Calpain secreted by lymphoid (MOLT-3, M.R.) or monocytic (U-937, THP-1) cell lines activated with PMA and A23187 degraded myelin antigens. The degradative effect of enzymes released in the extracellular medium was tested on purified myelin basic protein and rat central nervous system myelin in vitro. The extent of protein degradation was determined by SDS-PAGE and densitometric analysis. Various proteinase inhibitors were used to determine to what extent protein degradation was mediated by calpain and/or other enzymes. Lysosomal and serine proteinase inhibitors inhibited 20-40% of the myelin-degradative activity found in the incubation media of cell lines, whereas the calcium chelator (EGTA), the calpain-specific inhibitor (calpastatin), and a monoclonal antibody to m calpain blocked myelin degradation by 60-80%. Since breakdown products of MBP generated by calpain may include fragments with antigenic epitopes, this enzyme may play an important role in the initiation of immune-mediated demyelination.

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Granulocyte colony stimulating factor (G-CSF) regulates survival, proliferation, differentiation, and activation of myeloid cells. It binds to a high affinity receptor (G-CSF-R) expressed on myeloid cells, for which the signal transduction mechanisms other than protein tyrosine kinase (PTK) activation have not been completely identified. We explored the potential involvement of protein kinase-C (PKC) in G-CSF-R signal transduction. In this report, we provide direct evidence of PKC activation by G-CSF-R. G-CSF treatment of peripheral blood neutrophils, granulocytic cell lines (HL-60, NFS-60, KG-1), and monocytic cell lines (WEHI-3B,U-937) resulted in PKC activation. Chelerythrine chloride and HA-100, an isoquinolinesulfonamide derivative, the specific inhibitors of PKC, 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA), a chelator of intracellular calcium, and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester (TMB-8), an inhibitor of intracellular calcium release, blocked G-CSF-induced PKC activation in HL-60 cells, and reduced CD11b upregulation in neutrophils, but did not affect ligand-binding or down-modulation of G-CSF-R. Methyl 2,5-dihydroxycinnamate (MDHC), a potent inhibitor of protein tyrosine kinases (PTK), also inhibited PKC activation in response to G-CSF treatment, suggesting that PKC activation may occur downstream of PTK activation. Our results demonstrate the involvement of PKC in G-CSF-R signal transduction, and suggest a common signaling pathway in myeloid cells of granulocytic and monocytic lineages.

Calpain, a calcium-activated neutral proteinase, is ubiquitously present in human tissues. To determine if lymphoid cells implicated in pathogenesis of demyelination may harbor calpain in a functionally active form, we determined both muCalpain and mCalpain activities in human lymphoid cell lines. DEAE-cellulose and phenylsepharose column chromatography were used to isolate the enzyme from the natural inhibitor, calpastatin. Lymphocytic lines (CCRF-CEM, MOLT-3, MOLT-4, M.R.) showed predominance of muCalpain (55-80%) whereas the monocytic line (U-937) showed predominance of mCalpain (77%). Proportion and subcellular distribution of both isoforms varied among cell lines. Calpains isolated from U-937 cells degraded myelin basic protein. These results indicate that human lymphoid cells harbor functionally active calpain that can degrade myelin components in vitro. The study suggests a degradative role for calpain in demyelinating diseases.