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Aging is a multi-facetted process, but the deterioration of the immune function seems to play a central role in this context. Paradoxically, immune reactivity against exogenous antigens declines during aging while autoimmune reactivity increases. One of the aims of our investigations on the function of the senescent immune system was to define immune parameters of 'normal' aging, i.e. those not dependent on underlying diseases. Specifically, our interest was focused on the possible role of an altered lipid metabolism of cells of the immune system during aging. The known decrease of plasma membrane fluidity of lymphoid cells and monocytes in higher age may be one of the factors responsible for the nonoptimal functioning of the immune system. This property, in turn, seems to be based on an altered lipid metabolism. Specifically, we have evidence that the finely tuned balance between the transport of cholesterol to and from lymphoid cells via the environment and the intracellular cholesterol biosynthesis seems to be disturbed with increasing age. This conclusion is drawn from experiments where low-density lipoprotein (LDL) and high-density lipoprotein (HDL) receptor activity is assessed using fluorescently labeled lipoproteins in fluorescence-activated cell sorter (FACS) analyses. LDL receptor uptake is unexpectedly increased in the elderly, but LDL receptor regulation, and serum LDL composition itself seem to be normal. Preliminary data point out the possibility that the efflux of cholesterol via HDL may be insufficient. Attempts to modulate plasma membrane fluidity by means of the phospholipid mixture 'active lipid 721' (AL 721) showed that this drug, in contrast to literature reports, is not a 'membrane fluidizer' but rather exerts this effect as a nutrient for lymphocytes and monocytes.

The concept of an extrinsic regulation of the immune system through neuroendocrine signals is well established, as is the fact that the immune system in turn informs the brain about contacts with antigens via “immunotransmitters”, i.e. cytokines and/or hormones with central effects [1]. All these data that have accumulated during the last twenty years have contributed to the vision of the immune system as “the sixth sense” [2]. While there is certainly still more work needed to define the physiology of this concept in all details, strong evidence has been obtained that the immune-neuroendocrine dialogue is of relevance for the homeostasis of the immune response, as defects in the activation of the hypothalamo-pituitary-adrenal (HPA) axis by immune signals were found to be associated with and/or to predispose to spontaneously occurring [3] and experimentally induced autoimmune diseases in animal models [4, 5], and there is evidence that the same is true also in humans [6]. A large body of more recent literature data strongly suggests that this dialogue involves not only the hypothalamus, but several other brain areas, notably the structures of the “Limbic System” (for review see [7]).

Monoclonal antibody INN-CH 16 recognizes a surface determinant exclusively present on activated chicken T lymphocytes. The present experiments suggest that this chicken activated T lymphocyte antigen (CATLA) represents the chicken IL-2 receptor or an associated structure, as (i) the kinetics of CATLA expression during T cell activation are analogous to those described for the mammalian receptor for IL-2, (ii) the IL-2 dependent proliferation of mitogen prestimulated chicken lymphocytes is competitively inhibited by INN-CH 16, and (iii) pretreatment of T lymphoblasts with INN-CH 16 drastically reduces their capacity to absorb IL-2 activity from supernatants of mitogen activated chicken lymphocytes.

In about 10% of patients operated on a chronic sinusitis, an aspergilloma is found in the paranasal sinus. To detect possible underlying immunodeficiencies, patients with aspergilloma were subjected to an immunological screening programme. The data were compared with those of patients suffering from non-mycotic chronic sinusitis and healthy controls. Totale lymphocyte counts and immunological levels were normal in both groups of sinusitis. Leukocyte subset analyses by membrane fluorescence revealed a significant decrease of CD11+ cells, i.e. macrophages/monocytes and NK cells, in both types of sinusitis. Furthermore, a markedly enhanced frequency of CD25+-cells, i.e. IL 2-receptor bearing cells, was observed in patients with aspergilloma. Peripheral blood lymphocytes of both groups of patients showed a significant reduction in the proliferative response to both T and B-cell mitogens, the values for the mitogens ConA and PWM being significantly lower in aspergilloma patients than in those with non-mycotic sinusitis. This lack of lymphocyte stimulation in the aspergilloma group was also manifest in skin tests to recall antigens. These first data suggest an immunodeficiency in association with chronic sinusitis caused by Aspergillus fumigatus. Further studies are needed to clarify if this defect is cause or result of the mycotic infection.

The fluorescence kinetics of fluorescein isothiocyanate (FITC)-antibody conjugates during short (0.01 sec) laser excitations were analyzed for possible information about the immunological specificity of binding to antigenic substrates in direct immunofluorescence assays with antigen-coated Sephadex beads. First results of these investigations suggest marked differences in the bleaching characteristics of specifically and nonspecifically bound FITC conjugates: FITC-anti-bovine serum albumin (BSA) and FITC-anti-rabbit immunoglobulin (Ig) were found to fade significantly more slowly when bound to the respective homologous antigen (anti-BSA to BSA, and anti-rabbit Ig to rabbit Ig) as compared to nonspecifically adherent anti-BSA to rabbit Ig, and anti-rabbit Ig to BSA. Possible implications of these data for discrimination of nonspecific staining in immunofluorescence are discussed.

The B-L region of the chicken major histocompatibility complex (MHC), the so-called B -locus, corresponds to the murine H-2 I-region. Using alloantibodies and monoclonal antibodies to B-L we analyzed: (a) the tissue distribution of B-L + cells, (b) the function of B-L + cells, and (c) the possible role of B-L + cells in the development of spontaneous autoimmune thyroiditis (SAT) in Obese strain (OS) chickens. The tissue distribution of B-L + cells in peripheral blood and various lymphoid and nonlymphoid organs corresponds to what is known for mammals. In the bursa of Fabricius most lymphoid cells and the dendritic cells carry the B-L antigen; B-L + thymic nurse cells (TNC) first appear on day 17 of embryonic life; chickens possess dendritic B-L + cells in the skin resembling mammlian Langerhans cells; in addition we found that the microglia is unequivocally B-L + . B-L + peripheral blood lymphocytes (PBL) were separated with a fluorescence-activated cell sorter. Ten percent of unstimulated PBL and 60% of phytohemagglutinin (PHA) stimulated T-cell blasts are B-L + . In graft-vs-host (GvH) assays B-L − cells were identified as the effector cells. These cells respond to PHA and concanavalin A (Con A), but not to pokeweed mitogen (PWM). B-L + cells cannot be stimulated by Con A and PHA, but respond to PWM. They possess only a very low activity in GvH assays which can be inhibited by anti-T-cell sera. In OS chickens B-L + /non-B/, non-T and B-L + T (blasts?) cells are found in the “first line” of mononuclear cell infiltration in the thyroid glands. Most interesting, thyroid epithelial cells—which are normally B-L − —become B-L + in the neighbourhood of B-L + infiltrating mononuclear cells. This observation may be of significance for autoantigen presentation and perpetuation in autoimmune thyroiditis. Finally, OS thymuses contain significantly less TNC than normal controls.

Changes in the expression of surface determinants during maturation from central bursa and thymus cells to peripheral B and T lymphocytes in the spleen and peripheral blood in the chicken have been investigated by means of heteroantibodies. The results obtained in membrane immunofluorescence and lymphocytotoxicity assays can be summarized as follows: (1) Turkey antisera specific for bursa (ABS) and thymus (ATS) cells react exclusively with the respective central cell type after exhaustive absorption with peripheral splenic lymphocytes. (2) Antisera raised in rabbits against spleen cells (ASS) and exhaustively absorbed with both bursa and thymus cells recognize surface determinant(s) on a subpopulation of 30 – 40 % of peripheral lymphocytes in spleen and peripheral blood. (3) Double staining and cocapping experiments revealed this peripheral lymphocyte-specific antigen (Lp) on both B and T lymphocytes with no significant steric relationship to B or T lymphocyte markers.

A monoclonal antibody (INN-CH-16) was prepared which reacts with a cell surface antigen termed chicken activated T lymphocyte antigen. This antigen is expressed on antigen- or mitogen-activated T lymphocytes and is not present on nonstimulated lymphocytes. It has an apparent molecular mass of 48-50 kDa under reducing conditions. The value of this antibody for the immunohistochemical characterization of infiltrating cells in the thyroid glands from Obese strain chickens with spontaneous thyroiditis is demonstrated.

Previous work in this laboratory was dedicated to the serological classification of chicken lymphoid cells by means of specific heteroantisera. The original purpose of those attempts was to make use of such antibodies for the elucidation of the pathogenetic mechanisms in certain autoimmune phenomena, viz. the spontaneously occurring autoimmune thyroiditis in chickens of the Obese strain (1). Taking advantage of the clearcut anatomical separation of the bursa and thymus system in the chicken, antisera against bursa (ABS) and thymus cells (ATS) had been raised in turkeys, which — after proper absorptions — were shown to be strictly specific for the respective cell type (2). These reagents turned out to be perfectly suited for both the in vitro delineation of B and T cells in various lymphoid organs and the peripheral blood of chickens (3), as well as for the specific in vivo suppression of B and T cell functions most probably due to the ability of avian sera to fix chicken complement (4). In addition, these antisera have been successfully used for the analysis of the expression of antigenic surface determinants during the ontogeny of the lymphoid system (5).

Frozen sections of chicken lymphoid organs were examined for lymphocyte surface antigens by antisera to T and B lymphocytes (ATS;ABS), and for the presence of immunoglobulin (Ig) G Fc and complement receptors (FcR;CR) by hemadsorption with sheep erythrocytes (E) coated with chicken IgG(EA), and E coated with rabbit IgG and chicken complement (EAC). In the spleen FcR positive cells were confined to the periellipsoidal sheaths and the germinal centers. CR positive cells were found in the same spleen areas, as well as in the medulla of bursal follicles. These lymphoid areas reacted strongly with ABS, but they also stained with neutral alpha-naphthyl butyrate esterase, and phagocytosed carbon particles were found in the periellipsoidal sheaths. Furthermore, in vivo treatment with cyclophosphamide, which resulted in pronounced B-lymphocyte depletion, did not affect FcR activity, but reduced CR activity significantly. These data indicate that the FcR activity demonstrated in tissue sections is mainly confined to mononuclear phagocytes, while the CR positive cells are mainly B lymphocytes.

This paper describes a first attempt to correlate morphological data of chicken thrombocytoid cells obtained by phase contrast microscopy with the expression of surface determinants as recognized by heterologous anti-chicken lymphocyte sera. The following three morphological types of mononuclear cells in the peripheral blood of chickens have been distinguished: (I) small round cells, (II) small star shaped cells with short pseudopodia; both these types resembling lymphoid cells, and (III) platelet-like cells with the capacity for instantaneous adhesion and subsequent spreading, i.e., “chicken thrombocytes”. Fluid transitions of cells type I to type III have been observed to occur in earlier studies. In a combined phase contrast and membrane immunofluorescence assay on living cells using turkey anti-bursa cell and anti-thymus cell sera (ABS and ATS) and a rabbit anti-chicken immunoglobulin serum, cells of type I and II both turned out to be positively stained in 95% with ATS, whereas the vast majority (98%) of thrombocytoid cells reacted with ABS. The impact of these first data for a possible relationship between chicken B cells and thrombocytes will be discussed.

Autoimmune diseases, both organ-specific and systemic, have a multifactorial basis with a definite genetic component. Most investigations on the theoretically and practically important problem of autoimmunity have focussed on the genetic basis of the aberrant immunological reactivity in experimental animals and man afflicted with autoimmune disease (for Review see 1,2). Much less emphasis has been given to the role of hormones as facultative factors modulating the development and severity of autoimmune diseases, reflected, e.g., by the preponderance of most - but not all - of these conditions in females3,4. Our own group has advocated a role for genetically determined primary alterations of the target organ as an obligatory prerequisite for the emergence of spontaneous autoimmune thyroiditis (SAT) based on studies on the Obese strain (OS) of chickens. This new concept is schematically depicted in Figure 1 and has been presented in several recent reviews5,6. As can be seen in Figure 1 both obligatory and facultative factors, the latter having a modulatory role only, contribute to the final outcome of a given autoimmune disease. The OS chickens, which spontaneously develop an autoimmune thyroiditis early in life that closely parallels human Hashimoto thyroiditis, are especially suited as an animal model for the study of the relative contributions of the different factors outlined in Figure 1. Chickens have several advantages over mammalian species for this kind of investigation: (a) the extramaternal development of the embryo, (b) large numbers of offspring can be obtained from each pair of parents, (c) the expression of major histocompatibility complex (MHC = B locus in the chicken) antigens on the surface of the nucleated erythrocytes which allows easy and efficient tissue typing, (d) the characteristic morphological dichotomy of the avian immune system, and (e) the availability of inbred and even congenic normal strains of chickens for crossbreeding and backcross experiments.