Role of a histamine 4 receptor as an anti-inflammatory target in carrageenan-induced pleurisy in mice

Abstract

The histamine 4 receptor (H4R) is expressed primarily on cells involved in inflammation and immune responses. Despite much research into inflammatory diseases, no drugs with favourable safety profiles are yet available for their treatment. The aim of the present study was to determine the potential anti-inflammatory effect of 4-methylhistamine (4-MeH) or JNJ77777120 (JNJ) and to explore the role of H4R in a mouse model of carrageenan (Cg) -induced pleurisy. A single dose of 4-MeH or JNJ (30 mg/kg) was administered intraperitoneally 1 hr before Cg administration. The results illustrate that both the numbers of CD4(+) , CD25(+) , CD4(+) CD25(+) , GITR(+) , GITR(+) IL-17A(+) -expressing T cells and the levels of T helper type 1 (Th1)/Th17 cytokines were markedly increased in both the Cg-treated and 4-MeH-treated groups, whereas the cytokines produced by Th2 cells were significantly decreased in the same groups. However, JNJ treatment significantly decreased both the number of T-cell subsets and GITR(+) , GITR(+) IL-17A(+) -expressing T cells, and the production of Th1/Th17 cytokines. Further, JNJ up-regulated the expression of the Th2 cytokines. RT-PCR analysis revealed an increased expression of interleukin-1β, tumour necrosis factor-α, monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 in the Cg-treated and 4-MeH-treated groups, which was reduced by treatment with JNJ in lung tissues. Moreover, histological examinations revealed anti-inflammatory effects of JNJ, whereas 4-MeH worsened Cg-induced inflammation. In conclusion, the results of the present work clearly indicate that JNJ possesses important anti-inflammatory properties that are increased in 4-MeH-treated mice, suggesting that H4R are involved in pleurisy and that JNJ has an anti-inflammatory effect in associated disease conditions.

Keywords: 4-methylhistamine dihydrochloride; GITR-expressing cells; JNJ77777120; cytokines; histamine 4 receptor; λ-Carrageenan-induced inflammation.

Figure 1

Effect of 4-MeH and JNJ on CD4⁺, CD25⁺ and CD4⁺CD25⁺ levels. The levels were evaluated by flow cytometry in the exudate cells at 4 after the induction of pleurisy via Cg injection. Statistical analysis was performed using a one-way anova followed by the Tukey-Kramer post-test. Each value indicates the mean ± SEM of six animals. P < 0.05 is accepted as the level of significance; *P < 0.05 compared to the control group; ^aP < 0.05 compared to the Cg group; ^bP < 0.05 compared to the 4-MeH group.

Figure 2

Representative dot plots for CD4⁺, CD25⁺ and CD4⁺ CD25⁺ in the exudate from a mouse from each group at 4 h.

Figure 3

Effect of 4-MeH and JNJ on GITR⁺ and GITR⁺IL-17A⁺ levels. The levels were evaluated by flow cytometry using the exudate cells at 4 after the induction of pleurisy via Cg injection. Statistical analysis was performed using a one-way anova followed by the Tukey-Kramer post-test. Each value indicates the mean ± SEM of six animals. P < 0.05 is accepted as the level of significance; *P < 0.05 compared to the control group; ^aP < 0.05 compared to the Cg group; ^bP < 0.05 compared to the 4-MeH group.

Figure 4

Representative dot plots for GITR⁺ and GITR⁺IL-17A⁺ cells in the exudate from a mouse from each group at 4 h.

Figure 5

Effect of 4-MeH and JNJ on the levels of pro-inflammatory cytokines (a) IL-2, IL-6, IL-12 and (b) IL-17A, IL-23, IFN-γ. The levels were evaluated by ELISA from the exudates at 4 after the induction of pleurisy by Cg injection. Statistical analysis was performed using a one-way ANOVA followed by the Tukey-Kramer post-test. Each value indicates the mean ± SEM of six animals. P < 0.05 is accepted as the level of significance; *P < 0.05 compared to the control group; ^aP < 0.05 compared to the Cg group; ^bP < 0.05 compared to the 4-MeH group.

Figure 6

Effect of 4-MeH and JNJ on TGF-β1 and IL-10 anti-inflammatory cytokine levels. The levels were evaluated by ELISA in the exudates at 4 after the induction of pleurisy by Cg injection. Statistical analysis was performed using a one-way anova followed by the Tukey-Kramer post-test. Each value indicates the mean ± SEM of six animals. P < 0.05 is accepted as the level of significance; *P < 0.05 compared to the control group; ^aP < 0.05 compared to the Cg group; ^bP < 0.05 compared to the 4-MeH group.

Figure 7

Effect of 4-MeH and JNJ on the gene expressions of pro-inflammatory mediators (a) IL-1β and (b) TNF-α. mRNA expression was measured by quantitative RT-PCR in the lung tissue at 4 after the induction of pleurisy by Cg injection. Statistical analysis was performed using a one-way anova followed by the Tukey-Kramer post-test. Each value indicates the mean ± S.E.M. of six animals. P < 0.05 is accepted as the level of significance; *P < 0.05 compared to the control group; ^aP < 0.05 compared to the Cg group; ^bP < 0.05 compared to the 4-MeH group.

Figure 8

Effect of 4-MeH and JNJ on gene expression of the (a) ICAM-1 and (b) MCP-1 chemokines. mRNA expression was measured by quantitative RT-PCR in the lung tissue at 4 the induction of pleurisy by Cg injection. Statistical analysis was performed using a one-way anova followed by the Tukey-Kramer post-test. Each value indicates the mean ± SEM of six animals. P < 0.05 is accepted as the level of significance; *P < 0.05 compared to the control group; ^aP < 0.05 compared to the Cg group; ^bP < 0.05 compared to the 4-MeH group.

Figure 9

The effect of 4-MeH and JNJ on lung histology following 4 h of Cg administration in a mouse pleurisy model. (a) Shows a lung section of a normal mouse. (b,c) shows lung sections from a Cg treated mouse. (d,e,f) shows lung sections from a mouse treated with 4-MeH 4 h before Cg treatment. (g, h, j) shows sections from a mouse treated with JNJ 4 h before Cg treatment. The histological score (k) was made by an independent observer. Statistical analysis was performed using a one-way anova followed by the Tukey-Kramer post-test. Each value indicates the mean ± SEM of six animals. P < 0.05 is accepted as the level of significance; *P < 0.05 compared to the control group; ^aP < 0.05 compared to the Cg group; ^bP < 0.05 compared to the 4-MeH group.