Upregulation Of Cytokine mRNA Research Articles

The role of CD4 molecules in the induction phase of contact hypersensitivity cytokine profiles in the skin and lymph nodes

We have previously demonstrated that CD4 gene-targeted mutant mice (CD4- mice) demonstrate hyporesposiveness in contact hypersensitivity (CHS) suggesting that CD4 molecules are required for optimal induction of CHS. In the present study, we wished to examine the mechanisms of this hyporesponsiveness, in particular, we examined whether cytokines were altered in the skin and lymph nodes of CD4- mice following exposure to the contact allergen dinitrofluorobenzene (DNFB). Cytokine mRNA in the ear skin and draining lymph nodes (DLN) were examined by reverse transcription-polymerase chain reaction (RT-PCR) at various times after sensitization. Skin cytokine patterns revealed that in normal mice, interleukin (IL)-2, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha mRNA levels increased at 12 hr sensitization, whereas these cytokines were below the level of detection in CD4- mice. In the DLN of normal mice following the hapten application, sequential upregulation of cytokine mRNA including IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-10, IFN-gamma and TNF-alpha was found. No change was seen for IL-1 alpha, IL-1 beta, IL-10 and TNF-alpha and IL-2, IL-4 and IFN-gamma mRNA levels were below the level of detection in DLN from CD4- mice following the hapten application. However, IL-1 beta, IL-2 and TNF-alpha mRNA levels of lymph node cells from CD4- mice could be upregulated by phorbol myristate acetate in vitro. Flow cytometry study has revealed that the number of Langerhans' cells (LC) in DLN of CD4- mice was similar to that of normal mice, thus, inferring that the alterations of cytokine milieu in the ear skin did not have a significant effect on LC migration to DLN. These results suggest that CD4 molecules are crucial for the induction of certain cytokines in the skin and in inducing sequential cytokine signals in DLN required for optimal development of CHS, but that these changes in cytokines do not effect LC migration.

TOLERANCE TO RAT LIVER ALLOGRAFTS

Liver allografts in the fully allogenic combination of LEW donor liver to DA recipient (LEW-->DA) are spontaneously tolerated (TOL) with no requirement for immunosuppression, while DA-->LEW allografts are rejected in 12-15 days (REJ). We investigated the mechanism of tolerance induction by identifying differences between TOL and REJ grafts from day 1 to day 9 after transplantation and in normal livers and syngeneic liver graft controls. Infiltrating cell populations were counted after immunohistochemical staining of liver graft sections. There were occasional minor differences between TOL and REJ grafts in the T cell or CD11b/c+ (monocyte/macrophage/granulocyte) infiltrate. In contrast, there was a major difference in B cell infiltrate between TOL and REJ liver grafts. Membrane IgD+ cells were significantly greater in TOL (1796 +/- 225) versus REJ (569 +/- 281) (P = 0.004) portal tracts, as were B220+ cells (1086 +/- 100 vs. 181 +/- 105, P = 0.0004) and CD45RC+ cells (2317 +/- 456 vs. 597 +/- 194, P = 0.004). IgG1, IgG2a, IgM, and IgD deposition in liver allografts, identified by immunohistochemical staining of tissue sections, revealed no IgG or IgD in normal rat liver and low levels of IgM. Deposition of IgG1 was observed in REJ but not in TOL liver on days 7 and 9. IgM was increased in both TOL and REJ liver and appeared to be associated mainly with hepatocytes in REJ and with infiltrate in TOL liver. There was a parallel increase in IgG1-expressing plasma cells in the spleen and lymph nodes of REJ but not TOL animals. Cytokine mRNA was analyzed by reverse transcription and semiquantitative polymerase chain reaction amplification of liver RNA. Increased levels of IL-2, IL-4, IL-6, IL-10, TNF-alpha, transforming growth factor-beta, and IFN-gamma were observed, with similar levels of expression in TOL compared with REJ liver. Cytokine mRNA in syngeneic grafts was not different from normal except for IL-6 and transforming growth factor-beta, which were increased. There is no major difference in the T cell component of the infiltrate or in the extent of upregulation of cytokine mRNA between TOL and REJ grafts. There is a major difference in the B cell compartment, with more B cells in TOL livers and deposition of IgG1 in REJ grafts.