The subcellular distribution of antigen in macrophages.

The intracellular fate of phagocytosed antigens in cells from peritoneal exudate in CBA mice has been studied by using (126)I and (131)I labeled antigens. After uptake of labeled antigen, cells were homogenized and the subcellular fractions were analyzed by isopycnic centrifugation in a sucrose gradient. The uptake of heat-denatured BSA (c BSA) by these cells in vivo is 3.5 microg/mg c BSA injected/10(8) cells. The uptake by cells in animals which were exposed 2 days earlier to 900 r whole body irradiation is slightly lower but does not differ significantly. 90% of the phagocytosed material is degraded within 2-3 hr, the residual 10% is retained at least over an 8 hr periods. Using a pulse and chase technique, with (125)I and (131)I c BSA in vitro and in vivo it was shown that newly phagocytosed antigen is found mainly in a lysosomal turnover compartment of a density 1.19 g cm(-3). Antigen which has been in the cells for longer was found in a denser fraction (1.26 g cm(-3)). In a comparison of nhrmal and X-irradiated cells it can be shown that after irradiation with 900 r less c BSA is found in this storage compartment. Binding of the antigen to the subcellular fractions, and its behavior towards several detergents has been studied. Subcellular fractions do not have the increased immunogenic capacity of antigen enclosed in living macrophages. Two synthetic polypeptide antigens, poly(D-Tyr, D-Glu, D-Ala) and poly-(L-Tyr, L-Glu) have a different subcellular distribution from c BSA, BSA, or bovine gamma globulin. Apart from also being found in the 1.26 storage compartment the polypeptide antigens are mainly located in a 1.15 compartment and only to a small extent in the 1.19 compartment. The half-life of these antigens in the cells is much longer than the half-life of the protein antigens studied. The finding of several subcellular compartments is discussed in connection with the functions possibly performed by macrophages.

Aqueous humor alloreactive cell phenotypes, cytokines and chemokines in corneal allograft rejection.

As biopsies are not taken at the time of human corneal allograft rejection, most information on the early cellular changes in rejection is from animal models. We examined the phenotype of alloreactive cells present in the human anterior chamber during corneal graft rejection by flow cytometry and quantified aqueous humor levels of cytokines and chemokines using cytometric bead array. Aqueous and peripheral blood samples were taken from patients with graft endothelial rejection (n = 11) and from control patients undergoing cataract surgery (n = 8). CD45(+)CD4(+), CD45(+)CD8(+) and CD45(+)CD14(+) cells were found in aqueous during rejection; no CD45(+) cells were seen in control samples. Higher proportions of CD45(+) cells found in aqueous during rejection were CD14(+), denoting monocyte/macrophage lineage, than were CD4(+) or CD8(+). Large elevations were seen in aqueous levels of IL-6, MCP-1 and IP-10 during rejection compared with controls; smaller but still statistically significant increases were seen in MIP-1alpha and eotaxin. The role of CD14(+) cells in allorejection is unclear as is the potential of these chemokines and their receptors as therapeutic targets. Aqueous humor samples offer a unique opportunity to analyze components of the allogeneic response in direct contact with donor tissue but without artifacts inherent in examination of tissue.

Can unresolved infection precipitate autoimmune disease?

Autoimmune diseases are frequently postulated to arise as post-infectious phenomena. Here we survey the evidence supporting these theories, with particular emphasis on Crohn's disease and ankylosing spondylitis. Direct proof that infection establishes persistent autoimmunity remains lacking, although it may provoke a prolonged inflammatory response when occurring on a susceptible immunological background. The argument of infective causality is by no means trivial, since it carries important consequences for the safety of vaccine development.

Murine leukemia virus-associated cell surface antigens in rats neonatally infected with Gross murine leukemia virus.

The inoculation of newborn W/F, Lew, AS and DA rats with Gross murine leukemia virus (G-MuLV) resulted in the prompt appearance of cells with viral protein antigens (VPA) on their surfaces. These were first found in the bone marrow and spleen and later in the thymus gland. As the animals developed, the VPA-positive population expanded and the intensity of the fluorescence increased. In the spleen, the cells with the strongest fluorescence had the properties of T-cells, but in both spleen and bone marrow low levels of VPA were found on non-T-cells. The VPA-positive population expanded long before malignant cells could be detected and, in most animals, the entire T-cell compartment became antigen-positive. These animals were unable to respond to G-MuLV antigens and many eventually developed leukemia. However, some animals apparently broke the tolerance that followed neonatal infection and eliminated VPA-positive cells from their tissues

Conditional haploinsufficiency of NCF1 (encoding p47(phox)), a signaling gene with a heterozygous phenotype potentially subject to natural selection.

Even a minor degree of haploinsufficiency could eventually reduce the frequency of an autosomal immunodeficiency disease. Searching for such a condition, we have re-examined the phenotype of mice +/- for the NCF1 gene encoding p47(phox) and humans +/- for NCF1 and NCF2 using a procedure that allowed the respiratory burst of granulocytes and macrophages to be measured simultaneously. The mice showed significant haploinsufficiency in granulocytes but not in macrophages (i.e. conditional haploinsufficiency). Our human data were obtained from blister cells, and were too scattered to allow a firm conclusion. In view of recent re-evaluation of the role of the respiratory burst these findings are compatible with the view that haploinsufficiency occurs particularly among rate-limiting genes that operate in regulatory/signaling pathways.

Where are peptides taking us in T-cell biology?

T-cell epitopes are now well understood as amino-acid nonamers binding to major histocompatibility complex molecules. Powerful methods have been developed for their identification through screening of recombinant and synthetic peptides. Multiple epitopes from a single protein are valuable for detecting T-cell reactivity in disease, currently in human immunodeficiency virus infection, and in the future in autoimmune disease. Surprises are likely to be encountered while exploring the T-cell repertoire in this way, such as positive as well as negative selection of self-reactivity. T-epitopes are likely to find important applications in therapy, particularly in down-regulation of the immune response. Multiple mechanisms of down-regulation appear to operate, among which bystander suppression by TGF beta-producing T-cells from the gut is of great current interest.

The immunological synapse.

The immunological synapse plays a central role in organising the immune system. Through their synaptic activity both T and B cells usually, but not always, acquire the information that critically determines the level and nature of the responses that they make. For T cells much of that information comes from epicrine and paracrine cell-cell interactions in the cluster that forms around a dendritic cell. These interactions are being dissected by experiments in which two populations of TCR-transgenic T cells are combined in vivo. Another important aspect of synaptic activity is the way in which different levels of expression of MHC class II molecules influence Th1/Th2 balance. In exploring this form of control we are learning something of general importance about cis-regulation.

Single-step immunosorbent preparation of F-protein from mouse liver with conservation of the allo-antigenic site, and determination of concentration in liver and serum.

A single-step immunopurification procedure is described for murine protein F, in which the T-cell-defined allo-antigenic site on the protein is fully conserved. The procedure is based on the use of a newly developed monoclonal antibody. The protein is isolated as a 42,500 mol. wt (F.1) and a 43,000 mol. wt (F.2) monomer. The content in liver, as estimated by radioimmune inhibition assay, is 0.083% and the yield is approximately one third. An assay of immunogenic activity in adoptive transfer, which detects the T-cell-defined site, provides a similar estimate of content in liver. The adoptive transfer assay yields concns of F-protein in serum of young mice of 0.5-1.2 X 10(-9)M, the lowest concn of protein known to induce complete immunological tolerance.

Suppressor mechanisms in neonatally acquired tolerance to a Gross virus-induced lymphoma in rats.

Tolerance to a highly immunogenic Gross virus-induced tumour in Wistar/Furth rats (C58NT)D was produced by neonatal infection of the rats with the virus. These rats failed to reject the tumours when challenged 8 weeks after virus inoculation and to mount the appropriate cell-mediated immune response to the tumour. The mechanisms involved were studied in vivo by adoptive transfers into sub-lethally irradiated rats of tumour cells mixed with spleen cells and/or sera from normal, tolerant, or tumour immune rats, and in vitro by a 51Cr release assay involving similar mixtures. The results indicate the presence of a suppression mechanism which is sensitive to irradiation and abolished by trypsinisation. Weak blocking factors can also be detected in serum. An interpretation in terms of the release of virion proteins from infected cells is proposed, although participation of suppressor lymphocytes has not been excluded.

T cells in transplantation immunity.

This paper reviews the following problems in transplantation immunity: (i) short-lived ability to transfer immunity or suppression, in contrast to long-lived immunological memory in the autochthonous animal; (ii) short-lived ability to transfer graft-resistance, in contrast to long-lived ability to transfer helper activity for B-cells; (iii) the response to H-Y, as a system that might solve some outstanding problems in antigen presentation; and (iv) the contrast between live and killed allogeneic cells as immunogens. All of these problems, it is suggested, are amenable to study by modern methods. Students like me were drawn into Peter Medawar's orbit in the 1940s and 1950s by an irresistible mix of intellectual challenge and the glamour of experimental surgery. Much the same was happening elsewhere in the laboratories of Ray Owen, Milan Hasek, George Snell, Burnet, and Florey, and by 1960 the transplantation immunologists could justly claim to have opened up a whole new area of ideas in biology: we had discovered the lymphocyte as the antigen-sensitive cell, and the principles of immunological tolerance; we had revived interest in cellular immunity, and it was we who found the MHC (even if we had little idea of its real meaning). But by 1960 the first wave of success had passed, and the penetration of immunology by molecular biology had begun. Interest in transplantation immunity perceptibly declined, although many groups continued to address important problems, particularly in the field of organ transplantation. (ABSTRACT TRUNCATED AT 250 WORDS)

Thymic epithelial cells derived from the cultures of thymic nurse cells.

Thymic epithelial cells are derived from the cultures of thymic nurse cells. These cultures are free from fibroblasts and macrophages. The epithelial nature of these cells is confirmed by demonstrating the presence of keratin filaments in them. These epithelial cells show heterogeneity in shape, size and distribution of keratin filaments. They contain nonspecific esterase(s) molecules and express both I-A and H-2K antigens.

Progress in T cell biology.

We outline recent work in our laboratories on thymus progenitors, lineages within the thymus, interactions between regulatory and effector lymphocytes, splitting the CD4 (T4) T cell subset, and Ir and Is genes. We highlight the possibilities for future research opened up by the demonstration that certain marrow-derived cell lines can repopulate thymic lobes in culture, and also the deep insight into the logical structure of the lymph node provided by our ability to make an exact comparison between two-cell-type and three-cell-type immunoregulatory clusters.

Natural variation in immune responsiveness, with special reference to immunodeficiency and promoter polymorphism in class II MHC genes.

This review deals with natural selection operating on heterozygotes as a key factor controlling (a) the frequency of immunodeficiencies, and (b) promoter polymorphism in MHC class II genes. The known difference in frequency distribution of X-linked and autosomal deficiencies lend support to this possibility, and suggest that the frequency of neonatal defect may rise as old-established equlibria between entry and exit of deleterious mutations change. MHC class II gene promoters differ in their capacity to favor Th1 (or reciprocally Th2) responses, thus suggesting that promoter polymorphism is sustained by the greater flexibility in response that this confers on heterozygotes.

Relapsing arthritis induced by cell transfer from collagen-immunised mice.

Collagen-induced arthritis is a well-established model of chronic inflammatory arthritis. We here introduce a development of this model which combines the benefits of adoptive transfer and sequential relapse. DBA/1 x B10.Q F1 hybrid mice were immunised with bovine type II collagen, and those which developed a sufficiently high level of arthritis served as donors of spleen cells transferred into BALB/c SCID hosts. After boosting with 500 microg collagen, the development of host arthritis was monitored over a period of up to 256 days, during which up to three successive peaks were detected. In comparison with bovine collagen, mouse collagen used for boosting induced a lower initial peak but higher relapses. As expected, the transferred disease was more uniform than the freshly induced one. Previous information suggests that a shifting cytokine balance between protective and aggressive T cells may account for the relapse and remission. This study provides a model of relapsing polyarthritis, obtained with normal immunocytes boosted with a well-defined protein antigen in animals not themselves treated with adjuvant. As such it is relevant to the etiology of inflammatory arthritis in man, and, if further developed, could be of value for testing new therapeutic strategies.

Assessment of collagen type II induced arthritis in mice by whole blood chemiluminescence.

Lucigenin-enhanced chemiluminescence can be measured in 100 microliters samples of whole, unseparated mouse blood. A procedure for doing so is here described in detail, using a standard clinical luminometer. The assay measures the TPA-induced oxidative burst from granulocytes and macrophages, which is believed to depend on the overall level of inflammation in the body. It is here applied to mice suffering from type II collagen-induced arthritis, and its relation to overt disease symptoms (the arthritis score) is characterised during the course of the disease. A correlation between the assay and the arthritis score is found at the height of the disease (r = 0.42, p = .039), but not at early or very late time points, although there is a strong hint that the results of an early assay may predict the subsequent disease course. The assay provides a rapid, convenient, quantitative and economical method of assessing disease activity, which can be carried out repeatedly on the same individual. It should be applicable in other mouse models of chronic inflammatory disease. It may find application for rapid screening of novel anti-rheumatic drugs and treatments.

Genetic variation in the MHC II promoter: lessons for regulation and for comparative genomics.

Sequence data have been accumulating that reveal variation in gene promoters of the immune system, notably in MHC class II, cytokines and chemokines. The variation is non-random: it occurs most often in proximity to and within certain regulatory elements such as CRE and NFY (in MHC class II these are respectively the X2 and Y boxes). These are elements that are widely used elsewhere in the genome, and appear to act as rheostats (modulators of expression) in contrast to the type of on-off switch operated by the RFX element that is unique to a single family of promoters such as MHC class II. It is proposed that a complex mouse phenotype described in Prague and elsewhere may reflect this pattern of variation in/around CRE. Such rheostats are expected to operate in other promoters. Their identification will be facilitated by short-range comparisons (e.g. human-chimp), and indeed this is a motive for extending comparative genomics.

A survey of biotechnology.

This issue on immunology and biotechnology opens with a survey of the contribution of immunology to medicine. Proteins, growth and differentiation factors, and monoclonal antibodies to surface antigens will have an impact in the 1990's likely similar to that of antibiotics in the 1950's. The characterization of factors and cell-surface molecules opens the investigation of the role of "recessive oncogenes" in the development of cancer. A further impact of immunology in the classical field of prophilaxis is represented by the innovative development of contraceptive vaccines based on the use of episomal shuttle vectors for the expression of surface sperm proteins able to induce an effective immune response.