In vitro effects of Epstein-Barr virus on peripheral blood mononuclear cells from patients with rheumatoid arthritis and normal subjects.

Abstract

There are two reasons for studying the possible role of the Epstein-Barr virus (EBV) in relation to rheumatoid arthritis (RA). The first is the recent demonstration by Alspaugh and co-workers that EBV infection of normal peripheral blood lymphocytes induces the development in these cells of a nuclear antigen against which most patients with RA have precipitating antibodies (I). The second reason is that EBV is a naturally occurring, polyclonal activator of antibody production by human B lymphocytes (2, 3).In the present experiments, we have asked to what extent RA and normal subjects have B lymphoeytes which can be induced by EBV to secrete anti-IgG antibodies. At the same time, we have determined the degree to which lymphoeytes from RA patients spontaneously transform in the absence of EBV infection. EBV nucleic acid can be detected in most permanent lines of human B cells (4). In most cases, therefore, EBV plays an essential role in the in vitro transformation of human B cells to permanent lines. If RA lymphocytes undergo morphologic transformation and become permanent lines more readily than do normal lymphocytes, one might wonder whether or not they either contain more EBV genomes per cell than normal lymphocytes, or are abnormally responsive to EBV infection. Materials and MethodsSource. 50 ml of heparinized blood was obtained from I 1 patients with seropositive RA. These patients met the American Rheumatism Association criteria (8) for diagnosis and were all currently under treatment at the Scripps Clinic and Research Foundation. 10 other samples of the same volume were obtained from healthy persons having no prior diagnosis of a rheumatic disorder and using no medications.Virus. EBV was obtained from the B95-8 marmoset lymphoblastoid line, processed as described by and frozen until use (6,7).Cell Separation and Culture Procedure. Mononuclear cells were isolated from 50 ml heparinized blood by Ficoll-Hypaque sedimentation (8) followed by five washes in serum-free medium, and resuspension in RPMI-1640 and 10% fetal calf serum (Flow Laboratories, Inc., Roekville, Md.) at a concentration of 10 7 eells/ml. 1 ml of EBV was used to infect 1 × 107 cells. An equal volume of RPMI-1640 with 10% fetal bovine serum was added to duplicate control cells. Cells * Supported by National Institutes of Health grants AM 07144, AM 21175, GM 23200, AM 00369, RR 05514, and RR 00833. J. ExP. MED.

Analysis of immunoglobulins secreted by hybridomas derived from rheumatoid synovia

“…Another report of B cell hybridomas derived from rheumatoid synovium (Randen et al, 1989) illustrates that monoclonal RFs can be readily derived from this tissue. However, their methods included a 2-5 week stimulation with Epstein-Barr virus (EBV) prior to fusion and since EBV stimulation can induce RF in vitro (Slaughter et al, 1978) the higher frequency of RF-secreting hybridomas from RA synovium is not therefore directly comparable with our results which are based on fusion of cells that have been activated in the synovium as part ofthe disease process.…”

Restricted heterogeneity of polyclonal rheumatoid factors

“…Furthermore, RF molecules are known to share common idiotypes, as do experimental homogeneous antibodies. Alternatively, RF can also be compared with the oligoclonal antibodies of neurologic disorders since they are autoantibodies and since their synthesis can be initiated in vitro by a viral infection (21). Moreover, RF is partly synthetized in the synovium (22), and the local B lymphocytes were shown to be oligoclonal (23).…”

High levels of in vitro igm rheumatoid factor synthesis correlate with hla–dr4 in normal individuals

“…From this observation, it follows that the capability to synthesize IgM-RF is not in itself pathogenic. However, circulating cells that spontaneously synthesize this autoantibody are seen only in RA patients (9,12). Thus, it may be that HLA-DR4 or a closely linked gene has its effect at the level of control of RF-specific B cell activation.…”