Degree Name

PhD (Doctor of Philosophy)

Program

Biomedical Sciences

Date of Award

December 1997

Abstract

To date, the cellular mechanisms involved in the progression of diseases characterized by chronic inflammation, such as rheumatoid arthritis (RA), remain largely unknown. However, cell-to-cell contact interactions between CD4+ helper T (Th) cells and monocytes have been implicated in the induction and maintenance of pro-inflammatory cytokine synthesis that is characteristic to the pathogenesis of RA. One such cytokine produced during monocyte-Th cell contact is interleukin (IL)-1 β, a mediator directly involved in the characteristic tissue destruction that occurs in the synovia of individuals with RA. Previous studies in our laboratories have shown that ligation of CD40 on monocytes with CD40 ligand (CD40L) present on activated Th cells induces monocyte IL-1β synthesis and rescues monocytes from apoptosis. These findings suggest a role for CD40 signaling of monocyte activation in the exacerbation and maintenance of chronic inflammatory responses. This dissertation represents efforts to elucidate components of the CD40 signaling pathway critical to monocyte activation and how CD40-mediated signaling events are modulated by the anti-inflammatory cytokines IL-4 and IL-10. Using either monocytes isolated from human peripheral blood or a monocytic cell line (THP-1), cellular kinases and transcription factors activated upon CD40 ligation were examined by western blot analyses and electrophoretic mobility shift assays (EMSA), respectively. CD40-dependent interleukin-1β synthesis in monocytes was abrogated by inhibitors of protein tyrosine kinase (PTK) activity but not by inhibitors of protein kinase C (PKC). The extracellular signal-regulated kinases 1 and 2 (Erk1/Erk2) mitogen-activated protein kinases (MAPK's) were specifically activated upon CD40 ligation, and specific inhibition of Erk1/Erk2 activation diminished IL-10 production in a dose-dependent manner. Both IL-4 and IL-10 reduced Erk1/Erk2 activation and synergized in this effect. Finally, STAT3, a member of the family of transcription factors involved in cytokine signaling, was highly phosphorylated in monocytes treated with IL-10 or with IL-10 and IL-4 in combination but not with IL-4 alone. Together these results suggest that in monocytes (1) CD40-mediated IL-1β synthesis and NF-κB activation require PTK activity, (2) CD40-mediated IL-1β production is critically dependent upon Erk1/Erk2 activity, (3) both IL-4 and IL-10 target the Erk1/Erk2 signaling cascade in the downregulation of IL-1β synthesis, (4) IL-4 and IL-10 have divergent effects on the CD40 signaling pathway in that these cytokines are synergistic with respect to their ability to inhibit CD40-mediated Erk1/Erk2 activation and IL-1β synthesis, and differ in their ability to block CD40-mediated rescue from apoptosis, and (5) STAT3 activation may be directly involved in the downregulatory effects of IL-10 on CD40 signaling. (Abstract shortened by UMI.)

Document Type

Dissertation - unrestricted

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