Our laboratory has previously reported the isolation of a serum blocking factor (SBF) from infectious mononu- cleosis (IM) patients. The SBF has been purified by ...
0022-1 767/01/1271-0320$02.00/0 THE JOURNAL OF IUMUNOLOOV Copyright 0 1981 by The Williams (L Wilkins Co
Vol. 127, NO.1 , July 1981 Printed in U.S . A.
BIOLOGIC AND MOLECULAR CHARACTERIZATION OF THE IgG SERUM BLOCKING FACTOR (SBF-IgG) ISOLATED FROM SERAOF PATIENTS WITH EBV-INDUCED INFECTIOUS MONONUCLEOSIS' H. WAINWRIGHT, AND PHILIP M. SPRINKLE
ROBERT W. VELTRI,VALERIEANNKIKTA,WILLIAM
From the Departmentsof Otolaryngology and Microbiology, West Virginia University Medical Center, Morgantown, WV26506; a n d the Wyeth iaboratories, Inc., Marietta, PA 7 7547
Our laboratory has previously reported the isolation of Mangi etal. (10)demonstrated increased numbers of Bcells during the 1st week of IM, and a decline to normal numbers within 3 wk. a serum blocking factor (SBF) from infectious mononucleosis (IM) patients.The SBFhas beenpurified by a Hence, early in the disease there is a B cell proliferation, probably reflecting the replication of the infectedB lymphocyte series (5, 6), combinationofSephadexQAE-50ionexchangeand Sephadex G-200 molecular sieve chromatography. This followed by increased proliferation of T cells later in IM, reflecting an active response to the infection (1 0-1 2, 16, 17). material was found to be devoid of soluble immune com- However, in spite of these cellular immunologic dynamics, paplexes,andimmunochemicallyandbiochemicallywas tients with IM demonstrate a marked but transient depression in characterized as IgG and, hence, termed SBF-IgG. The cell-mediated immunity (CMI) (10, 12, 18-21). Two reports have SBF-IgG was shown to significantly (a = 0.05) suppress demonstrated the presence ofa IgG-like serum blocking factor antigen specific (InfluenzaA-l[HcN1])in vitro lymphocyte (SBF) capable of abrogating cell-mediated effector immune mechstimulation (LS) as well as leukocyte migration-inhibitionanisms (19, 21). We previously characterized the possible IgG (LMI)reactivity. Also, the SBF-IgGsignificantly sup- nature of the blocking factor(1 9); however, since patients with IM pressed the in vitro.LS response to phytohemagglutinin. also process circulatingsoluble immune complexes, further studies were required. Our laboratory reported on the further biochemical Inaddition,theSBF-lgGwhenboundtonormaldonor lymphocytes significantly reduced the high affinity E-ro- and immunochemical analysis of the SBF at the 64th Annual Meeting of the Federation of the American Society for Experimental sette (HAR) reactivity at 29°C. A purified T lymphocyte Biology in Anaheim, CA (22). This manuscript describes our bio; subpopulation of normal donor lymphocytes specifically chemical, immunochemical, and biologic characterization of the bound SBF-lgG, and the latter could be recovered using IgG SBF isolated from IM patients. glycine-HCI. It appearsthat SBF-IgGis anonspecific antibody; it binds neither lymphokines nor specific antiMATERIALSAND METHODS gen, but apparently elicits its in vitro cell-mediated suppressive effect at the level of the T lymphocytes. Study population. The SBF activity of 5 patients admitted to the West Primary infection with Epstein-Barr virus (EBV)' may manifest itself as a simple self-limiting heterophil-positive (1) or heterophilnegative case (2) of infectious mononucleosis (IM). Occasionally the IM may progress to a fatal disseminated disease process (3, 4). Also, the virus is notorious for assuming a permanent latent virus-host cell complex in man (1). The primary site of localization of latent EBV appears to be the B lymphocyte (5-7). Reactivation of such latently harbored EBV provide another source ofEBV infections and clinical diagnostic problems (8, 9). The immunologic impact of an acute EBV infection on the host is rather profound. There is a large increase in the number of T cells, which tends to be sustained into convalescence (1 0-1 2). The atypical lymphocyte, which is consideredto bea Tlymphocyte undergoing blastogenesis, is usually elevated early in the IM syndrome (13-15). Using sequential analysis of total T and B cells, Received for publication January 9,1981. Accepted for publication April 7. 1981. The costs of publication of this article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Thisworkwas supported by the West Virginia University Otolaryngology Foundation for Cancer Research. Abbreviations used in this paper: BLMI. blocking leukocyte migration inhibition; CMI. cell-mediated immunity; EBV. Epstein-Barr virus; EBV-EA. EpsteinBarr virus early antigen; F1, InfluenzaA-1 (H,N,) subunit vaccine; FITC. fluorescein isothiocyanate; HAR. high-affinity rosette; IC. immune complex; IM. infectious mononucleosis; LMI. leukocyte migration inhibition; LS. lymphocyte stimulation; MNL. mononuclear lymphocytes; NDL. normal donor lymphocytes; NHS, normal human serum: NTE, neuraminidase-treated sheep erythrocytes; PES-H. PBS with heparin (1 U/1 ml): PHA. phytohemmagglutinin-P: PMN. polymorphonuclear leukocytes; SEF, serum blocking factor; SEF-IgG. IgG fraction of serum blocking factor; SNL, sensitized normal lymphocytes; SPA, staphylococcal protein A; VCA, viral capsid antigen.
Virginia University Student Health Service with clinically diagnosed and hematologically confirmed IM was studied. The criteria for diagnosis of IM included clinicalfeatures, atypical lymphocytes in the bloodsmear, and the presence of heterophil antibody (23). None of the patients was receiving medication to cause depression ofCMI. Five healthy graduate students volunteered to serve as control subjects. These normal donors had been previously exposed toEBV as evidenced by titers of preexisting antibody to EB viral capsid antigen (VCA). All controlsubjects were enjoying good health at the time of the study. Preparation ot antigen. A 1 :160 dilution of commercially prepared Influenza A-1 (HI, N,) subunit vaccine (F1 ; Wyeth Labs, Marietta, PA) as well as previously reported tetanus toxoid (19) were used as specific recall antigens. This dilution was determined to elicit an optimal in vitro immune response from normal lymphocytes without being cytotoxic. The mitogen phytohemagglutinin-P (PHA; Difco, Detroit, MI) was used at a concentration of 20 &ml. Preparation of leukocytes. Peripheral blood leukocytes from an antigensensitive donor were obtained by Dextran T-500 (Pharmacia, Piscataway, NJ) sedimentation. Fiftymilliliters of whole blood were diluted1:2 with phosphate-buffered saline, 0.1 M (PES). pH 7.2, containing heparin (1 U/ ml) (PBS-H) and were incubated at 37°C for 45 min. The leukocyte-rich plasma was removed by aspiration and diluted 1 :4 in PBS-H, then was washed 3 times with this buffer. The cells were resuspended in RPMl 1640 tissue culture medium at a final concentration of 2 x 10' cells/ml after the final wash. Direct assayof leukocyte migration inhibition(LMI). The direct assay for Horvat human LMI factorwas performed accordingto McCoy etal. (21) and e t a/. (%).The LMI index was calculated from the formula: average area of migration with antigen/average area of migration without antigen. An LMI (>20% inhibition) was considered significantfor the inhibiindex of
