Richard Rudick, M.D. | Research | Publications | Laboratory

Research

Clinical Approach Tests Interferon, CNS Antigen Impact on Thwarting MS

Work in our lab has focused on response to immunotherapy in multiple sclerosis (MS), and autoreactivity to CNS antigens in the disease.

In the first project, we have investigated response to interferon (IFN) b-1a treatment in MS patients, to understand the mechanisms of IFNb action. We have hypothesized that IFNb has an anti-inflammatory effect in the CNS. This was formally tested in a phase III study of IFNb-1a by analyzing cerebrospinal fluid from patients before and after treatment. 262 subjects had lumbar puncture at entry into the trial, and 137 of these subjects had repeat LPs after 2 years of therapy. There was a significant association between CSF WBC counts at baseline and gadolinium lesions at baseline and between CSF WBC counts at baseline, and subsequent clinical and MRI disease activity in placebo recipients. IFNb-1a treatment significantly reduced CSF WBC counts, confirming that IFNb has an intrathecal anti-inflammatory effect. Studies to investigate the mechanisms underlying this include:

  • Following both the 1st and 5th weekly IFNb-1a injection, there was a significant increase from baseline serum IL-10 concentrations (p<0.001 at 48 hours, 96 hours, and 168 hours).
  • Analysis of PBMC cytokine specific RNA by RT-PCR showed significant upregulation in IL-10 and IL-4, a trend for increased IL-5, but no significant change in IFNg or TNFa message.
  • CSF analysis from the phase III study patients showed significantly increased IL-10 concentration (p<0.001) together with a trend toward reduced levels of IL-2 and b-2 microglobulin. CSF/serum IL-10 ratios increased with IFNb-1a treatment, suggesting intrathecal IL-10 synthesis accounted in part for the CSF findings.

There was a statistically significant relationship between a favorable clinical response to therapy and increased CSF IL-10 levels. The results document in vivo systemic and intrathecal induction of immunosuppressive cytokines by IFNb-1a in relapsing MS patients, suggest that this response is linked to the therapeutic effect of IFNb, and provide one mechanism for the observed effects on MRI lesion enhancement and CSF pleocytosis.

The second project ongoing in the lab is aimed at identifying patterns of cytokine responses to myelin determinants in MS patients. Specific questions include:

  • Which determinants are targeted at a given time in the course of the disease and do these change over time?
  • What is the clonal size of T cells targeting that determinant?
  • What is the effector function of the T cells that are targeting the determinant as assessed by cytokine production (Th1 or Th2)?
  • What are the MHC class I or class II restriction elements of the effector T cells that respond to peptides of MBP and PLP? Do MBP or PLP peptide-reactive Th1-type or Th2-type cytokine responses represent mixtures of single-cytokine secreting cells or individual cells that are capable of producing more than one cytokine simultaneously?

In this study, we used the cytokine ELISPOT assay to study proteolipid protein (PLP) peptide reactivity as measured by IFNg and IL-5 secretion in freshly isolated peripheral blood mononuclear cells from MS patients and controls. Using overlapping PLP peptides, MS patients demonstrated 12 times as many multiple-adjacent-positive peptides compared to controls. Comparing the total number of IFNg -positive cytokine-secreting cells, multiple-adjacent-positives were 43 times as high in MS patients as controls. The average number of IL-5 single positives was almost identical between MS patients and controls and no IL-5 multiple-adjacent positives were observed, suggesting that PLP-specific IL-5 responses do not differ significantly between MS patients and controls. Our data suggest that MS patients give a much stronger MHC Class II-restricted immune response to PLP peptides compared to controls, which is characterized by secretion of IFNg, an inflammatory cytokine that has been strongly implicated in the disease process.