A. van Oudenaren

Improvement of the protein A plaque assay for immunoglobulin secreting cells by using immunoglobulin-depleted guinea pig serum as a source of complement.

This paper describes a modification of the protein A hemolytic plaque assay for the enumeration of immunoglobulin (Ig)-secreting cells independent of antibody specificity of the Ig. This assay was originally developed by Gronowicz et al. (1976), and is based upon binding of the Fc portion of IgG to protein A. Ig-secreting cells are mixed with protein A-coated sheep erythrocytes, developing rabbit anti-Ig antiserum and guinea pig serum as a source of complement. This mixture is either pipetted between two microscope slides, or added to agarose and plated on a petri dish or microscope slide. The hemolytic plaques are enumerated after incubation at 37 degrees C. Here we show that purification of the guinea pig complement over a Sepharose-protein A column in order to eliminate the IgG fraction facilitates plaque formation. This modification reduces the incubation period required for plaque formation, and yields a higher number of, and more discrete plaques, than the original method.

The effect of corticosteroids upon the number and organ distribution of “background” immunoglobulin-secreting cells in mice

The influence of the synthetic corticosteroid dexamethasone sodium phosphate (DEXA) upon the immunoglobulin (Ig)-secreting cells was studied in not intentionally immunized BALB/c mice. This was done for IgM-, IgG-, and IgA-secreting cells in spleen, mesenteric lymph nodes (MLN), and bone marrow (BM). A single injection of DEXA (16 to 144 mg/kg body wt) markedly reduced the number of Ig-secreting cells in spleen and MLN within 1 day, but hardly affected their number in the BM. The decrease was immediately followed by a recovery and, at the highest doses and especially in MLN, by an overshoot. Two weeks after the initial decrease a second decrease was found. When mice were subjected to daily treatment with DEXA during 1 week, initially a recovery pattern was found in spleen and MLN similar to that found after a single injection of a high dose. In this case, however, the effects were less dose dependent, and the overshoot reaction was followed by a period of subnormal numbers of Ig-secreting cells which lasted at least 1 week. This late effect of DEXA not only occurred in spleen and MLN, but also in the BM. The most prominent effect of daily treatment with DEXA was the long-lasting decrease of the number of IgG-secreting cells starting 1 week after withdrawal of treatment. This decrease was associated with a severely decreased serum IgG level.

Quantitative Aspects of the Nonspecific Humoral Immune Response to Sheep Erythrocytes

The kinetics and magnitude of the nonspecific humoral immune response was studied at the cellular level in mice immunized with sheep erythrocytes (SRBC). Intravenous injection of the antigen evoked, in addition to a specific anti-SRBC response, a nonspecific response of all immunoglobulin (Ig) classes and subclasses. This nonspecific response peaked on day 4 or 5 after immunization, irrespective of the Ig class. The absolute number of nonspecific Ig-secreting cells induced by immunization varied with the different Ig-classes, and it was not proportional to the background level of Ig-secreting cells of the various classes. The nonspecific IgM-IgG1- and IgG2-response was 3 to 4 times as large as the antigen-specific responses of these classes. The nonspecific IgA-response, however, was many times greater.

Regulation of delayed type hypersensitivity to host histocompatibility antigens during graft-versus-host reactions.

During GvH reactions in irradiated mice a variety of specific anti-host immune responses may occur. One of these is the occurrence of DTH to the host histocompatibility antigens, which may account for the inflammatory aspects of GvH. During acute as well as delayed GvH reactions the occurrence of anti-host DTH precedes the clinical symptoms of GvH disease. The anti-host DTH is mediated by long-lived, recirculating Lyt-1 + 2- T cells that need to proliferate in the irradiated recipients in order to display maximum activity. Not all host histocompatibility antigens can elicit anti-host DTH. H-2I and Mls-locus coded alloantigens do, but H-2K/D coded alloantigens and non-H-2 alloantigens other than Mls-locus coded alloantigens do not. This correlates with the ability to elicit proliferative mixed lymphocyte reactions in vitro but is in contrast to the response of nonirradiated mice to sc administered alloantigens. Under the latter conditions, all histocompatibility antigens induce a state of antigen-specific DTH. While the anti-host DTH is mediated by long-lived, recirculating Lyt-1 + 2- T cells, their response can be amplified by short-lived, sessile Lyt-1 + 2 + T cells. The latter T cell subset reacts to the host H-2K/D alloantigens and/or to non-H-2 alloantigens other than Mls-locus coded products. These cells alone cannot mount an anti-host DTH response. The anti-host DTH can be mitigated by Ts cells and non-T suppressor cells. Appropriate Ts cells can be readily induced by iv preimmunization of the donors with 5 X 10(7) irradiated, recipient-type spleen cells. Non-T suppressor cells can be induced by iv injection of bacterial LPS and simultaneous sc injection of 1 X 10(7) recipient type spleen cells. The suppression induced by these protocols shares several characteristics. In both cases the suppression is long-lasting, i.e., lasts at least 50 d, is transferable to syngeneic mice by spleen and lymph node cells, and both suppressive systems affect the induction of anti-host DTH as well as already activated anti-host DTH reactive T cells. Furthermore, while Ts cells and non-T suppressor cells are specific with regard to their antigen recognition, they are both able to suppress the DTH to a completely different set of host alloantigens. This, however, only occurs if the latter are inherited by the irradiated recipients as bystanders to the type of alloantigens that had activated the suppressor cells in the lymphoid cell donors.

Modulation of Total IgE Levels in Serum of Normal and Athymic Nude BALB/c Mice by T Cells and Exogenous Antigenic Stimulation

Several different grades of T-system impairment were studied for their effects on the total serum IgE concentration in BALB/c mice. Homozygous athymic nu/nu mice and their heterozygous nu/+ littermates were compared for serum IgE levels while kept under either barrier-maintained or conventional conditions. The results show a paradox between the T-cell dependency of the IgE immune response and the increased levels of serum IgE in the absence of T cells. Both barrier-maintained and conventionalized nu/nu mice have at least twofold increased serum IgE levels as compared to nu/+ mice. With age, IgE levels increased faster and reached higher plateau values in nu/nu than nu/+ mice. Moreover, after adult thymectomy of BALB/c mice the serum IgE levels increased up to 15-fold at 4 months of age, while infusion of immunocompetent T cells in nude mice resulted in a 2- to 5-fold decrease of the IgE level.

Serum immunoglobulin levels in mice. Determination of the low IgA level in AKR mice by an irradiation-resistant factor.

A comparison was made between the serum immunoglobulin (Ig) levels in H-2 compatible AKR and C3H mice. The IgG1 and especially the IgA level in preleukemic AKR mice was much lower than in age-matched C3H mice, while the IgM concentration was hardly different for AKR and C3H. Lethally irradiated AKR and C3H mice reconstituted with syngeneic bone marrow (BM) cells showed a return to serum Ig levels which are normal for these strains. In AKR mice reconstituted with C3H BM cells low IgA levels were observed. On the other hand, in C3H mice reconstituted with AKR BM cells high quantities of IgA appeared, showing the AKR allotype. It is concluded that the low serum IgA concentration in AKR mice is not a reflection of a genetically determined inability of the B cell line to produce IgA, but rather a manifestation of a genetically determined capability to prevent IgA synthesis.

Comparison of the sensitivity of the protein A plaque assay and the immunofluorescence assay for detection of immunoglobulin producing cells.

This paper reports a comparative study on the sensitivity of the protein A plaque assay and the cytoplasmic immunofluorescence assay for detection of immunoglobulin (Ig) producing cells in cell suspensions of murine lymphoid organs. The protein A plaque assay was found to detect as many or several times more Ig producing cells than the immunofluorescence assay, depending on the age and antigenic load of the mice, and upon the Ig class and organ studied.

Cytokines in lethal graft-versus-host disease

Graft-versus-host disease (GVHD) is caused by donor T lymphocytes that recognize foreign antigens on host tissues. This leads to T cell activation, which involves a cascade of events including the transcription of genes for cytokines and their receptors and the production of cytokines. One of the first cytokines to appear is interleukin 2 (IL-2). IL-2 production enhances the IL-2 receptor expression and leads to T cell proliferation. As a further step, differentation of T cells occurs, which results in the production of a certain pattern of cytokines. These cytokines influence the expression of cell surface antigens and adhesion molecules, and are able to activate other cell types such as cytotoxic T cells, macrophages and natural killer cells, which might act as effector cells in tissue destruction. Insight into the sequential expression of the various cytokines involved might enable a more effective treatment of GVHD. Therefore, we investigated the occurrence of cytokines in a murine model for acute GVHD. We addressed in particular the period early after allogeneic reconstitution.

The Influence of the Thymus upon the Number and Class Distribution of Immunoglobulin Containing Cells in the Bone Marrow and Other Lymphoid Organs of the Mouse

Due to the lack of mature T-cells, congenitally athymic (nude) mice have severely decreased serum IgG and IgA levels, together with normal or enhanced serum IgM levels (1–3). The cells responsible for the synthesis of these immunoglobulins are demonstrable as cytoplasmic immunoglobulin containing cells (C-Ig cells) by means of immunofluorescence (4). C-Ig cell numbers in the spleen are comparable in young and adult nude and heterozygous mice, whereas the age-related increase of the C-Ig cell population in the other lymphoid organs is retarded in nude mice (5). This is especially true for the bone marrow, which contains the majority of all C-Ig cells in thymus-bearing mice at adult age (5–7). In this paper the effect of thymus transplantation was studied upon the number and class distribution of C-Ig cells in spleen, bone marrow, mesenteric lymph node and Peyer’s patches of nude mice.