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Comparative Study
.2004 Nov 15;200(10):1289-97.
doi: 10.1084/jem.20041789. Epub 2004 Nov 8.

T cell-specific inactivation of the interleukin 10 gene in mice results in enhanced T cell responses but normal innate responses to lipopolysaccharide or skin irritation

Affiliations
Comparative Study

T cell-specific inactivation of the interleukin 10 gene in mice results in enhanced T cell responses but normal innate responses to lipopolysaccharide or skin irritation

Axel Roers et al. J Exp Med..

Abstract

Interleukin (IL)-10 is a regulator of inflammatory responses and is secreted by a variety of different cell types including T cells. T regulatory cells have been shown to suppress immune responses by IL-10-dependent, but also IL-10-independent, mechanisms. Herein, we address the role of T cell-derived IL-10 in mice with an inactivation of the IL-10 gene restricted to T cells generated by Cre/loxP-mediated targeting of the IL-10 gene. Splenocytes from this T cell-specific mutant secrete increased amounts of proinflammatory cytokines after activation in vitro compared with show enhanced contact hypersensitivity reactions, and succumb to severe immunopathology upon infection with Toxoplasma gondii. Despite intact IL-10 genes in other cell types, the dysregulation of T cell responses observed in the T cell-specific IL-10 mutant closely resembles the phenotype in complete IL-10 deficiency. However, in contrast to complete IL-10 deficiency, sensitivity to endotoxic shock and irritant responses of the skin are not enhanced in the T cell-specific IL-10 mutant. Our data highlight the importance of T cell-derived IL-10 in the regulation of T cell responses and demonstrate that endotoxic shock and the irritant response of the skin are controlled by IL-10 from other cell types.

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Figures

Figure 1.
Figure 1.
Conditional targeting of theIL-10 gene. (A) Scheme showing the WTIL-10 genomic locus (WT) and the mutantIL-10 alleles after homologous recombination of the targeting vector (Neo), after Cre-mediated deletion of the neomycin resistance gene (FL), and after Cre-mediated deletion of the loxP-flanked promoter region and exon1 (D). The location of the probe for Southern blot analysis as well as restriction enzyme sites (P, PstI) are indicated. PstI fragments are in kilobases (kb). Filled boxes represent exons. LoxP sites are depicted as arrowheads. (B) Southern blot analysis of DNA from tail biopsies from a WT, anIL-10FL/FL, and anIL-10D/D mouse.
Figure 2.
Figure 2.
T cell–specific deletion of theIL-10 gene inIL-10FL/FL CD4-Cre+ mice. Southern blot analysis of DNA extracted from different FACS-sorted cell populations and tissues after PstI digest. (A) CD3+ T and CD19+ B cells sorted to a purity of 98 and 96%, respectively, from splenocyte suspension of a 3-mo-old mouse. The result is representative of five animals. (B) F4/80+ macrophages from peritoneal lavage fluid (purity 97%, representative of four animals) and splenic T cells. (C) Splenic CD11c+ dendritic cells sorted to a purity of 95% (representative of three animals) and splenic T cells. (D) Various tissues and splenic T cells from an 8-wk-old mouse.
Figure 3.
Figure 3.
Cytokine and chemokine secretion by splenocytes from mice with complete or T cell–specific IL-10 deficiency after stimulation by plate-bound anti-CD3 and anti-CD28 antibodies in vitro. (A) Concentrations of IFN-γ, TNF-α, IL-12, IL-6, and MCP-1 in 48-h culture supernatants of splenocytes fromIL-10FL/FL CD4-Cre+ animals (n = 9; □, unstimulated; ▪, stimulated),IL-10FL/FL Cre− controls (n = 8; ○, unstimulated; •, stimulated), and (B) in supernatants of splenocyte cultures fromIL-10D/D animals (n = 5; □, unstimulated; ▪, stimulated) andIL-10WT/D controls (n = 5; ○, unstimulated; •, stimulated).
Figure 4.
Figure 4.
(A) Histology of colonic mucosa. Note massive hyperplasia and edema of the mucosa in the T cell–specificIL-10 mutant. Paraffin sections, hematoxylin and eosin stain. Bar, 40 μm. (B) Cumulative incidence of rectal prolapse indicating severe intestinal inflammation inIL-10D/D mice (•,n = 90) andIL-10FL/FL CD4-Cre+ mice (□,n = 142).
Figure 4.
Figure 4.
(A) Histology of colonic mucosa. Note massive hyperplasia and edema of the mucosa in the T cell–specificIL-10 mutant. Paraffin sections, hematoxylin and eosin stain. Bar, 40 μm. (B) Cumulative incidence of rectal prolapse indicating severe intestinal inflammation inIL-10D/D mice (•,n = 90) andIL-10FL/FL CD4-Cre+ mice (□,n = 142).
Figure 5.
Figure 5.
Infection of T cell–specificIL-10 mutants withT. gondii. (A) Survival ofIL-10FL/FL CD4-Cre+ mice (n = 7) andIL-10FL/FL Cre− control animals (n = 8). Two and three mutants were killed because of severe, overt disease on days 7 and 10 after infection, respectively. Two and three control mice, which never showed symptoms or signs of disease, were killed in parallel on days 7 and 10, respectively. Three controls were killed 10 wk after infection. (B) Cryostat liver sections. On the top, hematoxylin and eosin stain is shown at a magnification of 25. Note the more intense infiltration of liver from T cell–specific mutants with inflammatory cells 7 d after infection. On the bottom, immunostaining for CD4 (Hemalum counterstain) is shown at a magnification of 100. Note the increased number of CD4+ cells in mutant liver compared with the controls.
Figure 6.
Figure 6.
Contact hypersensitivity reactions of (A)IL-10FL/FL CD4-Cre+ (n = 28) andIL-10FL/FL Cre− control mice (n = 26), and (B)IL-10D/D (n = 28) andIL-10D/WT control mice (n = 21). The increase in ear thickness of sensitized mice was measured daily after challenge with DNCB.
Figure 7.
Figure 7.
Irritant response of (A)IL-10FL/FL CD4-Cre+ (n = 36) andIL-10FL/FL Cre− control mice (n = 35), and (B)IL-10D/D (n = 17) andIL-10D/WT control mice (n = 16). The percent increase in ear thickness was measured 24 and 48 h after application of croton oil.
Figure 8.
Figure 8.
Response of mice with complete or T cell–specific IL-10 deficiency to LPS. 20 μg of LPS/g body weight were injected i.p. (A) Survival ofIL-10FL/FL CD4-Cre+ mice (□,n = 10) andIL-10FL/FL Cre− control littermates (▪,n = 10), andIL-10D/D animals (○,n = 10) andIL-10D/WT control littermates (•,n = 10). (B) Additional animals were bled 6 h after LPS injection and serum TNF-α, IFN-γ, and IL-12 levels were determined (symbols as in A).
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