Impaired defense mechanism against inflammation, hyperalgesia, and airway hyperreactivity in somatostatin 4 receptor gene-deleted mice

Abstract

We have shown that somatostatin released from activated capsaicin-sensitive nociceptive nerve endings during inflammatory processes elicits systemic anti-inflammatory and analgesic effects. With the help of somatostatin receptor subtype 4 gene-deleted mice (sst(4)(-/-)), we provide here several lines of evidence that this receptor has a protective role in a variety of inflammatory disease models; several symptoms are more severe in the sst(4) knockout animals than in their wild-type counterparts. Acute carrageenan-induced paw edema and mechanical hyperalgesia, inflammatory pain in the early phase of adjuvant-evoked chronic arthritis, and oxazolone-induced delayed-type hypersensitivity reaction in the skin are much greater in mice lacking the sst(4) receptor. Airway inflammation and consequent bronchial hyperreactivity elicited by intranasal lipopolysaccharide administration are also markedly enhanced in sst(4) knockouts, including increased perivascular/peribronchial edema, neutrophil/macrophage infiltration, mucus-producing goblet cell hyperplasia, myeloperoxidase activity, and IL-1beta, TNF-alpha, and IFN-gamma expression in the inflamed lung. It is concluded that during these inflammatory conditions the released somatostatin has pronounced counterregulatory effects through sst(4) receptor activation. Thus, this receptor is a promising novel target for developing anti-inflammatory, analgesic, and anti-asthmatic drugs.

Fig. 1.

Generation of sst₄ receptor gene–deficient mice. (A) Targeting strategy used for gene disruption of the mouse sst₄ genomic locus. The 1155-bp coding sequence of sst₄ resides on a single exon (white box). A replacement vector was designed to delete the entire sst₄ coding sequence and replace it with a cassette comprising a nuclear localized lacZ reporter gene (lacZpA) and loxP-flanked selectable markers uracil3 (Ura3) and neomycin resistance (neo) (shaded boxes). The initiation codon of lacZ is inserted, in frame, into the initiation codon of sst₄. (B) Southern blot analysis of genomic DNA prepared from tail biopsies of an sst₄ mutant pedigree shows correct targeting. DNAs were digested with BclI and hybridized with probe A or digested with SacI and hybridized with probe B (sst₄^+/+: +/+; sst₄^{+/lacZUra3neo}: +/-). (C) RT-PCR analysis of sst₄ and lacZ transcription. RT-PCR from total brain RNA using multiplexed sst₄ and lacZ primer sets demonstrates the presence of sst₄ mRNA in sst₄^+/+ and sst₄^+/lacZ but an absence in sst₄^lacZ/lacZ mice. Conversely, lacZ mRNA is detected in sst₄^+/lacZ and sst₄^lacZ/lacZ but is absent from sst₄^+/+ animals (sst₄^+/+: +/+; sst₄^+/lacZ: +/-; sst₄^lacZ/lacZ: -/-). No amplification was observed when RT-PCR was performed in the absence of reverse transcriptase (No RT Control) or when PCR was performed in the absence of cDNA template (No DNA).

Fig. 2.

(A) Decrease of the mechanonociceptive thresholds and (B) increase of the paw volume 6 h after intraplantar carrageenan (50 μL, 3%) injection in sst₄^+/+ and sst₄^−/− mice. Percentage changes are calculated by comparing the data with the initial self-control values. Results obtained in sst₄^+/+ and sst₄^−/− mice bred as separate lines, as well as littermate WT and knockouts, are indicated. The effect of the selective sst₄ receptor agonist J-2156 (100 μg/kg i.p.) is also shown in both group (means ± SEM of n = 6–8 experiments; *P < 0.05, **P < 0.01 vs the sst₄^+/+, group, +P < 0.05 vs solvent treated sst₄^+/+ mice determined by one-way ANOVA followed by Bonferroni's modified t test).

Fig. 3.

Histopathologic examination, MPO activity, and inflammatory cytokine concentrations of the lung in LPS-induced inflammation. (A) Representative histopathologic pictures of the lung samples of sst₄^+/+ and sst₄^−/− mice (periodic acid-Schiff staining; ×200; Br, bronchioles; V, vessels; open arrows indicate goblet cells, black arrowheads indicate edema formation, and double black arrowheads indicate granulocyte accumulation). (B) Semiquantitative evaluation and scoring of the lung on the basis of perivascular edema, perivascular/peribronchial granulocyte accumulation, goblet cell hyperplasia, and alveolar mononuclear cell infiltration. Columns represent medians with upper and lower quartiles of n = 8–10 mice; ⁺P < 0.01 LPS-treated inflamed vs. respective PBS-treated noninflamed mice; *P < 0.05 LPS-treated sst₄^−/− vs. LPS-treated sst₄^+/+ group (Kruskal-Wallis test plus Dunn's posttest). (C) MPO activity, as a quantitative indicator of the number of accumulated granulocytes, and (D) concentrations IL-1β, IFN-γ, and TNF-α determined from homogenized lung samples. Columns show means ± SEM of n = 8–10 mice; **P < 0.01 vs. the respective noninflamed mice; ⁺⁺P < 0.01 vs. the sst₄^+/+ group (one-way ANOVA plus Bonferroni's t test).

Fig. 4.

Carbachol-evoked bronchoconstriction and inflammatory airway hyperreactivity in sst₄^+/+ and sst₄^−/− mice. The panels demonstrate the percentage increases of (A) Penh and (B) RL above baseline [(mean values in response to the respective carbachol concentration- baseline values)/baseline values × 100] calculated in each 15-min period after respective carbachol stimulations. (C) Inflammatory cells accumulated in the BALF 24 h after intranasal endotoxin administration in sst₄^+/+ and sst₄^−/− littermate mice. Data show means ± SEM of n = 6 mice per group; *P < 0.05, **P < 0.01 vs. the respective PBS-treated mice; ⁺P < 0.05, vs. the sst₄^+/+ group (one-way ANOVA plus Bonferroni's modified t test).

Fig. 5.

Oxazolone-induced (A) swelling and (B) inflammatory cytokine concentrations of the ear of sst₄^+/+ and sst₄^−/− mice. Edema data points represent means ± SEM percentage increase of ear diameter compared with the initial values of n = 8–10 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. respective ethanol-treated ears; ⁺⁺⁺P < 0.001 vs. oxazolone-treated sst₄^+/+ mice. Columns showing cytokine concentrations in the ear homogenates 48 h after oxazolone application are means ± SEM of n = 6–8 per group. *P < 0.05 vs. sst₄^+/+ (one-way ANOVA followed by Bonferroni's modified t test).