Interleukin 10 (IL-10) is released during the induction phase of contact sensitivity and was shown in prior functional studies to convert epidermal Langerhans cells (LC) from potent inducers of primary immune responses to specifically tolerizing cells in vitro. To investigate whether IL-10 also subserves the function of a tolerizing agent in vivo ears of BALB/c or C3H mice were injected intradermally with 1-2 mug of recombinant mouse (rm)IL-10 8 h before epicutaneous application of 3% trinitrochlorobenzene (TNCB; a contact allergen). As a control, mice were injected with phosphate-buffered saline or IL-10 plus neutralizing amounts of anti-IL-10 mAb. 5 d later, mice were challenged with 1% TNCB on contralateral ears and ear swelling response was measured 24 h later. Whereas control-treated mice showed a normal ear swelling response to epicutaneous challenge (DELTA mm-2 = 25 +/- 5), ear swelling response of IL-10-treated animals was significantly inhibited (DELTA mm-2 = 3 +/- 2). Coinjection of IL-10-specific mAb together with rmIL-10 completely abrogated this effect. To differentiate between a state of nonresponsiveness and induction of tolerance by IL-10, mice initially treated with IL-10 and TNCB were resensitized with 3% TNCB in the absence of any treatment after 14 d of rest (group 1). Again mice were challenged 5 d later and ear swelling responses were tested. Whereas control mice treated with allergen alone (group 2) showed a good swelling response (DELTA mm-2 = 28 +/- 6), IL-10-treated mice (group 1) showed a minimal response towards application of allergen (DELTA mm-2 = 4 +/- 2). To show that anergy induction by IL-10 was antigen-specific, mice initially treated with IL-10 plus TNCB were exposed to 0.5% dinitrofluorobenzene (DNFB) 14 d later (group 1). After challenge with 0.1% DNFB, IL-10-treated mice showed an ear swelling response (DELTA mm-2 = 13 +/- 3; group 1) simILar to that of control mice only sensitized with DNFB (DELTA mm-2 = 14 +/- 3; group 3). In an attempt to show the induction of antigen-specific tolerance in these mice in vitro, regional lymph nodes of mice initially treated with TNCB plus IL-10 (group 1) and control-treated mice (groups 2 and 3) were prepared and cultured in the presence of TNBS, dinitrobenzene sulfonate (DNBS), or medium to measure antigen-specific proliferation. Lymph node cells from animals sequentially treated with IL-10 plus TNCB and afterwards DNFB in the absence of IL-10 (group 1) showed a low, but significant proliferation (approximately 15,000 cpm) to DNBS, but only background proliferation (approximately 3,000 cpm) to TNBS, or medium. In contrast, lymph node cells from animals treated with TNCB or DNFB in the absence of IL-10 (groups 2 and 3) proliferated to the sensitizing agent, but not control allergens. To elucidate the mechanism of action of IL-10, the epidermal cytokine pattern was analyzed on the mRNA level after injection of IL-10 or controls and application of allergen. Injection of IL-10 (but not controls) significantly impeded the induction of proinflammatory cytokines IL-1beta, tumor necrosis factor alpha and IL-1alpha. In aggregate our data indicate that in vivo application of IL-10 before allergen treatment induces antigen-specific tolerance in mice and that IL-10 might act via inhibition of proinflammatory cytokines.
