Autogenic thymocytotoxic IgG antibody in normal guinea pigs.

Normal guinea pig serum (GPS) was confirmed to be cytotoxic for guinea pig thymocytes at 4 degrees C. Approximately 50% of the cells were sensitive for the cytotoxic action of GPS (4 h incubation). Binding of IgG and, to a smaller extent, IgM to the thymocyte surface was shown after incubation with GPS. Absorption of GPS with protein A agarose (which removes immunoglobulins), heat inactivation (56 degrees C, which removes complement activity) or treatment with 2-mercaptoethanol (2-me) (which affected binding of IgG and IgM to thymocytes) abolished the cytotoxic activity. After gel filtration chromatography with Sephadex G-150, the cytotoxic activity was found in fractions with molecular weights comparable to that of IgG. This is different from reports of others on natural thymocytotoxic antibodies (NTA) where IgM was found to be the dominant thymocytotoxic antibody. The toxic activity in GPS was readily absorbed by autogenic bone marrow, spleen, lymph node and thymus cells. In contrast to normal thymocytes, mitogen stimulated thymus cells were totally resistant to the cytotoxic activity. In summary, it is suggested that the thymocytotoxic activity in GPS at 4 degrees C is mediated by IgG and complement and is directed against immature thymocytes. The antigenic determinant is also present on cells in other lymphoid organs.

Thymocytotoxic antibodies in synovial fluid.

Synovial fluid (SF), obtained from patients with juvenile rheumatoid arthritis (JRA), osteochondritis, traumatic synovitis and septic arthritis were tested for the presence of thymocytotoxic activity. Such activity, directed against guinea pig thymocytes, was earlier demonstrated in human serum and shown to be mediated by IgM antibodies and a heat labile factor, most likely complement. The cytotoxic activity was demonstrated in all SFs tested, and was abolished by either one of the following procedures: depletion of IgM by use of anti-IgM-antibody coated protein A-Sepharose, or heating to 56 degrees C for 30 min. Activity was regained when samples inactivated by these two procedures were recombined. The cytotoxic SFs produced similar distortion of cell volume distribution of the target thymocytes as previously has been shown with human serum. The findings indicate that the two cytotoxic activities are identical. In most cases the IgM levels and the cytotoxic activity were lower in SF than in normal serum. Although the material does not permit correlation of cytotoxic activity with specific joint conditions, a high SF cytotoxicity was noted in two out of four patients with JRA. The demonstration of IgM-associated thymocytotoxic activity is of interest both in connection with earlier observations of various specific antibodies in SF and in connection with speculations regarding local immune regulatory activity in the joint.

On the nature of natural thymocytotoxic antibodies. A screening in neonatal, young, adult and pathological human sera.

The thymocytotoxic activity of human sera against guinea pig cells was earlier shown to be mediated by IgM and a heat-labile serum factor, presumably complement. It is not known if such natural cytotoxic activity represents background activity of preexisting clones of immunoglobulin-producing cells, cross-reacting antibodies appearing after immunization, physiological immune regulatory molecules, or components of an immune network. We have therefore examined the presence of thymocytotoxic IgM molecules in normal adult and neonatal sera and in a number of diseases which affect the lymphoid and immune system. The thymocytotoxic effect of serum was measured in different dilutions, both directly and after heat inactivation of the sera and supplementation with a standard amount of IgM-depleted serum, which is inactive in itself but provides a fixed amount of the heat-labile cofactor. The cytotoxic IgM was present in various amounts in all sera tested, although in neonates very small amounts were found. No specific aberration in toxic activity was seen in rheumatoid arthritis or a number of hematological diseases. In general, the cytotoxic activity correlated well with the total amount of IgM. However, in cases of chronic lymphocytic leukemia, idiopathic thrombocytopenic purpura and immunocytoma aberrant cytotoxic activities were found, but to ascertain a connection between these diseases and the factor would require a more extensive follow-up study. The results indicate that the naturally occurring thymocytotoxic IgM is widespread and may reflect a clone of B cells which is activated by an endogenous stimulus, or by some ubiquitous exogenous immunogen.

Demonstration of C3d and the terminal complement complex on thymic large granular lymphocytes and common thymocytes after incubation with naturally thymocytotoxic serum.

Guinea pig thymocytes were incubated in vitro with normal human or rabbit serum, which resulted in lysis of a major part of the cells, or with autologous serum causing lysis of a fraction (30%) of the cells. By using antibodies against human C3d and a neoepitope on the terminal complement complex (TCC), activation of both the initial and terminal part of the complement cascade was demonstrated on the surface of thymocytes incubated in the presence of serum. With human serum both types of antigen were detected. With rabbit serum only TCC was detected since immunoglobulins were bound to thymocytes and prevented specific demonstration of C3d by the antirabbit secondary antibody. With autologous serum only C3d was demonstrated, due to lack of cross-reactivity of the monoclonal anti-TCC antibody with guinea pig. Heat-inactivated sera or human serum devoid of IgM neither caused lysis nor resulted in complement activation. Addition of heat-inactivated serum restored the complement activating ability of IgM-depleted serum, indicating that heat stabile IgM is an obligate but sufficient requirement for complement activation in this system. The TCC epitope was also identified on a considerable number of granulated cells, on the basis of morphology classified as large granular lymphocytes.

Studies on thymocyte subpopulations in guinea pigs. VIII. Characterization of a thymocyte population resistant to the cytotoxic effect of normal rabbit serum.

Guinea pig thymocytes were incubated with normal rabbit serum, which resulted in the death of a great majority of the cells. The remaining rabbit serum-resistant cells, representing less than 10% of the thymocytes, contained euchromatic DNA and were of intermediate size and low density. Functional tests indicated that they were enriched in immunologically mature cells, which responded to the mitogenic lectins phytohemagglutinin and concanavalin A, and were depleted of immature, spontaneously proliferating cells and in cells responding to the thymocyte growth peptide. The described procedure for enrichment of immunologically mature thymus cells in guinea pigs may become useful since glucocorticoid treatment, used in mice for enrichment of mature thymocytes, cannot be used for this purpose in guinea pigs.

Studies on a serum factor which blocks the effect of human natural thymocytotoxic antibodies.

Normal human serum contains an IgM associated cytotoxic activity acting on guinea pig thymocytes. The inhibition of this cytotoxic effect by normal guinea pig serum was investigated. The blocking effect could be detected within 1 min and seemed to act by interfering with the cytotoxic factor in solution, since preincubation of human serum with autologous guinea pig serum potentiated the blocking effect, whereas no irreversible blocking effect was obtained by pre-incubation of target cells with the blocking factor. The inhibitory factor was not absorbed by incubation with thymocytes. The blocking effect was heat stabile (56 degrees C, 30 min) and detectable at both 4 degrees C and 37 degrees C. The clear effect on the cytotoxic effect of heterologous serum at 4 degrees C was in contrast to the much lower effect on the cytotoxicity of autologous serum at this temperature.

Purification, identification and elimination of a natural human serum antibody with cytotoxic effect on guinea pig thymocytes.

Normal human sera contain one or several factors cytotoxic for normal guinea pig thymocytes, and when serum is precipitated with ammonium sulphate (60% saturated) and the precipitate dissolved and dialyzed, the activity is preserved. Gel chromatography with Sephadex G-150 and Sepharose CL-6B indicated a molecular weight of approximately 900,000 daltons. The active fractions contained a high amount of IgM according to single radial immunodiffusion and two-dimensional gel electrophoresis. Quantitation of the IgM band in one-dimensional gel electrophoresis preparations by gel scanning indicated that IgM accounted for 65% of the eluted proteins in active fractions. Purified human IgM from myeloma patients eluted as the active factor during gel chromatography. Elimination of IgM from serum by affinity chromatography eliminated the cytotoxic activity. The serum could also be inactivated by heating. The mixing of IgM-depleted serum with either polyclonal IgM or heat-inactivated serum restored the activity. Thus, the cytotoxic activity is due to IgM antibodies plus a heat-labile component (presumably complement). The presence of the cytotoxic activity in autologous (guinea pig) serum was recently demonstrated. The possible functional role of these antibodies in the elimination process of a large number of cortical thymocytes is suggested.

Cytotoxic effect of heterologous and autologous serum factor on guinea pig thymocytes assayed by electronic cell volume distribution analysis.

The cytotoxic effect of serum on guinea pig thymocytes was studied by dye exclusion and by analysis of cell volume distribution curves obtained by means of a computer-assisted electronic cell volume distribution analyzer. Guinea pig, rabbit and human sera were shown to be cytotoxic for guinea pig thymocytes (incubation for 1 h at 37 degrees C or overnight at 4 degrees C). A similar, but less potent, activity was found in autologous guinea pig serum (4 degrees C). The activity was dialysable and lyophilizable but was destroyed by heating at 56 degrees C for 30 min. It was recovered after ammonium sulphate precipitation followed by dialysis. The effect of this dialysate was dose-dependent. A tentative definition of viable and dead cells was made on the basis of 'electronic' cell volume. The following results indicate that by this definition electronic cell volume distribution allowed detection of the cytotoxic serum factor: (1) sera and dialysates produced marked distortion of cell volume distribution curves, not seen in controls treated with heat-inactivated preparations; (2) the dose-response relationship was similar when dye exclusion and cell volume distribution was used as assay: (3) both methods detected similar differences in sensitivity of different thymocyte subpopulations to serum; (4) the electronic method could be used on separate thymocyte subpopulations although they differed in cell volume. This rapid, objective and reproducible method for studying the cytotoxic effect of sera may be useful during attempts to purify and identify the active factor(s).