and the effects of Keishi-bukuryo-gan with those of Toki-shakuyaku-san, which has been shown to have an effect on the ovary. We cultured whole ovarian dispersates from immature (3-week-old) female rats with Keishi-bukuryo-gan, Toki-shakuyaku-san and crude ingredients of Keishi-bukuryo-gan. The contents of CINC/gro, IL-1beta and TNFalpha in the cultured media were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Keishi-bukuryo-gan stimulated the secretion of CINC/gro in a dose-dependent manner, and the secretion of CINC/gro into the culture medium increased significantly at concentrations of Keishi-bukuryo-gan of 10 and 100 microg/mL (P < 0.001). The stimulatory effect of Keishi-bukuryo-gan on the production of CINC/gro is significantly (P < 0.001) stronger than that of Toki-shakuyaku-san at the same concentrations of 100 microg/mL. In addition, Keishi-bukuryo-gan stimulated the secretion of IL-1beta in a dose-dependent manner, while it did not stimulate the secretion of TNFalpha even at a concentration of 100 microg/mL. Moutan Cortex, Paeoniae Radix and Persicae Semen, which are crude ingredients of Keishi-bukuryo-gan, enhanced the secretion of CINC/gro significantly (P < 0.01) in cultured whole ovarian dispersates. CONCLUSIONS: These results show that Keishi-bukuryo-gan can stimulate the secretion of CINC/gro as well as the production of IL-1beta and that this stimulatory effect of Keishi-bukuryo-gan was significantly stronger than that of Toki-shakuyaku-san in immature rat ovarian cell culture.     

The herbal medicine Unkei-to stimulates the secretion of a cytokine-induced neutrophil chemoattractant,CINC/gro,in the rat ovarian cell culture

PROBLEM AND METHOD OF STUDY: We investigated the ovulation-inducing effects of Unkei-to, a Japanese herbal medicine, in relation to the production of sex steroid hormones (17beta-estradiol and progesterone), cytokine-induced neutrophil chemoattractant (CINC/gro), interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) in the rat ovarian cell culture. RESULTS: Unkei-to at a concentration of 100 microg/mL significantly stimulated the secretions of 17beta-estradiol and progesterone (P < 0.01) in cultured whole ovarian dispersates. Unkei-to also enhanced the secretion of CINC/gro in a dose-dependent manner, and the secretions of CINC/gro increased significantly at concentrations of 10 and 100 microg/mL (P < 0.01). These stimulatory effects of Unkei-to on steroidgenesis and CINC/gro production are very similar to those of another Japanese herbal medicine, Toki-Shakuyaku-san. In addition, Unkei-to significantly (P < 0.01) enhanced the secretions of both IL-1beta and TNF-alpha, which are known to stimulate the secretion of CINC/gro in the ovulatory process, at concentrations of 10 and 100 microg/mL. The stimulatory effect of Unkei-to at a concentration of 100 microg/mL on IL-1beta/was significantly (P < 0.01) lower than that of Toki-Shakuyaku-san, while the stimulatory effects of these two herbal medicines at a concentration of 100 microg/mL on TNF-alpha were similar. CONCLUSIONS: These results show that Unkei-to can stimulate ovarian steroidgenesis and the ovulatory process by inducing the secretion of CINC/gro with IL-1beta and TNF-alpha in vitro. Unkei-to has stimulatory effects on both steroidgenesis and the ovulatory process in the ovary as well as a stimulatory effect on the hypothalamus-pituitary axis, and it may be useful for treating patients with ovulatory disorders.     

Biological activities of C3 beta c, a novel neutrophil chemoattractant derived from the beta-chain of rat complement C3.

Biological activities of C3 beta c, which is a C-terminal fragment of the beta-chain of rat complement C3, have been studied by in vivo and in vitro experiments. C3 beta c was purified as a novel neutrophil chemoattractant from the exudate of the chronic phase of rat carrageenin-induced inflammation. The purified C3 beta c induced neutrophil chemotaxis in vivo when C3 beta c was injected into the preformed air-pouch on the back of rats. C3 beta c transiently increased the intracellular free Ca2+ concentration of neutrophils and enhanced the adhesion of neutrophils to fibrinogen in vitro, suggesting that C3 beta c has the ability to express an adhesion molecule of rat neutrophils. In addition, C3 beta c at low concentrations (10(-10)-10(-11) M) stimulated rat macrophages to produce cytokine-induced neutrophil chemoattractant-2, a member of the interleukin-8 family. Furthermore, C3 beta c enhanced vascular permeability in vivo, which is suppressed by cyproheptadine, suggesting that C3 beta c may have the characteristics of an anaphylatoxin. Our results suggest that C3 beta c contributes to oedema formation and neutrophil accumulation at inflammatory sites in rats.     

Appearance of cytokine-induced neutrophil chemoattractant isoforms and immunolocalization of them in lipopolysaccharide-induced acute lung inflammation in rats

OBJECTIVE AND DESIGN: Recently, rat cytokine-induced neutrophil chemoattractant (CINC), which belongs to the interleukin-8 family, was grouped into four isoforms, CINC-1, CINC-2a, CINC-2beta, and CINC-3. To determine the major component and the source of CINC in airways, we investigated the change in appearance of CINC isoforms after exposure of rats to lipopolysaccharide. METHODS: Male Sprague-Dawley rats, 8-10 weeks old, were used in the present study. Bronchoalveolar lavage (BAL) was performed at 1, 2, 4, 6, 12, and 24 h after lipopolysaccharide inhalation (4 mg/ml for 30 min). The concentrations of each specific rat CINC in the BAL supernatant were measured by use of commercially available kits. Furthermore, lung tissue was employed for immunohistochemical staining of CINCs (CINC-1, -2alpha,-2beta, and -3) using the streptavidin-biotin technique. RESULTS: Inhalation of lipopolysaccharide caused increases in CINC-1, CINC-2aalpha, and CINC-3 in BAL fluids, whereas CINC-2beta was not detected. The increases in CINC-2a and CINC-3 were less than the increase in CINC-1. Positive immunohistochemical staining for CINC-1 was detected in bronchial noncilliated cells and in certain neutrophils that had infiltrated into the submucosa. CONCLUSIONS: These findings suggest that CINC-1 is the major isoform among the four CINCs in lipopolysaccharide-induced acute lung inflammation in rats. Its sources are likely to be bronchial noncilliated cells and certain infiltrating neutrophils.     

Induction of neutrophil infiltration by rat chemotactic cytokine (CINC) and its inhibition by dexamethasone in rats

In vivo effects of cytokine-induced neutrophil chemotactic factor (CINC) derived from rats on neutrophil infiltration were investigated using an air-pouch-type inflammation model in rats, and effects of dexamethasone on neutrophil infiltration induced by CINC was also examined in order to gain further insight into the mechanism of antiinflammutory activity of glucocorticoids. Injection of CINC into the air pouch made on the dorsum of rats induced a marked infiltration of neutrophils into the pouch fluid but not mononuclear cells and eosinophils during a 30-min interval after the injection. Maximum effect was induced at a dose of 1.4g/pouch. Treatment with dexamethasone 3 h before the injection of CINC suppressed the neutrophil infiltration in a dose-dependent manner, but no complete inhibition was observed. CINC injection into the air pouch of rats that had been sacrificed by bleeding in order to minimize neutroph il infiltration from blood stream also stimulated neutrophil infiltration into the pouch fluid when the carcass was incubated at 37C for 30 min, but the number of infiltrated neutrophils was about 35% of CINC-induced neutrophil infiltration in intact ruts. CINC-induced neutrophil infiltration in the carcass, which is supposed to be a reflection of neutrophil migration from extravascular space in subcutaneous tissues to pouch fluid, was not inhibited by dexamethasone treatment. Therefore, the inhibition of neutrophil infiltration by dexamethasone might be due to inhibition of the extravasation of peripheral neutrophils but not due to inhibition of neutrophil chemotaxis from subcutaneous extravascular space to pouch fluid. These findings suggest that clinical effects of steroidal antiinflammatory drugs on neutrophil infiltration in inflammatory disease is partly due to inhibition of neutrophil extravasation induced by preformed neutrophil chemotactic factors in the inflammatory site.     

Aging Reduces the Expression of Lung CINC and MCP-1 mRNA in a P. aeruginosa Rat Model of Infection

We investigated dynamic changes of inflammatory cell infiltration and expression of cytokine-induced neutrophil chemoattractant (CINC) and monocyte chemoattractant protein-1 (MCP-1) mRNA in aged rats with Pseudomonas aeruginosa pulmonary infection. Disease manifestation and lung tissue pathology (lesion dispersion, inflammatory reactions, tissue edema and bleeding) were more severe in aged rats than young rats. At various time points, lung tissue polymorphonuclear neutrophil and mononuclear macrophage numbers were lower in the aged group than the young group (P < 0.05), and at 24 h there was no difference in mononuclear macrophage numbers. After inoculation with P. aeruginosa, CINC and MCP-1 mRNA expression increased in both groups, but the peak lagged in old rats compared with young. Thus, aging can reduce the expression of CINC and MCP-1 mRNA in lung tissues, and reduce the infiltration of neutrophils and monocyte–macrophages induced by CINC and MCP-1. This might lead to increased risk of pneumonia in elderly patients.     

Role of interleukin-17 and the neutrophil in asthma.

Recent clinical evidence shows that acute, severe exacerbations of asthma are associated with recruitment and activation of neutrophils in the airways. There is also experimental evidence from rodents that T-lymphocytes are involved in the recruitment of neutrophils following allergen challenge in sensitised airways. This review addresses the potential role of neutrophils and the cytokine interleukin-17 (IL-17) in severe asthma. IL-17 is produced and released as a free protein from T-lymphocytes of the memory (CD45RO+) subset. Evidence from rats in vivo suggests that IL-17 can recruit and activate neutrophils in the airways; the recruitment is mediated by the rat correlate to the neutrophil chemoattractant interleukin-8 (IL-8) macrophage inflammatory protein-2 (MIP-2). Endogenous peptidases modulate neutrophil recruitment by acting on NK-1 receptors in rat airways in vivo. Human bronchial epithelial cells in vitro respond to stimulation with IL-17 by increasing the production and release of the human neutrophil chemoattractant IL-8. This release of IL-8 is functionally significant; it causes neutrophil chemotaxis in vitro. Furthermore, this IL-8 release is sensitive to a glucocorticoid and is potentiated by the pro-inflammatory cytokine, tumour necrosis factor-alpha (TNF-alpha) in vitro. In addition, IL-17 stimulates human bronchial epithelial cells in vitro to release the neutrophil-activating factor IL-6. This effect of IL-17 on IL-6, and IL-8 is in part mediated via mitogen-activated protein kinases. In conclusion, as indicated in rat airways in vivo and in human bronchial epithelial cells in vitro, IL-17 may constitute a link between the activation of certain T-lymphocytes and mobilisation of neutrophils in the airways, via induced release of C-X-C chemokines and tachykinins. Further studies are required to answer the question whether free, soluble IL-17 protein plays this role in the airways of patients with severe asthma.     

Morphological Alteration of Cultured Tracheobronchial Epithelial Cells is Accompanied by the Expression of Chemokines, MCP-1 and CINC/gro, in Rats

Morphologically altered epithelial cells are generally observed in fibrotic lung conditions and have been reported to produce several cytokines. To examine the relationship between morphological changes of the tracheobronchial epithelial cells (TBECs) and their chemokine production, we investigated, (1) the mRNA expression and protein secretion of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant/gro (CINC/gro), (2) morphological changes by electron microscopy, and (3) cytokeratin (CK) expression, using a primary culture system of rat TBECs. The constitutive secretion of MCP-1 in the culture supernatant of TBECs increased in a time-dependent manner, whereas the CINC/gro secretion was not changed. These results were consistent with the chemokines'' mRNA expression observed by in situ hybridization. The constitutive secretions of MCP-1 and CINC/gro were inhibited partially but significantly by dexamethasone. With the extension of the culture period, the morphology of the TBECs became flat and spindle in shape, similar to squamous metaplasia, as observed on electron microscopy, and with strong expression of CK 14. Sequential staining using immunocytochemistry and in situ hybridization revealed the coexpression of MCP-1 mRNA and CK 14. These data indicate a significant relationship between the morphological squamoid alteration and the constitutive expression of MCP-1 but not of CINC/gro. It is thought that the squamous metaplasia of TBECs may accompany the alteration of cytokine production and play an important role in chronic lung inflammation.     

Eosinophil accumulation induced by human interleukin-8 in the guinea-pig in vivo.

Interleukin-8 (IL-8) is a neutrophil chemoattractant cytokine. Initially IL-8 appeared to exhibit specificity for neutrophils over other cells of the immune system. However, several recent studies have shown that this mediator can also activate other leucocyte types in vitro. In this study we have used an in vivo model of local [111In]leucocyte accumulation in the guinea-pig and an in vitro assay of leucocyte activation (changes in cytosolic-free Ca2+) to investigate the eosinophil chemoattractant activity of IL-8. The intradermal injection of recombinant human (rh)IL-8 induced a dose-dependent accumulation of intravenously administered [111In]eosinophils into the skin sites over 4 hr. Time-course experiments revealed that this cell infiltration was delayed in onset, occurring between 1 and 2 hr after injection of IL-8. The delay may indicate that IL-8 operates via an indirect mechanism. In contrast, eosinophil accumulation induced by the complement fragment C5a occurred within the first hour following injection. Other human cytokines, IL-1, IL-3, IL-5, tumour necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), were not eosinophil chemoattractants in this in vivo test system. Direct activation of eosinophils by IL-8 was demonstrated in vitro by a transient elevation in cytoplasmic-free Ca2+ levels where it was less potent than either rhC5a or leukotriene B4 (LTB4). Experiments using [111In]neutrophils in vivo indicated that rhIL-8 and rhC5a were similar in potency in inducing local neutrophil infiltration into guinea-pig skin. The demonstration of the eosinophil chemoattractant activity of IL-8 in vivo raises the possibility that this cytokine, or a structurally related molecule, contributes towards eosinophil infiltration in a number of inflammatory conditions such as asthma, helminthic infections and adult respiratory distress syndrome.     

Investigations on the biology, epidemiology, pathology and control of Tunga penetrans in Brazil. V. Cytokine concentrations in experimentally infected Wistar rats

Tungiasis is caused by the penetration of the female sand flea Tunga penetrans into the skin of its host. This parasitic skin disease is almost invariably associated with an intense inflammation around embedded fleas, the underlying mechanisms being unknown. A study was undertaken to determine whether Wistar rats can be used as an animal model to assess cytokine kinetics during the natural course of the infection. Laboratory-raised Wistar rats were exposed in cages put on the soil in an area with high human attack rates. Rats were examined daily and blood samples were taken before exposure and at 2, 6, 10, 13, 16 and 20 days after flea penetration. TNF-, IL-1, IFN-, IL-4, IL-10 and CINC (a rat cytokine- induced neutrophil chemoattractant and member of the IL-8 family) were determined by enzyme immunoassay. The results showed an increasing serum concentration of TNF- and IL-1 10–13 days after penetration and a rapid increase in IL-4 2 days after fleas became embedded. During the natural course of the infection, the ratio of the serum concentration of TNF- to that of IL-10 decreased, indicating a relative increase in the secretion of the anti-inflammatory cytokine. The treatment of lesions with silicone oil abrogated the natural disease course and changed the pattern of cytokine secretion. We conclude that the Wistar rat is an appropriate model to study immune responses in tungiasis.     

Neutrophil chemorepulsion in defined interleukin-8 gradients in vitro and in vivo

We report for the first time that primary human neutrophils can undergo persistent, directionally biased movement away from a chemokine in vitro and in vivo, termed chemorepulsion or fugetaxis. Robust neutrophil chemorepulsion in microfluidic gradients of interleukin-8 (IL-8; CXC chemokine ligand 8) was dependent on the absolute concentration of chemokine, CXC chemokine receptor 2 (CXCR2), and was associated with polarization of cytoskeletal elements and signaling molecules involved in chemotaxis and leading edge formation. Like chemoattraction, chemorepulsion was pertussis toxin-sensitive and dependent on phosphoinositide-3 kinase, RhoGTPases, and associated proteins. Perturbation of neutrophil intracytoplasmic cyclic adenosine monophosphate concentrations and the activity of protein kinase C isoforms modulated directional bias and persistence of motility and could convert a chemorepellent to a chemoattractant response. Neutrophil chemorepulsion to an IL-8 ortholog was also demonstrated and quantified in a rat model of inflammation. The finding that neutrophils undergo chemorepulsion in response to continuous chemokine gradients expands the paradigm by which neutrophil migration is understood and may reveal a novel approach to our understanding of the homeostatic regulation of inflammation.     

Rat Macrophage Inflammatory Protein-1α, a CC Chemokine, Acts as a Neutrophil Chemoattractant in Vitro and in Vivo

Recombinant rat macrophage inflammatory protein-1 (rMIP-1) at a concentration of 3 × 10^–8 M had strong neutrophil chemotactic activity, though the potency of rMIP-1 was less than that of cytokine-induced neutrophil chemoattractant (CINC)-1 at lower concentrations. In addition, rMIP-1 induced neutrophil chemotaxis in vivo when rMIP-1 was injected into the preformed air-pouch on the back of rats. The adhesion of rMIP-1-treated neutrophils to fibrinogen significantly increased, reaching a maximum adhesion at 10^–8 M. Stimulation of neutrophils with rMIP-1 induced a transient increase in intracellular free [Ca²⁺] dose-dependently. rMIP-1 still induced an increase in the intracellular [Ca²⁺] of rat neutrophils stimulated first with CINC-1, CINC-3 or C5a, suggesting that rat neutrophils have a specific receptor for rMIP-1. Supporting these findings, an additive increase in chemotactic potency was found when both rMIP-1 and CINC-1 were added to the lower wells of Boyden chamber in vitro. In addition, high levels of rMIP-1 were detected in the inflammatory site of air-pouch/carrageenan-induced inflammation in rats. Our results suggest that rMIP-1 acts as a neutrophil chemoattractant and, together with CINCs, plays an important role in infiltration of neutrophils into inflammatory sites in rats.     

Crucial role of neutrophils in the development of mechanical inflammatory hypernociception

Neutrophil migration is responsible for tissue damage observed in inflammatory diseases. Neutrophils are also implicated in inflammatory nociception, but mechanisms of their participation have not been elucidated. In the present study, we addressed these mechanisms in the carrageenan-induced mechanical hypernociception, which was determined using a modification of the Randall-Sellito test in rats. Neutrophil accumulation into the plantar tissue was determined by the contents of myeloperoxidase activity, whereas cytokines and PGE(2) levels were measured by ELISA and radioimmunoassay, respectively. The pretreatment of rats with fucoidin (a leukocyte adhesion inhibitor) inhibited carrageenan-induced hypernociception in a dose- and time-dependent manner. Inhibition of hypernociception by fucoidin was associated with prevention of neutrophil recruitment, as it did not inhibit the hypernociception induced by the direct-acting hypernociceptive mediators, PGE(2) and dopamine, which cause hypernociception, independent of neutrophils. Fucoidin had no effect on carrageenan-induced TNF-alpha, IL-1beta, and cytokine-induced neutrophil chemoattractant 1 (CINC-1)/CXCL1 production, suggesting that neutrophils were not the source of hypernociceptive cytokines. Conversely, hypernociception and neutrophil migration induced by TNF-alpha, IL-1beta, and CINC-1/CXCL1 was inhibited by fucoidin, suggesting that neutrophils are involved in the production of direct-acting hypernociceptive mediators. Indeed, neutrophils stimulated in vitro with IL-1beta produced PGE(2), and IL-1beta-induced PGE(2) production in the rat paw was inhibited by the pretreatment with fucoidin. In conclusion, during the inflammatory process, the migrating neutrophils participate in the cascade of events leading to mechanical hypernociception, at least by mediating the release of direct-acting hypernociceptive mediators, such as PGE(2). Therefore, the blockade of neutrophil migration could be a target to development of new analgesic drugs.     

Differential production of MCP-1 and cytokine-induced neutrophil chemoattractant in the ischemic brain after transient focal ischemia in rats.

Chemokines have been shown to play an important role in leukocyte infiltration into ischemic lesions. Recently, the increased expression of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant (CINC) was observed in experimental stroke models where infiltrated leukocytes were supposed to induce tissue injury, however, the protein level and time course of these chemokines have not been fully elucidated. Therefore, we analyzed the time-dependent production of MCP-1 and CINC in the rat brain after transient middle cerebral artery occlusion (MCAO) by means of specific enzyme-linked immunosorbent assay systems. The MCP-1 levels in the ipsilateral hemispheres increased from 6 h, peaked at 2 days, and thereafter gradually decreased. The peak MCP-1 concentration was 89.2+/-28.2 ng/g tissue wet weight (mean +/- SEM, n = 5, 49.3-fold greater than the contralateral value at the same time, P < 0.05), which is supposed to be high enough to exert its biological effects. In contrast, the maximum CINC concentration that corresponded to 2.9+/-0.7 ng/g tissue wet weight (mean +/- SEM, n = 5, 55.0-fold greater than the contralateral value at the same time, P < 0.05), was observed at 6 h. In addition, we confirmed the temporal profile of leukocyte subtypes that infiltrated into the ischemic brain, thus, neutrophil infiltration occurred at early stages (1-3 days), followed by massive infiltration of macrophages at later stages (2-7 days). These studies suggest that MCP-1 in cerebral ischemia actually plays a significant role in the migration of macrophages into the lesion and that the differential temporal production of these chemokines contributes to the regulation of infiltrated leukocyte subtypes.