Direct crosstalk between mast cell-TNF and TNFR1-expressing endothelia mediates local tissue inflammation

Abstract

Signaling through tumor necrosis factor receptor 1 (TNFR1) controls bacterial infections and the induction of inflammatory Th1 cell-mediated autoimmune diseases. By dissecting Th1 cell-mediated delayed-type hypersensitivity responses (DTHRs) into single steps, we localized a central defect to the missing TNFR1 expression by endothelial cells (ECs). Adoptive transfer and mast cell knockin experiments into Kit(W)/Kit(W-v), TNF(-/-), and TNFR1(-/-) mice showed that the signaling defect exclusively affects mast cell-EC interactions but not T cells or antigen-presenting cells. As a consequence, TNFR1(-/-) mice had strongly reduced mRNA and protein expression of P-selectin, E-selectin, ICAM-1, and VCAM-1 during DTHR elicitation. In consequence, intravital fluorescence microscopy revealed up to 80% reduction of leukocyte rolling and firm adhesion in TNFR1(-/-) mice. As substitution of TNF(-/-) mice with TNF-producing mast cells fully restored DTHR in these mice, signaling of mast cell-derived TNF through TNFR1-expressing ECs is essential for the recruitment of leukocytes into sites of inflammation.

Figure 1

DNFB induces strong tissue necrosis in TNFR1^+/+ mice. (A) DNFB-sensitized and -challenged TNFR1^+/+ mice developed reduced ear swelling responses 24 hours after ear challenge compared with TNFR1^−/− mice (n = 5-7). (B) TNCB-sensitized and -challenged TNFR1^−/− mice developed reduced ear swelling responses 24 hours after ear challenge compared with TNFR1^+/+ mice (n = 16-27). (C) Enhanced tissue necrosis in TNFR1^+/+ mice after DNFB application. Hematoxylin and eosin–stained abdominal skin sections from TNCB-treated (top left) or DNFB-treated (top right) TNFR1^+/+ and from TNCB-treated (bottom left) or DNFB-treated (bottom right) TNFR1^−/− mice at day 5 after hapten application on the abdomen (n = 3-5).

Figure 2

Impaired TNCB-specific DTHRs in TNFR1^−/− mice. (A) TNCB-sensitized and naive TNFR1^−/− () and TNFR1^+/+ (■) mice were challenged with TNCB. Ear thickness was measured before and at the indicated time points after TNCB challenge. Differences in ear thickness between TNFR1^−/− () and TNFR1^+/+ (■) were significant (P < .05) 24, 48, and 72 hours after ear challenge (24 hours: n = 16-27; 48 hours and 72 hours: n = 6 or 7). (B) Reduced PMN infiltrates, tissue necrosis, and edema in ear tissue from TNFR1^−/− mice 24 hours after TNCB challenge. Hematoxylin and eosin–stained ear sections from TNFR1^+/+ (left: top represents overview; bottom represents detail) and TNFR1^−/− mice (right: top represents overview; bottom represents detail; n = 13-15). (C) PMN recruitment is TNFR1-dependent. MPO activity in protein extracts from ear tissue from TNFR1^−/− and TNFR1^+/+ mice (n = 3).

Figure 3

Only TNF^+/+ mast cells reconstitute PMN recruitment in Kit^W/Kit^W-v ears. MPO activity in protein extracts of ear tissue from Kit^W/Kit^W-v mice reconstituted with either TNF^−/− () or TNF^+/+ (■) mast cells. Ear tissue was harvested 24 hours after TNCB challenge from sensitized and naive Kit^W/Kit^W-v mice (n = 2).

Figure 4

TNF^+/+ mast cells reconstitute DTHRs in TNF^−/− ears but not in TNFR1^−/− ears. (A) Representative visualization of PMN infiltrates, tissue necrosis, and edema. Ear sections from TNF^−/− (top left), TNFR1^−/− (top right), mast cell-reconstituted TNF^−/− mice (bottom left), and TNFR1^−/− mice (bottom right) were fixed 24 hours after ear challenge and then stained with hematoxylin and eosin. (B) Hapten-specific DTHRs in C57BL/6 wild-type, TNF^−/−, TNFR1^−/−, mast cell–reconstituted TNF^−/−, or mast cell–reconstituted TNFR1^−/− mice. Ear swelling was measured 24 hours after TNCB challenge (n = 4-6). (C) Confocal microscopy of intracutaneously engrafted Cy5 (left) and red fluorescent protein (right) stained mast cells in ears of TNFR1^−/− mice 5 days after mast cell engraftment (top represents overview; bottom represents detail).

Figure 5

Altered adhesion molecule expression and reduced leukocyte adhesion in TNFR1^−/− mice. (A) mRNA expression of TNFR1 in mouse heart ECs, brain ECs, and mouse ear (data were normalized to aldolase expression; relative mRNA expression was calculated by dividing TNFR1 mRNA expression of TNFR1^+/+ mice, mouse heart ECs, and brain ECs though the background TNFR1 mRNA in TNFR1^−/− mice, corresponding to the detection limit, set as 1). (B) mRNA expression of P-selectin, E-selectin, ICAM-1, and VCAM-1 in TNFR1^−/− mice and TNFR1^+/+ mice, 4 hours after elicitation of DTHR (data were normalized to aldolase expression). Data are given in a decade log scale (n = 9 or 10). (C) Immunofluorescence staining of P-selectin, E-selectin, ICAM-1, and VCAM-1 (red) 2.5 hours and 4.0 hours after TNCB challenge in TNFR1^+/+ and TNFR1^−/− mice (green represents nuclei; blue represents type IV collagen; n = 3). (D) Leukocyte adhesion to vascular endothelia in TNFR1^+/+ and TNFR1^−/− mice 3.5 hours after elicitation of DTHR as determined by intravital fluorescent microscopy (n = 6 or 7). (E) Noninvasive intravital microscopy images of leukocyte adhesion to vascular endothelia in TNFR1^+/+ and TNFR1^−/− mice, 4.5 hours after elicitation of DTHR (supplemental Videos 1,2). (F) Leukocyte rolling and (G) firm adhesion of rhodamine-stained leukocytes to vascular endothelia in TNFR1^+/+ mice after application of P-selectin, E-selectin, ICAM-1, and VCAM-1 blocking Abs or istotype control 2.5 and 3.5 hours after elicitation of DTHR. Analysis was performed by intravital fluorescent microscopy (n = 5-7).

Figure 6

TNFR1 expression is not needed for T-cell priming in vivo. (A) Proliferation of 2.5 × 10⁵ (gray and black bars) or 1.25 × 10⁵ (□ and ) CD4⁺ T cells from either naive TNFR1^+/+ or TNFR1^−/− mice, or primed TNFR1^+/+ or TNFR1^−/− mice were stimulated for 72 hours with 5 × 10⁵ of either unmodified ( and □) or TNCB-modified (■ and ) APCs; [³H] thymidine was added for the final 6 hours. (B) Frequency of IFN-γ–producing TNCB-specific CD4⁺ T cells, 5 × 10⁵ (■), or 2.5 × 10⁵ () T cells from either naive or sensitized TNFR1^+/+ or TNFR1^−/− mice were stimulated on anti–IFN-γ mAb-coated 96-well plates with 5 × 10⁵ either unmodified APCs (, third column) or TNBS-modified APCs (■ and ). After 48 hours of incubation, ELISPOT assay was developed. Control T cells were stimulated with 10 μg/mL concanavalin A and APCs (□); 3 independent experiments.

Figure 7

TNFR1 expression is needed neither for T-cell imprinting nor for T-cell effector functions. (A-D) Proliferation and IFN-γ production by TNFR1^+/+ or TNFR1^−/− Th1 and Tc1 cells. (A-B) TNFR1^+/+ (■) or TNFR1^−/− () TNCB-specific T cells (10⁵) of the indicated origin were stimulated for 24 hours with 5 × 10⁵ hapten-modified APCs. [³H] thymidine was added for the final 6 hours. (C-D) TNFR1^+/+ (■) or TNFR1^−/− () T cells (10⁵) from the indicated origin were stimulated for 24 hours with 5.0 × 10⁵ hapten-modified APCs. Supernatants were harvested after 24 hours, and the IFN-γ content was determined by enzyme-linked immunosorbent assay. (E-F) Efficient DTHRs require TNFR1-expressing resident cells. Th1 or Tc1 cell lines that were either TNFR1^−/− or TNFR1^+/+ were transferred intracutaneously into ears of naive TNFR1^−/− or TNFR1^+/+ mice, 0.5 hours before challenge with TNCB (n = 3-7). Ear swelling was determined 24 hours later.