The role of platelets in leukocyte recruitment in chronic contact hypersensitivity induced by repeated elicitation

Abstract

Platelets have been shown to be important in inflammation, but their role in chronic allergic dermatitis remains unclear. To investigate the role of platelets in a mouse model of chronic contact hypersensitivity induced by repeated elicitation, mice were sensitized and repeatedly elicited in ears with hapten, with or without platelet depletion, by administering antiplatelet antibody or busulfan. Ear thickness, leukocyte infiltration, serum IgE, and scratching behavior significantly decreased in thrombocytopenic mice. cDNA microarray of ear tissue showed reduced gene expression associated with Th2 lymphocytes. Flow cytometry showed increased P-selectin expression on platelets and an increased number of platelet-leukocyte aggregates in blood of repeatedly elicited mice, compared with sham-sensitized mice. In thrombocytopenic mice, inflammation was restored by platelet infusion, which was blocked by platelets from P-selectin-deficient mice or by pretreating platelets with anti-P-selectin antibody. Moreover, injection of activated platelet supernatant into ears led to increased leukocyte infiltration, which was blocked by pretreating platelets with antiplatelet compounds or neutralizing several chemokines in the platelet supernatant. These results suggest that platelets induce leukocyte recruitment into skin by forming platelet-leukocyte aggregates via P-selectin in blood and secreting chemokines at inflamed sites. Therefore, controlling platelet activity may be useful for treatment of chronic allergic dermatitis.

Figure 1

Platelet levels in peripheral blood of mice during chronic busulfan or APAS administration. A: Schemes showing the timing of busulfan (top panel) and APAS (bottom panel) administration and repeated hapten application. B: Mice were depleted of platelets by intraperitoneal busulfan administration. C: Mice were depleted of platelets for up to 72 hours after the first injection of APAS. D: Platelet depletion was maintained with APAS administration every 3 days over the chronic period. Data are expressed as the mean platelet number ± SD of five mice per group. **P < 0.01.

Figure 2

Increase in ear thickness following repeated application of TNCB. TNCB was repeatedly applied to the ears of mice at 2-day intervals for 8 weeks with administration of busulfan or vehicle (A) or APAS or CS (B). Repeated application of acetone/olive oil alone served as a control. Ear thickness was measured before challenge every 7 days. The increase in ear thickness is defined as the difference between ear thickness on day 0 and that on days 7, 14, 21, 28, 35, 42, 49, and 56. Data are expressed as the mean ± SD of five mice per group. **P < 0.01 versus vehicle or CS administration in repeatedly elicited mice.

Figure 3

Time course of ear swelling responses after application of TNCB. TNCB was repeatedly applied to the ears of mice at 2-day intervals for 8 weeks with busulfan or vehicle administration. Repeated application of acetone/olive oil alone served as the control. Ear swelling responses were determined before and at 6-hour intervals after challenge on days 0 (A), 7 (B), 14 (C), 28 (D), and 56 (E), as the difference in ear thickness before and after elicitation. Data are expressed as the mean ± SD of five mice per group. **P < 0.01 versus vehicle administration in repeatedly elicited mice.

Figure 4

Histology in the ears of busulfan-treated mice and controls. Skin tissue was excised 6 hours after the final application of TNCB with busulfan or vehicle administration, and formalin-fixed/paraffin-embedded sections were stained with hematoxylin and eosin. Repeated application of acetone/olive oil alone served as the control. A and E: Repeated application of acetone/olive oil alone with vehicle administration. B and F: Repeated application of TNCB with vehicle administration. C and G: Repeated application of acetone/olive oil alone with busulfan administration. D and H: Repeated application of TNCB with busulfan administration. Original magnifications: A–D, ×100; E–H, ×400. Scale bar = 50 μm. The numbers of CD4⁺ T cells, CD8⁺ T cells, eosinophils, and mast cells in the dermis per 10 high-power fields were counted (I). Data are expressed as the mean (per mm²) ± SD of six mice per group. **P < 0.01.

Figure 5

cDNA microarray analysis of gene expression in the ears of busulfan- and APAS-treated mice and controls. mRNA was prepared and hybridized with a GEArray S Series Mouse Autoimmune and Inflammatory Response Gene Array, as described in Materials and Methods. Gene expression levels in busulfan- and APAS-treated mice are represented as a gradation of red, and those in the controls are represented as a gradation of green. Genes expressed commonly in both appear in yellow (n = 10 mice per group).

Figure 6

Effects of platelet restoration on ear swelling responses and leukocyte recruitment in the skin. Washed platelets were isolated from blood of repeatedly elicited mice and sham-sensitized mice. Busulfan-treated mice were injected intravenously with platelet suspension or vehicle before the final challenge. A: Ear swelling responses were determined before and at 6-hour intervals after challenge. B: The skin tissue was examined 6 hours after challenge. The numbers of CD4⁺ T cells, CD8⁺ T cells, eosinophils, and mast cells in the dermis per 10 high-power fields were counted. Data are expressed as the mean (per mm²) ± SD of six mice per group. **P < 0.01 versus mice injected with vehicle.

Figure 7

Platelet P-selectin is required for cutaneous leukocyte recruitment in chronically challenged mice. Blood was obtained from repeatedly challenged or sham-sensitized mice 6 hours after the final challenge and analyzed for P-selectin expression on platelets (A) and platelet-leukocyte complexes (B). Data are shown as the percentage of basal values in sham-sensitized mice. In studies of platelet restoration, washed platelets were isolated from P-selectin^−/− mice and control wild-type C57/BL6J mice (C). In some experiments, washed platelets were isolated from normal BALB/c mice and treated with anti-P-selectin blocking antibody or isotype control antibody (D). Platelet suspension or vehicle was injected intravenously into busulfan-treated mice before the final challenge. Skin tissue was examined 6 hours after challenge. The number of infiltrated cells in the dermis per 10 high-power fields was counted. Data are shown as the percentage of the basal number of cells after injection of vehicle. Bars indicate the mean ± SD of five mice per group. *P < 0.05; **P < 0.01.

Figure 8

Effects of platelet supernatant on leukocyte recruitment in the skin. Washed platelets were isolated from blood of normal mice and stimulated with thrombin. In studies using antiplatelet drugs, washed platelets were pretreated with 1 mmol/L aspirin or vehicle before stimulation with thrombin. In some experiments, mice were orally dosed with clopidogrel (30 mg/kg/day) or vehicle for 2 days, and blood was collected 1 hour after the final dose. Washed platelets were isolated and stimulated with thrombin. In further studies, washed platelets were mechanically lysed by means of ultrasonic treatment. The supernatant of platelets fixed without stimulation with thrombin served as the control. A: The levels of MIP-1α, RANTES, and TARC were measured in platelet supernatant and lysates. Data are shown as the percentage of the maximum number of chemokines in platelet lysates. Bars indicate the mean ± SD of four independent experiments. B: Platelet supernatant was injected into the ears of busulfan-treated mice just before the final challenge, after adding anti-MIP-1α, anti-RANTES, anti-TARC, or isotype control antibody to the supernatant. C: The supernatant of platelets treated with aspirin, clopidogrel, or vehicle was injected into the ears of busulfan-treated mice just before the final challenge. Skin tissue was examined 6 hours after challenge. The number of infiltrated cells in the dermis per 10 high-power fields was counted. Data are shown as the percentage of the basal number of cells after injection of fixed platelet supernatant. Bars indicate the mean ± SD of five mice per group. **P < 0.01.