Tieno Germann

The role of IL-12 in the induction of organ-specific autoimmune diseases

The concept that T cells are subdivided into T helper 1 (Th1) and Th2 subsets was recently extended to suggest that Th1 cells contribute to the pathogenesis of several organ-specific autoimmune diseases, whereas Th2 cells inhibit disease development. Here, Sylvie Trembleau and colleagues examine the role of interleukin 12 (IL-12), a key cytokine guiding the development of Th1 cells, in the induction of autoimmune diseases, and discuss potential immunointervention strategies based on administration of IL-12 antagonists.

Induction of cytokine production in naive CD4+ T cells by antigen-presenting murine liver sinusoidal endothelial cells but failure to induce differentiation toward Th1 cells

BACKGROUND & AIMS Murine liver sinusoidal endothelial cells (LSECs) constitutively express accessory molecules and can present antigen to memory Th1 CD4(+) T cells. Using a T-cell receptor transgenic mouse line, we addressed the question whether LSECs can prime naive CD4(+) T cells. METHODS Purified LSECs were investigated for their ability to induce activation and differentiation of naive CD4(+) T cells in comparison with bone marrow-derived antigen-presenting cells and macrovascular endothelial cells. Activation of T cells was determined by cytokine production. LSECs were further studied for expression of interleukin (IL)-12 by reverse-transcription polymerase chain reaction, and the unique phenotype of LSECs was determined by flow cytometry. RESULTS We provide evidence that antigen-presenting LSECs can activate naive CD62Lhigh CD4(+) T cells. Activation of naive CD4(+) T cells by LSECs occurred in the absence of IL-12. In contrast, macrovascular endothelial cells from aorta could not activate naive CD4(+) T cells. The unique functional characteristics of microvascular LSECs together with a unique phenotype (CD4(+), CD11b+, CD11c+, CD80(+), CD86(+)) make these cells different from macrovascular endothelial cells. Furthermore, LSECs did not require in vitro maturation to activate naive CD4(+) T cells. Most importantly, LSECs failed to induce differentiation toward Th1 cells, whereas conventional antigen-presenting cell populations induced a Th1 phenotype in activated CD4(+) T cells. Upon restimulation, CD4(+) T cells, which were primed by antigen-presenting LSECs, expressed interferon gamma, IL-4, and IL-10, which is consistent with a Th0 phenotype. Exogenous cytokines (IL-1beta, IL-12, or IL-18) present during T-cell priming by antigen-presenting LSECs could not induce a Th1 phenotype, but neutralization of endogenously produced IL-4 during T-cell priming led to a reduced expression of IL-4 and IL-10 by CD4(+) T cells upon restimulation. The addition of spleen cells to cocultures of LSECs and naive CD4(+) T cells during T-cell priming led to differentiation of T cells toward a Th1 phenotype. CONCLUSIONS The ability of antigen-presenting LSECs to induce cytokine expression in naive CD4(+) T cells and their failure to induce differentiation toward a Th1 phenotype may contribute to the unique hepatic microenvironment that is known to promote tolerance.

Identification and induction of human keratinocyte-derived IL-12.

Interleukin 12 is a heterodimeric molecule that serves as a potent co-stimulator enhancing the development of Th1 cells. As one of the classical Th1 cell-mediated responses is contact sensitivity in skin, we wondered whether IL-12 might be produced by epidermal cells and serve as a mediator of this immune response. Using a sensitive, quantitative PCR technique we demonstrate that p35 chain mRNA of IL-12 is produced constitutively by human epidermal cells, whereas p40 chain mRNA can only be detected in epidermis treated with contact allergen, but not epidermis exposed to irritants or tolerogens. Time course studies showed a dramatic induction of IL-12 p40 mRNA 4 h after in vivo allergen treatment reaching peak strength after 6 h. In cell depletion assays we show that epidermal keratinocytes are the major source of this cytokine in the epidermis. This was further supported by analysis of mRNA derived from the human keratinocyte cell line HaCat expressing IL-12 p35 and p40 mRNA upon stimulation. The presence of bioactive IL-12 in supernatants derived from allergen-stimulated epidermal cells was demonstrated by IL-12-specific bioassay. Additional evidence for the functional importance of IL-12 in primary immune reactions in skin was obtained in allogeneic proliferation assays using human haptenated epidermal cells containing Langerhans cells as APC and allogeneic CD4+ T cells as responders. Anti-IL-12 mAb inhibited the proliferation of T cells by approximately 50%. In aggregate our data demonstrate that nonlymphoid keratinocytes are capable of producing functional IL-12 and provide evidence for the functional significance of IL-12 in primary immune responses in skin.

Characterization of the adjuvant effect of IL-12 and efficacy of IL-12 inhibitors in type II collagen-induced arthritis.

A destructive joint disease can be induced in susceptible DBA/1 mice by immunization with type II collagen emulsified with oil and either killed Mycobacterium tuberculosis or IL-12 as adjuvant. Cellular and humoral anti-collagen immune mechanisms appear to be involved in the pathogenesis of arthritis. We have characterized the adjuvant effect or IL-12 in more detail and addressed the question whether mycobacteria might act via the induction of endogenous IL-12. Injections of IL-12 into collagen-immunized DBA/1 mice promoted the development of IFN-gamma-producing CD4+ T cells and strongly upregulated the production of complement-fixing IgG2a and IgG2b antibodies resulting in severe arthritis. Neutralization of IFN-gamma in vivo largely inhibited the increase in antibody synthesis and prevented joint disease in IL-12-treated mice. However, collagen-specific IFN-gamma synthesis by T cells was further enhanced in these animals. Furthermore, IL-12 treatment promoted the development of IFN-gamma-producing T cells but failed to enhance antibody synthesis and to induce arthritis in C57BL/6 or BALB/c mice immunized with collagen in oil. These results indicate that the induction (by IL-12) of a strong collagen-specific T-cell response alone is not sufficient to trigger arthritis. Attempts to show a role for endogenous IL-12 in DBA/1 mice immunized with collagen with mycobacteria as adjuvant gave no reliable results. Whereas anti-IL-12 treatment delayed the onset and ameliorated the disease in some experiments, it failed to do so in other experiments, or, control reagents also had some effect. A slight inhibition of collagen-specific IgG2a synthesis was observed in most experiments in the sera of anti-IL-12-treated mice. Taken together, the results show that exogenous IL-12 can promote arthritis via its direct effect on T cells and its effect on antibody production, which is at least in part IFN-gamma-dependent. On the other hand, whether or not endogenous IL-12 is involved in the adjuvant effect of mycobacteria needs further clarification.

Co-development of naive CD4+ cells towards T helper Type 1 or T helper type 2 cells induced by a combination of IL.-12 and IL-4

Cytokines were found to play a key role in Th cell differentiation. Among them IL-12 was shown to be a potent differentiation factor for Th1 cells, whereas IL-4 is the only known cytokine that promotes the development of Th2 cells. Upon addition of comparable amounts of IL-4 and IL-12 to a primary culture of naive CD4+ T cells activated by immobilized anti-CD3 mAb, it was found that the Th1-inducing capacity of IL-12 is dominated by the Th2-promoting effect of IL-4. However, high amounts of IL-12 (10,000 U/ml) in combination with low amounts of IL-4 (100 U/ml) led to the development of a Th cell population that, upon rechallenge, showed a substantial secondary IFN-gamma (Th1 cytokine) production concomitantly with the production of high amounts of IL-4 (Th2 cytokine). This can be due to the coexistence of Th1 and Th2 cells or to the development of Th0 cells producing a mixed pattern of cytokines. Immunofluorescence double staining of intracellular IL-4 and IFN-gamma in combination with flow cytometry (FACS) revealed that most of the emerging Th cells produced either IL-4 or IFN-gamma. Only a few double producers could be detected. This finding indicates that individual naive CD4+ T cells can differentiate under the same conditions towards Th1 or Th2 cells and implicates that the development of Th1 and Th2 cells is not necessarily mutually exclusive.

Human Keratinocyte-Derived IL-12 Affects LC-Induced Allogeneic T-Cell Responses

Our laboratory has recently defined the cytokines involved in the early induction phase of contact sensitivity that help LC to become more potent APC and that help to direct this classical Thl immune response1,2.

The Host Response to Listeria monocytogenes Mutants Defective in Genes Encoding Phospholipases C(plcA, plcB)and Actin Assembly(actA)

Several genes involved in the determination of Listeria monocytogenes pathogenesis have been identified. Among them, plcA gene encodes phosphatidylinositol‐specific phospholipase C (PI‐PLC), plcB gene encodes a broad‐range phospholipase C (PC‐PLC), and actA encodes a protein contributing to actin assembly in infected cells. The interaction of L. monocytogenes wild type (LO 28) strain and two derivative mutants, plcA− (BUG 206) and actA−/plcB− (LUT 12), with macrophages and T lymphocytes was investigated in a mouse model of listeriosis. Both mutants showed evidence of attenuation. The plcA− mutant, but not the plcB− mutant, expressed an increase in susceptibility to the anti‐listerial activity of macrophages. Both mutants showed a decreased ability to induce IL‐12 production by bone marrow macrophages when co‐stimulated with E. coli LPS or IFN‐γ. In vivo, L. monocytogenes plcA− mutant was found to be a more effective stimulator of T cells than the wild LO 28 strain.