Platelet, not endothelial, P-selectin expression contributes to generation of immunity in cutaneous contact hypersensitivity

Abstract

Leukocyte extravasation is a prerequisite for host defense and autoimmunity alike. Detailed understanding of the tightly controlled and overlapping sequences of leukocyte extravasation might aid development of novel therapeutic strategies. Leukocyte extravasation is initiated by interaction of selectins with appropriate carbohydrate ligands. Lack of P-selectin expression leads to decreased contact hypersensitivity responses. Yet, it remains unclear if this is due to inhibition of leukocyte extravasation to the skin or due to interference with initial immune activation in lymph nodes. In line with previous data, we here report a decreased contact hypersensitivity response, induced by 2,4,-dinitrofluorobenzene (DNFB), in P-selectin-deficient mice. Eliciting an immune reaction towards DNFB in wild-type mice, followed by adoptive transfer to P-selectin-deficient mice, had no impact on inflammatory response in recipients. This was significantly reduced in wild-type recipient mice adoptively transferred with DNFB immunity generated in P-selectin-deficient mice. To investigate if platelet or endothelial P-selectin was involved, mice solely lacking platelet P-selectin expression generated by bone marrow transplantation were used. Adoptive transfer of immunity from wild-type mice reconstituted with P-selectin-deficient bone marrow led to a decrease of inflammatory response. Comparing this decrease to the one observed using P-selectin-deficient mice, no differences were observed. Our observations indicate that platelet, not endothelial, P-selectin contributes to generation of immunity in DNFB-induced contact hypersensitivity.

Figure 1

P-selectin deficiency leads to a diminished inflammatory response in DNFB-induced contact hypersensitivity. A: Ear swelling evaluated 24 hours after challenge with DNFB of sensitized wild-type (+/+) or P-selectin-deficient mice (P-sel^−/−). Data are from at least eight mice per group; P < 0.001, t-test. B: Representative H&E stained specimen from either wild-type or P-selectin-deficient mice 24 hours after challenge with DNFB. Numbers in the upper right indicate the severity of leukocyte infiltration of all mice investigated as determined by a semiquantitative score (ranging from 1: no infiltration to 4: servere infiltration ²³). C: Furthermore, a decrease in lymphocyte, neutrophil, and macrophage expression was noted in mice lacking P-selectin expression. In detail, sections were stained with the markers indicated in the figures as outlined in the Materials and Methods section. Infiltration of respective leukocyte subsets was scored as detailed above. CD3 expression was significantly lower in P-selectin-deficient (1.8 ± 0.3) compared with wild-type mice (2.8 ± 0.3, P = 0.008). Likewise, a notable lower expression of neutrophils and macrophages was observed in mice lacking P-selectin expression (Neu: 2.6 ± 0.3 (wild-type)/2.1 ± 0.1 (P-sel^−/−), P = 0.04; F4/80: 2.6 ± 0.4 (wild-type)/1.3 ± 0.1 (P-sel^−/−); t-test, n = 3 mice per group).

Figure 2

P-selectin is required for generation of immunity, but is not involved in extravasation of leukocytes to the skin. Wild-type (+/+), L-selectin-deficient (L-sel^−/−), and P-selectin-deficient (P-sel^−/−) mice were sensitized with DNFB. Five days after DNFB-exposure, mice were sacrificed and lymphocytes were isolated from peripheral lymph nodes and spleens. Isolated cells were injected i.v. into recipient wild-type mice, which were subsequently exposed to DNFB on the right ear. Twenty-four hours later, ear swelling response and leukocyte infiltration was evaluated in recipient mice. Ear swelling response 24 hours after exposition to DNFB in recipient wild-type mice: As previously described, L-selectin significantly contributes to sensitization in the model of DNFB-induced cutaneous contact hypersensitivity, as L-selectin deficiency leads to reduced rolling interactions of lymphocytes with order I-III venules in lymph nodes. We here however provide evidence that P-selectin also is critically involved in generating immunity toward DNFB. By comparing the relative contribution of L- and P-selectin in this process, the contribution of P-selectin seems more detrimental (analysis of variance on ranks—multiple comparisons according to the Dunn‘s method).

Figure 3

Reduced ear swelling response in wild-type mice after adoptive transfer of leukocytes from DNFB sensitized P-selectin-deficient mice. 24 hours after adoptive transfer and challenge with DNFB, ears were homogenized. From homogenates total cell number was counted, and subsets of infiltrating leukocytes were evaluated using flow cytometry. As indicated below, total cell number is reduced in the ears of mice, which received lymphocytes from P-selectin-deficient mice. This reduction in infiltration leukocytes is due to decreased numbers of CD8+ cells and neutrophils. Data shown is from three mice. Statistical evaluation was performed using the t-test.

Figure 4

Primarily platelet, not endothelial, P-selectin is required to generate immunity. Using BMT mice as donors for adoptive transfer experiments into wild-type C57Bl/6 mice, ear swelling responses were evaluated. Lack of platelet P-selectin expression had a similar effect as mice fully deficient in P-selectin expression. Hence, platelet P-selectin expression seems more crucially involved in generating immunity than endothelial P-selectin. Data from at least six mice per group; statistical comparisons performed using t-test.