Göran Möller

Do Suppressor T Cells Exist

The notion of suppressor T cells has had a great impact in immunology and clinical medicine, A number of diseases, such as allergy and autoimmunity, are now considered to be caused by a relative lack of suppressor T cells, while other conditions, such as immunodeficiencies, are thought to be due to too many suppressor cells. Even fundamental immunological concepts, like immunological tolerance and the distinction between self and non-self, are often thought to be regulated by a delicate balance between suppressor cells and potentially autoreactive T and B cells. The concept of suppressor T cells has markedly affected the way immunologists interpret their findings. A depressed response is generally considered to be caused by suppression and implicitly by suppressor T cells. It is quite interesting to observe the changes that have occurred during the last 15 years: suppressor eells are now considered to be not only the essential, but also the only regulating elements of the immune system. The previous concept of powerful and specific regulating mechanisms, such as antibody feed-back inhibition ofthe immune response that efficiently regulates both T and B cells, now appears to be of little interest, I should like to analyse in general terms the plausibility of the eoneept that there is a separate T cell subpopulation with a suppressor function, which exists to control other cells of the immune system, I am not questioning the existence of suppressive phenomena or findings that T cells can mediate suppressive effects, but I am sceptical ofthe notion of suppressor Tcells as a separate subpopulation of T cells.

Potent suppression of the adaptive immune response in mice upon dietary exposure to the potent peroxisome proliferator, perfluorooctanoic acid

In a previous investigation, we demonstrated that severe thymus and spleen atrophy occurs in mice upon dietary exposure to several potent peroxisome proliferators (PPs). In the present investigation, the effects of the potent PP perfluorooctanoic acid (PFOA) on the adaptive immunity of mice was evaluated both in vivo and ex vivo. The in vivo immune response examined involved immunization of mice with horse red blood cells (HRBCs), displaying T-cell-dependent antigens after pre-treatment with a PFOA-containing diet for 10 days. Subsequent quantitation of the primary humoral response was performed employing both the plaque-forming cell (PFC) assay and determination of the antibody titer by ELISA. The results clearly demonstrate that oral administration of PFOA prevents both the increases in plaque formations by anti-IgM and -IgG and in serum levels of IgM and IgG normally evoked by such immunization. Ex vivo spleen cells proliferation (assayed as incorporation of 3H-thymidine) in response to both T- and B-cell activators was attenuated by dietary treatment with PFOA, although the analogous in vitro treatment of mouse spleen cells with this same compound had no such effects. Thus, the relatively metabolically inert PP PFOA may exert adaptive immunosuppression in mice by an indirect mechanism. The possible relevance of this immunosuppression to the alterations in plasma lipids caused by PPs is discussed.

Enumeration of IFN-γ-producing cells by flow cytometry: Comparison with fluorescence microscopy

Abstract A new intracytoplasmic immunofluorescence staining to detect and quantify human interferon-γ (IFN-γ)-producing cells by means of flow cytometry is described. Mononuclear leukocytes, stimulated in vitro to produce IFN-γ, were fixed and made permeable to antibodies by sequential exposure to paraformaldehyde and the detergent n-octyl-glucoside. Cytoplasmic IFN-γ was demonstrated by indirect immunofluorescence using IFN-γ-specific mouse monoclonal antibodies. The staining exhibited a very characteristic morphology and was localized in the Golgi apparatus. An excellent agreement between the enumeration of cytoplasmic IFN-γ-positive cells by immunofluorescence microscopy and flow cytometry was noted. However, the latter has the advantage of a standardized control, is less labor consuming and is observer independent.

Mechanism of mercury-induced autoimmunity: both T helper 1- and T helper 2-type responses are involved

Mercury can induce a systemic autoimmune disease in susceptible mouse strains. H‐2s mice are particularly susceptible to mercury‐induced autoimmunity and other mouse strains are more or less resistant. T helper 1/T helper 2 (Th1/Th2) dichotomy has been proposed for resistance or susceptibility, respectively. In the current study we show that mercury treatment induced a full autoimmune response in both C57BL/6 (H‐2b) wild‐type and interleukin‐4 (IL‐4)‐deficient mice. Antibody production of all isotypes were induced, except that in IL‐4‐deficient mice there was no immunoglobulin E (IgE) and very low levels of immunoglobulin G1 (IgG1) antibody synthesis. Autoantibodies of different specificities were produced. The granular pattern of all IgG subclasses deposits were detected in the kidneys. In contrast to mercury‐treated H‐2s seconds mice, we did not detect any anti‐nucleolar autoantibodies in the sera of mercury‐treated wild‐type or IL‐4‐deficient mice. To further explore the role of Th1/Th2 cytokines in the mercury model, we performed anti‐interferon‐γ antibody treatment in IL‐4‐deficient mice together with mercury treatment and found that the production of IgG2a and IgG3, but not IgG2b, antibodies was downregulated. This indicated that besides Th2‐type cytokines, Th1‐type and other cytokines were involved as well in mercury‐induced autoimmune response. Thus, C57BL/6 mice with H‐2b genotype are highly susceptible to mercury‐induced autoimmunity, and the genetic susceptibility to mercury involves more than a predisposition of a Th1‐or Th2‐type response.

Regulation of IgG1 and IgE Synthesis by Interleukin 4 in Mouse B Cells

Mouse interleukin 4 (IL‐4) has been shown to act on B cells as an induction factor for Ig class switch. We studied the characteristics of IL‐4‐regulated Ig isotype production in lipopolysaccharide (LPS)‐stimulated splenic B‐cell cultures with emphasis on the comparison between the IgG1 and IgE responses. The results show that the kinetics for the appearance of IgG1 and IgE isotypes are similar, but that the dose of IL‐4 required for the induction of an IgE response is 3–10 times higher than that for an IgG1 response. No requirement for T cells was found for the induction of either isotype. Pre‐incubation of cells for 24 h with IL‐4 alone was sufficient 10 induce an IgG1 response when cells were recultured with LPS from days 1 to 6. However, the simultaneous presence of both IL‐4 and LPS for at least 24 h was required for a detectable IgE response. For an optimal IgE response. IL‐4 needed to be present for more than 72 h in LPS‐activated cultures. The possible reasons for the different regulation of IgG1 and IgE responses are discussed.

Characterization of individual tumor necrosis factor α- and β-producing cells after polyclonal T cell activation

Abstract Mononuclear cells from human blood were stimulated to tumor necrosis factor α (TNFα) or β (TNFβ) production by the T cell mitogens anti-CD3 antibody (OKT3) or staphylococcal enterotoxin A (SEA). The cells were then fixed and subsequently permeabilized in suspension by the detergent saponin in order to enable TNFα- or TNFβ-specific antibodies to enter the cells and interact with cytoplasmic TNF in producer cells. A characteristic morphology of the staining pattern of the two cytokines was noted, with a local accumulation in the cytoplasm in a perinuclear position reflecting the presence of TNFα or -β in the Golgi system. TNFα-producing cells appeared 2–3 h after activation of the cultures and increased up to 6 h. The majority of these early TNFα-producing cells were monocytes as judged by two-color staining and morphology, but a small fraction of CD4- and CD8-positive T cells was found up to 72 h. TNFβ production started later and peaked 18 or 48 h after OKT3 or SEA stimulation, respectively. The number of TNFβ-producing cells was much larger than that of TNFα-producing cells, and approximately 90% of them were CD4-positive T cells. The remaining TNFβ production occurred in CD8-positive T cells and in B cells. Almost every second CD4-positive T cell made TNFβ at the peak of the SEA-induced synthesis. The cytotoxic activity found in the supernatants correlated well with the number of TNF-producing cells found in the cultures. Cells from fresh blood or unstimulated cultures showed no or very few TNF-producing cells.

Autoantibody Formation after Bone Marrow Transplantation

Clinical myasthenia gravis has been reported in an increased frequency after bone marrow grafting. The number of bone marrow transplanted patients making IgG autoantibodies directed against the autoantigens cardiolipin, SS-B (La) and thyroglobulin was found to be significantly lower as compared to the autoantigen acetylcholine receptor protein. The occurrence of antibodies to single-stranded DNA was found in a lower frequency than acetylcholine receptor antibodies but the difference was not statistically significant. Antibodies to cardiolipin were frequently observed prior to grafting. The G1m1,2 and G3m5 phenotype frequency did not differ in individuals who developed receptor antibodies from that found in the normal population. Analysis of HLA antigens in this patient group revealed no association to HLA B8/DR3 or B35/DR1. This may indicate that the etiology of myasthenia gravis induced by bone marrow grafting differs as compared with the spontaneous form of myasthenia gravis and the penicillamine-induced disease.

Macrophage depletion decreases IgG anti-DNA in cultures from (NZB · NZW)F1 spleen cells by eliminating the main source of IL-6

We have studied ihe rote of macrophages in the produclion of IgG anli‐DNA autoanlibodies by (NZB × NZW)F1 tnice(B/W). One of the main features of the syslemic lupus cryibematosus(SLE)‐like disease that aftects these mice, h ihe presence of circuialing IgG autoantibodies and immune complexes, which lead lo renal failure and death by the age of 8–9 months. IgG autoantibodies are produced without in vitro siimulallon by lolal spleen eells from these miee when they reach tbe age of 6 monlhs. We have demonstrated ihat IL‐6 increases ihe production of IgG autoantibodies in cultures of splenic purified B cells from the old B/W miee. The aim of this study was to show the involvement of macrophages in the production of IL‐6 and consequently in the produclion of IgG anti‐DNA antibodies in ritro. We show thai elimination of the macrophages by different treatments led to reduction of the content of IL‐6 in ihe supernalants as well as of IgG anti‐DNA autoantibodies. Addition of fresh, splenic or peritoneal macrophages restored the produclion of autoantibodies in macrophage‐depleted cultures from old B/W miee. There were no ditferences in the capacity of IL‐6 produelion between macrophages from old or young B/W miee. but an imporiani difference was observed between peritoneal and splenic maerophages, where the former produced mueh higher levels ol IL‐6, and eonsequently were more potent inducers of IgG autoantibodies. The present results reinforee Ihe role of macrophages and lL‐6 in Ihe production of IgG anti‐DNA autoantibodies in B/W mice. The implieations of these results in the pathogenesis of the disease are discussed.

Pretreatment of lymphocytes with mercury in vitro induces a response in T cells from genetically determined low‐responders and a shift of the interleukin profile

Mercury can induce autoimmune disease in susceptible mouse strains. We found that in vitro mercuric chloride induced a high proliferative response in spleen lymphocytes from mercury‐susceptible SJL mice, but a low response in resistant mice, such as C57BL/6 (H‐2b), A/J (H‐2a) and CBA (H‐2k) mice. However, a high proliferative response was obtained with lymphocytes from all tested low‐responder mice by pretreating them in vitro for 1–3 days with mercuric chloride and then wash away the excess mercury. Both CD4+ and CD8+ T cells were activated in the restored response, but CD4+ T cells was the major responding cell population, as in high‐responder mice. We also measured the cytokine production at the protein level after mercury stimulation in vitro. We found that in mercury stimulation the different culture conditions resulted in different patterns of cytokine production. The continuous presence of mercury induced interleukin‐2 (IL‐2) and interferon‐γ, but not IL‐4 production in spleen cells from both high‐ and low‐responder mice. In contrast, by pretreating the cells with mercury and then washing, spleen cells from both high and low‐responder mice produced IL‐4. Our results suggest that spleen cells from both mercury‐susceptible and ‐resistant mice have the potential to respond to mercury in vitro and produce both Th1‐ and Th2‐type cytokines. But the mercury‐induced cytokine profile can shift depending on the conditions for activation.

Mercury-induced renal immune complex deposits in young (NZB x NZW)F1 mice: characterization of antibodies/autoantibodies

It is well demonstrated that mercury induces a systemic autoimmune disease in susceptible mouse strains. One of the major characteristics of mercury‐induced autoimmune disease in mice is the development of renal immune complex deposits. We have previously shown that continual injection of mercury into young autoimmune prone (NZB × NZW)F1 mice induced an increase in antibody/autoantibody production as well as development of early renal immune complex deposits. In the present study, we characterized the isotype, the specificity and the possible pathogenicity of deposited immunoglobulins in the kidneys of mercury‐injected (NZB × NZW)F1 hybrids. We found that young (NZB × NZW)F1 mice injected with mercuric chloride (HgCl2) for 6 weeks developed intense antibody formation of all immunoglobulin isotypes (except for IgG2b) as well as high levels of granular deposits of IgM, IgG1, IgG2a and IgG3 antibodies in the renal mesangium. Increased levels of the same antibody isotypes were also found in the kidney eluate of mercury‐ but not saline‐injected mice. The dominant antibody in the kidney eluate of mercury‐injected mice was of IgG1 isotype and found to be directed against double‐stranded DNA, collagen, cardiolipin, phosphatidylethanolamine, and the hapten trinitrophenol, but not against nucleolar antigens. Further studies demonstrated that mercury‐induced renal immune complex deposits in young (NZB × NZW)F1 mice did not lead to a severe kidney injury. Thus, in response to mercury, young (NZB × NZW)F1 mice develop renal immunoglobulin deposits with an isotype and specificity pattern correlating with that seen in the spleen and in the serum.