Tumour necrosis factor-alpha (TNF-alpha) transcription and translation in the CD4+ T cell-transplanted scid mouse model of colitis

Abstract

The adoptive transfer of activated CD4+ alpha/beta T cell blasts from the spleens of immunocompetent C.B-17+/+ or BALB/cdm2 mice into C.B-17scid/scid (scid) mice induces a colitis in the scid recipient within 8 weeks, which progresses to severe disease within 16 weeks. T cells isolated from recipient colon show a Th1 cytokine phenotype. We have examined the relationship between the phenotype of the cellular infiltrate and the transcription and translation of the proinflammatory cytokine TNF-alpha. The techniques of double indirect immunohistology and in situ hybridization using digoxigenin-labelled riboprobes were used. The prominent myeloid cell infiltrate in diseased tissues comprised F4/80+, Mac-l+ macrophages, neutrophils, dendritic cells and activated macrophages. TNF-alpha transcription and translation were associated with activated macrophages in the lamina propria. Activated macrophages transcribing and translating TNF-alpha were clustered in areas of tissue destruction. Crypt epithelium of inflamed tissues transcribed TNF-alpha at a very early stage of the disease process, but translation of TNF-alpha protein could only be found in advanced epithelial dysplasia. This indicates differential post-transcriptional control of TNF-alpha in activated macrophages and the epithelium.

Fig. 1

Immunohistochemical localization of myeloid cells in the normal scid mouse colon (a) and in the inflamed colon of a scid mouse transplanted with CD4⁺ T cells (b,c). Stained for CD11b, MoAb M1/70 and F4/80, MoAb C1:A3-1 (a); CD11b only (b); and F4/80 only (c). In colitis, tissue hypertrophy and increased infiltration by myeloid cells: arrow, infiltration of submucosa; arrowhead, CD11b⁺ neutrophils crossing epithelium. (All ×10 objective.)

Fig. 2

Scid mouse colitis, demonstrating distribution of (a) activated macrophages, MoAb 158.2 and (b) CD3⁺ T cells, MoAb KT3. Activated macrophages are present with equivalent numbers of T cells (closed arrows), or as foci without T cells (open arrows). CD3⁺-dominant foci can also be seen (arrowheads). (Both ×10 objective.)

Fig. 5

Correlation of TNF-α transcription and protein synthesis with presence of activated macrophages in a region of crypt destruction in scid mouse colitis. Serial sections: (a) TNF-α sense riboprobe, in situ hybridization (ISH); (b) TNF-α antisense riboprobe, ISH; (c) activated macrophages, MoAb 158.2, immunohistochemistry; (d) TNF-α protein, MoAb XT3, immunohistochemistry. Coincidence of TNF-α mRNA and protein and activated macrophages in pericryptal sheath. (All ×40 objective.)

Fig. 3

Transcription of TNF-α in colonic tissues of normal scid mouse (a) (×10 objective) and C.B-17^+/+ mouse (b) (×20 objective), by in situ hybridization using TNF-α antisense riboprobe. Positive staining in lower crypt epithelium of scid only.

Fig. 4

Correlation of TNF-α mRNA in scid colitis by in situ hybridization (a) with distribution of activated macrophages (MoAb 158.2) by immunohistochemistry (b). Note especially corresponding areas of transcription and activated macrophage infiltration highlighted by insets (slight differences between inset size and tissue included are caused by serial sectioning and differential tissue shrinkage artefact using the two methods). (Both ×10 objective.)

Fig. 6

TNF-α protein in damaged epithelium in scid colitis does not correlate with TNF-α mRNA expression and is not derived from activated macrophages. (a) TNF-α mRNA, antisense riboprobe. (b) TNF-α protein, MoAb XT3, immunohistochemistry. (c) Immunohistochemistry for activated macrophages, MoAb 158.2. Serial sections. (All × 100 oil objective.)