Int. J. Immunopharmac., Vol. 4, No, 1, pp. I-7, 1982. 0192-0561/82/010001-07 $02.00/0 Printed in Great Britain. : 1982 I ntcrnational Societ y for lmmunopharmacolog~,, THE EFFECT OF PROTEASE INHIBITORS ON THE POLYCLONAL B CELL ACTIVATOR FROM THE SERUM OF PATIENTS WITH RHEUMATOID ARTHRITIS* MARIUS TEODORESCU,~" DOINA GANEA, TIEH T. LEE, JOHN L. SKOSEY and GABRIEL Rt3TTER~ Department of Microbiology and Immunology and the Department of Medicine, University of lllinois at the Medical Center, Chicago, IL 60612, U.S.A (Received 20 February 1981 and in final form 22 May 1981) Abstract--It has been reported that polyclonal B cell stimulation results in formation of autoantibodies and immune complexes. We have previously reported that a polyclonal B cell activator (PBA) associated with a,-macroglobulin (a,M) is present in the serum of patients with rheumatoid arthritis and related diseases. Here we studied the-possibility that patient a:M (Pt-azM) carries a trypsin-like protease responsible for the PBA activity. This activity was determined by the Ig-turnover assay developed in our laboratories. The small molecular weight protease inhibitors, aprotinin (Trasylol, Bayer) and phenylmethylsulfonylfluoride (PMSF), and the large molecular weight soybean trypsin inhibitor (SBTI) were used. These inhibitors did not affect the PBA activity of dextran sulfate or LPS. However, as expected, trypsin had a PBA-activity which was blocked by all of the above mentioned inhibitors. A trypsin--normal a2M complex (Tr-Na2M)) had PBA activity which was inhibited by PMSF or aprotinin but not by SBT1. The PBA associated with Pt-a2M was also inhibited by PMSF or aprotinin but not by SBTI. Moreover, the Tr-Na2M complex and the Pt--a,M, but not that from normal donors, had esterase activity for p-toluenesulfonyl-L-argininemethyl ester. Tl~ese data suggest a similarity between the Pt-a,M and Tr-Na:M complex. Thus, we concluded that the esterolytic activity is sufficient for PBA activity, that Pt--a,M has esterolytic activity and that this PBA activity can be blocked by small molecular weight protease inhibitors. In vitro stimulation of B cells with polyclonal B cell activators (PBA) results in the production of auto- antibodies (HammarstrOm, Smith, Primi & MOiler, 1976). Also, when mice are inoculated i.v. with PBA, autoantibodies and immune complexes develop (FourniG Lambert & Miescher, 1974; Fishbach, Roubinian & Talal, 1978). These experiments, as well as the observation that peripheral blood lymphocytes from patients with systemic lupus erythematosus are polyclonally activated (Budman, Merchant, Stein- berg, Daft, Gershwin, Lizzio & Reeves, 1977), sug- gested that polyclonal B cell activation may be in- volved in the pathogenesis of autoimmune diseases (see Fauci, 1980). We have recently investigated the serum of patients with rheumatoid arthritis and related diseases for the presence of a PBA in their serum. We have found that over 90°70 of the patients with active disease had PBA in their serum, PBA associated exclusively with serum a: macroglobulin (a.,M) (Teodorescu, Chang & Skosey, 1981). These results suggested that the a:M associated PBA may be involved in the pathogenesis of autoimmune diseases (Teodoreseu, 1981). In the present work, we attempt to determine the nature of the PBA associated with a:M. We started from two pieces of information: (a) proteolytic en- zymes act as PBA and can replace the helper factor in antibody formation (Gisler, Vischer & Dukor, 1976) and (b) normal a2M (Na:M) is known to bind proteo- lyric enzymes resulting in the inactivation of their proteolytic activity but not of their esterolytic func- tion (Barrett & Starkey, 1973). Hence, we put forward two hypotheses: (a) that the esterolytic acti- vity of the enzymes complexed with a2M is sufficient to cause polyclonal B cell activation and (b) that patient a2M (Pt-a:M) is similar to trypsin-Na2M complex (Tr-N%M). If these were true, Tr-N%M was expected to have PBA activity and the small * Supported by the NCI Grant CA No. 29552, NIH Grant AM 26094, and by a grant from the Illinois Chapter of the Arthritis Foundation. Presented in parl at the Southern Regional Meeting of the Arthritis Foundation, Charleston, South Carolina, December 1980. t Correspondence and requests for reprints to: M. Teodorescu, Department of Microbiology and Immunology, University of Illinois at the Medical Center, Chicago, IL 60612, U.S.A. Senior Investigator in the Heinrich Pette Institute of Virology, Hamburg University, Hamburg, West Germany. 1