Recombinant Production and Biological Properties of Rat Cytokine-Induced Neutrophil Chemoattractants, GRO/CINC-2alpha, CINC-2beta and CINC-3

Shibata, Futoshi; Kawai, Kiyoshi; Kato, Hideko; Komorita, Naruyasu; Al-Mokdad, Maher; Fujioka, M.; Nakagawa, Hideo

doi:10.1111/j.1432-1033.1995.tb20701.x

Cited by 62 publications

(33 citation statements)

References 23 publications

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“…5,6) CINC-3 at a concentration of 10 nM induced calcium mobilization to an extent similar to that induced by CINC-1 and CINC-2 at a concentration of 100 nM. Stimulation of neutrophils with CINC-1 (100 nM) caused a loss of sensitivity to CINC-1 and CINC-2 (100 nM) but not to CINC-3 (10 nM).…”

Section: Discussionmentioning

confidence: 94%

“…5) Neutrophils were incubated with the calcium indicator fura-2 acetoxymethyl ester (2.5 mM) in Ca 2ϩ /Mg 2ϩ -free phosphate-buffered saline containing 10 mM HEPES, 0.25% bovine serum albumin, and 10 mM glucose (pH 7.4) for 30 min at 37°C. After incubation, the cells were washed twice and resuspended at 10 6 cells/ml with the buffer.…”

Section: Measurement Of Intracellular Free Calcium Levelsmentioning

confidence: 99%

“…5) As an index of chemotaxis, the migration rate was expressed as the percentage of the number of neutrophils migrating into the lower wells during an 80-min incubation to that of the neutrophils applied to the upper wells.…”

Section: )mentioning

confidence: 99%

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Chemokine Receptor CXCR2 Activates Distinct Pathways for Chemotaxis and Calcium Mobilization.

Shibata

Kawai

Nakagawa

2002

Biological & Pharmaceutical Bulletin

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Cytokine-induced neutrophil chemoattractants (CINCs) are potent neutrophil chemotactic factors in rats and major mediators of acute inflammation.1-3) Four members of the CINC family have been identified: CINC-1, CINC-2a, CINC-2b, and CINC-3/macrophage inflammatory protein-2 (MIP-2).1) CINC-2a and CINC-2b differ only in the sequence of carboxy-terminal three amino acid residues and are produced by alternative splicing.4) CINCs show high degrees of sequence similarity to human GROs, interleukin 8 (IL-8), mouse KC, and mouse MIP-2 and belong to the CXC chemokine family. CINC-1 and CINC-2 show chemotactic activity comparable with that of CINC-3.5) Treatment of neutrophils with CINC-1 or CINC-2 inhibited the chemotactic activity of CINC-1 and CINC-2, but not that of CINC-3, whereas treatment with CINC-3 inhibited the activity of all CINCs.6) On the other hand, CINC-3 induced calcium mobilization more potently than CINC-1 and CINC-2, and desensitized calcium mobilization in neutrophils induced by CINC-1 and CINC-2.5) In addition, CINC-3 potently competed with the binding of 125 I-labeled CINC-3 Tyr, although CINC-1 competed only weakly. 7) To determine the biochemical basis for the differences in neutrophil responses to CINC-1/-2 versus CINC-3, we have cloned the gene encoding the receptor and identified rat CXCR2 as the common receptor for all CINCs.8) CXCR2 belongs to the family of G-proteincoupled receptors with seven transmembrane-spanning domains. Herein, we report the relationship between the biological activities of CINCs and G-protein usage. MATERIALS AND METHODSPreparation of Neutrophils Neutrophils were harvested from the peritoneal exudate 16 h after intraperitoneal injection of Krebs/Ringer bicarbonate solution containing 1% casein (120 ml/kg body weight of male Wistar rats), washed twice with Gey's balanced salt solution and twice with the assay buffer and then resuspended at a concentration of 10 7 cells/ml. 5)Pertussis Toxin Treatment Neutrophils were incubated with pertussis toxin (PTX, 1 mg/ml) at 37°C for 2 h in a humidified 5% CO 2 atmosphere and washed twice with the assay buffer. Measurement of Intracellular Free Calcium LevelsThe intracellular free calcium level in neutrophils was measured by the method described previously.5) Neutrophils were incubated with the calcium indicator fura-2 acetoxymethyl ester (2.5 mM) in Ca 2ϩ /Mg 2ϩ -free phosphate-buffered saline containing 10 mM HEPES, 0.25% bovine serum albumin, and 10 mM glucose (pH 7.4) for 30 min at 37°C. After incubation, the cells were washed twice and resuspended at 10 6 cells/ml with the buffer. In the presence of 1 mM CaCl 2 , fluorescence changes were monitored using a Hitachi F4500 fluorescence spectrophotometer at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Fura-2-loaded cells were stimulated with CINCs, then lysed with 0.1% Triton-X, and finally Ca 2ϩ was chelated with 6 mM EGTA. Intracellular calcium levels were calculated as described by Grynkiewicz et al. 9)Chemotaxis Assay in Vitro Chemotactic a...

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Section: Discussionmentioning

confidence: 94%

Section: Measurement Of Intracellular Free Calcium Levelsmentioning

confidence: 99%

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Chemokine Receptor CXCR2 Activates Distinct Pathways for Chemotaxis and Calcium Mobilization.

Shibata

Kawai

Nakagawa

2002

Biological & Pharmaceutical Bulletin

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“…As members of the CXC chemokine family, rat CINC-1, CINC-2␣, CINC-2␤, and CINC-3 are neutrophil-specific chemokines that bind to a common receptor (CXCR2), show structural and functional homology to human IL-8, and are potent neutrophil chemoattractants in vitro and in vivo. [23][24][25][26][27] In a variety of acute vascular injury models, including a rat stroke model of middle cerebral artery occlusion with reperfusion and a rat liver transplant model with prolonged cold ischemia, upregulation of CINC has been demonstrated and correlated with neutrophil infiltration. 28,29 These acute-injury responses may play important roles in many pathological conditions, including acute coronary syndromes, as demonstrated by experimental and autopsy histological studies that show neutrophil infiltration.…”

Section: Discussionmentioning

confidence: 99%

Estrogen Modulates Inflammatory Mediator Expression and Neutrophil Chemotaxis in Injured Arteries

et al. 2004

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Background-We have previously shown that estrogen (17␤-estradiol; E2) inhibits neointima formation and migration of leukocytes, particularly neutrophils, into rat carotid arteries after acute endoluminal injury. This study tested the hypothesis that E2 inhibits expression of adhesion molecules, chemokines, and proinflammatory cytokines in rat carotid arteries in the early hours after balloon injury, thus attenuating the stimulus for leukocyte entry and negatively modulating the injury response. Methods and Results-Ovariectomized (OVX) rats were randomly assigned to treatment with E2 or vehicle (V) and subjected to balloon injury of the right carotid artery. After 2, 6, and 24 hours, rats were euthanized, and both carotid arteries were processed for real-time reverse transcription-polymerase chain reaction (2 and 24 hours), ELISA (6 hours), or neutrophil chemotaxis assay (24 hours). Expression of mRNA for adhesion molecules (P-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1), chemoattractants (cytokine-induced neutrophil chemoattractant [CINC]-2␤ and monocyte chemoattractant protein [MCP]-1), and proinflammatory cytokines (interleukin [IL]-1 and IL-6) was markedly increased (2 to 5000 times) in injured arteries of OVXϩV rats at 2 hours and was reduced by 24 hours. E2 significantly attenuated expression of the proinflammatory mediators (by 60% to 80%) at 2 hours. ELISA confirmed injury-induced upregulation of neutrophil and monocyte/macrophage chemoattractants (CINC-2␣, MCP-1) in OVXϩV arteries and E2-induced inhibition of CINC-2␣ expression. E2 significantly (by 65%) inhibited neutrophil chemotactic activity of arterial homogenates. Conclusions-E2 attenuates the early vascular injury response, at least in part, by negatively modulating proinflammatory mediator expression and the resultant chemotactic activity of injured vessels for neutrophils.

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“…Stimulation of neutrophils with CINCs also induces a dose-dependent increase in intracellular calcium levels, among which CINC-3 is more potent than are the other CINCs. CINC-2α, CINC-2β, and CINC-3 have been shown to induce a response similar to that induced by CINC-1 in terms of the release of a serine protease, cathepsinG, from rat neutrophils [3]. Among the CINCs, CINC-1 has been most extensively investigated.…”

Section: Introductionmentioning

confidence: 99%

Rat Cytokine-Induced Neutrophil Chemoattractant-1 (CINC-1) in Inflammation

Handa¹,

Naito²,

Yoshikawa³

2006

J. Clin. Biochem. Nutr.

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Summary Rat cytokine-induced neutrophil chemoattractant-1 (CINC-1) belongs to the CXCchemokine family and is a counterpart of human growth-related oncogene (GRO) in the interleukin-8 (IL-8) family. In rats, CINC-1 plays a key role in neutrophil-mediated inflammatory diseases by attracting neutrophils to the site of inflammation. Although IL-8 is a well-known chemokine in humans, it remains difficult to investigate human inflammatory diseases in detail. Therefore CINC-1 is expected to be an excellent tool for investigating neutrophil-mediated inflammatory diseases, and it has served extensively as a target chemokine in studies performed to date. However, little is known regarding CINC-1 signaling pathways. In this review, we focused on the signal transduction of CINC-1 production/ expression.

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Recombinant Production and Biological Properties of Rat Cytokine-Induced Neutrophil Chemoattractants, GRO/CINC-2alpha, CINC-2beta and CINC-3

Cited by 62 publications

References 23 publications

Chemokine Receptor CXCR2 Activates Distinct Pathways for Chemotaxis and Calcium Mobilization.

Chemokine Receptor CXCR2 Activates Distinct Pathways for Chemotaxis and Calcium Mobilization.

Estrogen Modulates Inflammatory Mediator Expression and Neutrophil Chemotaxis in Injured Arteries

Rat Cytokine-Induced Neutrophil Chemoattractant-1 (CINC-1) in Inflammation

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