Polycystic kidney disease in the CBA/N immunodeficient mouse.

We describe a polycystic lesion of the kidney in the CBA/N mouse with an X-linked recessive immunodeficient syndrome. There is progressive cystic dilatation affecting all parts of the nephron. The cyst lining is composed of a single layered epithelium with focal nuclear crowding and the formation of micropapillary structures. The cystic epithelial cells show subnuclear vacuolation. Focal basement membrane thickening is also a feature. There is no significant inflammatory infiltrate present within these kidneys. Electron microscopic examination reveals that the subnuclear vacuolation is due to loss of the membrane infoldings at the basal pole of the epithelial cell with fluid accumulation within the extracellular space. The basement membrane thickening is due to expansion of the lamina densa. These changes are not present at birth but develop progressively with age. The finding of a polycystic kidney lesion in these mice offers an opportunity to investigate the relationship between the immune system and renal cyst formation.

Transmembrane electrical potential of lymphocytes in ageing mice. Flow cytometric analysis of mitogen-stimulated cells.

The changes in transmembrane electrical potential (TMP) of Concanavalin A (Con-A)-stimulated lymphocytes from young adult and aged CBA/Ca mice were studied with a potential-sensitive fluorescent oxonol probe. The initial effect of Con-A was to depolarise lymphocytes from young mice and abrogated in the presence of tetraethylammonium chloride (TEA), an inhibitor of K(+)-selective channels. Young and old T lymphocytes both responded to the calcium ionophore A23187 by becoming hyperpolarized, but this occurred more slowly in the old cells. While treated with the ionophore, old B cells appeared to be limited in their ability to depolarize in the presence of high external K+ concentrations, which did not hold for T cells of old animals. One or more defects in the mechanisms of monovalent ion transport across the membrane of old lymphocytes are probably responsible for these differences and may be associated with the known age-related dysfunction of the immune system.

Further evidence for the importance of parental source of the Xce allele in X chromosome inactivation.

Using mice that were mosaics for both Xce and phosphoglycerate kinase (Pgk-1) alleles, we present further evidence that the parental source of the X chromosome may affect the probability of that X chromosome remaining active. The reciprocal cross differences in PGK-1 activity described here are intermediate between those published previously for other alleles of Xce.

Development of B lymphocytes in mice heterozygous for the X-linked immunodeficiency (xid) mutation. xid inhibits development of all splenic and lymph node B cells at a stage subsequent to their initial formation in bone marrow.

CBA/N mice were crossed with CBA/Ca-Pgk-1a to produce female F1 hybrids that were heterozygous for both xid and the phosphoglycerate kinase 1 (PGK-1) allozymes. PGK acted as a quantifiable marker for the frequency of cells in which the xid-bearing X chromosome was active in lymphocytic and other cell populations. In adults, such cells (termed xid cells) were virtually absent in FACS-sorted splenic and lymph node B cells, and in all three splenic subpopulations distinguished on the basis of their relative expression of membrane mu and delta chains. Thus, the xid mutation appeared to compromise the development of all B cells. Erythrocytes, thymocytes, T cells, and granulocytes were unaffected. Selection against xid cells was less pronounced in the spleens of 2-6-wk-old mice. In the bone marrow, there was evidence for selection against xid in the production of B cells (except at 2 wk of age), but not at the pre-B cell level. These data suggest that, in competition with normal non-xid cells, newly-formed xid B cells were less likely to be incorporated into the peripheral B cell pool.

Numbers and dispersion of repopulating hematopoietic cell clones in radiation chimeras as functions of injected cell dose.

Lethally irradiated mice were repopulated with low (10(5)), medium (10(6)) or high (10(7)) doses of congenic bone marrow cells. Marrow donors were heterozygous for the X-chromosome-encoded allozyme marker phosphoglycerate kinase (PGK-1). A second allozyme marker, phosphoglucose isomerase (GPI-1), distinguished between donor and radioresistant host cells. Use of these markers allowed the numbers and dispersion of repopulating hematopoietic clones to be estimated by binomial statistics. The number of major repopulating clones was related to the injected cell dose in a linear fashion, the inferred frequency of clonogenic cells in donor bone marrow being about 1:40,000. In high-dose recipients, the clones grew locally, with little or no dispersion between bones. Low-dose recipients, in contrast, carried widely dispersed clones; these tended to become reduced in number with increasing time after repopulation. Most of the (few) bone marrow clones present in low-dose recipients were also present in the thymus. In contrast, only about 10% of bone marrow clones in high-dose recipients were substantially represented in the thymus at any one time--about 16 clones in each lobe.

Influence of reserpine on in vivo localization of injected lymph node cells in the mouse.

The effects of reserpine, and other agents that affect the storage and availability of 5-hydroxytryptamine (5HT), on the localization of injected 51Cr-labelled syngeneic lymph node cells have been investigated. A high dose (5 mg/kg) of reserpine to the recipients reduced localization in the lymph nodes and prevented the usual accumulation of lymphocytes in lymph nodes draining the site of an antigen (sheep erythrocytes: SE) injection. These effects were partially reversible by the monoamine oxidase inhibitor nialamide. This dose of reserpine produced deep sedation throughout the period of the experiment. Lower doses, up to 2.5 mg/kg, produced little sedation and had no effect on the localization of lymphocytes. Other workers had previously reported reduced localization of cells in delayed-type hypersensitivity (DTH) lesions after treatment of the recipients with 5 mg/kg reserpine, and had interpreted this in terms of a role of 5HT in promoting vascular permeability and egress of blood cells. The effect of lower doses of reserpine was not reported. We suggest that the effects on cell localization in both sets of experiments may have been secondary to the general state of sedation and not attributable to a direct local influence of 5HT. Other effects of reserpine included prolonged retention of lymphocytes in lungs and blood, and a reduction of cellularity and DNA synthesis in the thymus, spleen and lymph nodes.

Stromal cells in long-term murine bone marrow culture: FACS studies and origin of stromal cells in radiation chimeras.

Adherent layers from hematopoietically active long-term bone marrow cultures (LTBMC), incubated with fluorescent beads, were analyzed for autofluorescence and phagocytic ability, using a fluorescence-activated cell sorter (FACS). Four groups of cells were separated from the adherent layers, including a group of large polygonal fibroblastoid stromal cells. Long-term chimeras were made by lethal irradiation of CBA/Ca (CBA) and C57Bl6/J (B6) mice and repopulation with phosphoglycerate kinase (PGK-1) alloenzyme-congenic bone marrow cells. Hematopoietically active LTBMC were established from such chimeras, and donor and host contributions of FACS-sorted adherent-layer cells were measured. While macrophages and other hematopoietic cells were of donor origin, the fibroblastoid stromal cells were mainly or entirely host derived.

Decreased membrane potential of T lymphocytes in ageing mice: flow cytometric studies with a carbocyanine dye.

The membrane potential of lymphocytes from young (1-4-month-old) and old (25-37-month-old) CBA/Ca mice was studied with the aid of the fluorescent dye 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)). In young mice, most B and T lymphocytes showed a high, and equal, degree of polarization. In old animals most, and in the older individuals almost all, T lymphocytes were found to be depolarized; both Lyt-2+ and Lyt-2- subsets were affected. B cells were largely unaffected. Since changes in transmembrane potential, including temporary hyperpolarization, are known to accompany lymphocyte activation, the depolarized state of T cells in old mice may be related to the decline of T-cell function that occurs during senescence.

Lymphocyte subsets in renal carcinoma--a sequential study using monoclonal antibodies.

Using monoclonal antibodies in conjunction with flow cytometry, circulating lymphocyte subsets with distinct functions in the regulation of the immune response were enumerated in 32 patients with proven renal carcinoma. Analyses were performed at presentation and sequentially during the clinical course of the patients. Untreated patients with advanced disease had a deficit of T cells with the "helper/inducer" phenotype (Leu-3a+) and this resulted in abnormal T "helper/suppressor" (Leu-3a+/Leu-2a+) ratios. Following nephrectomy, performed in 26 patients, there was a significant increase in the number of T cells with the "helper/inducer" phenotype and a significant increase in T "H/S" ratios. Subsequent follow-up at a minimum of 2 months after nephrectomy showed that the increase in T cells with the "helper/inducer" phenotype was maintained (with the exception of 6 patients with disease progression) and was then accompanied by a significant increase of the T cell subset with the "suppressor/cytotoxic" phenotype (Leu-2a+). Pre-operative renal arterial embolisation resulted in an early transient lymphopenia. The response to embolisation combined with nephrectomy was little different when compared with nephrectomy alone. These observations represent a novel view of the immunosuppressive effects of renal carcinoma and their relation to anaemia and disease progression are discussed.

Specificity of tumour associated transplantation antigens (TATA) of different clones from the same tumour.

The TATA of two clones from the same murine methylcholanthrene-induced fibrosarcoma have been investigated by immunizing syngeneic mice with irradiated cells of one or both clones and challenging them 14 days later with viable cells. The tumour had been induced in a female backcross CBA mouse heterozygous for the A and B alloenzymes of phosphoglycerate kinase-1 (PGK-1). One clone expressed A and the other B, and both A and B hosts were used in the experiments. Each clone was found to possess strong TATA but there was no demonstrable cross reactivity. The clonal composition of tumours produced by inoculating mice with a mixture of the two clones was profoundly altered by prior immunization with one of them. A second experiment was performed with 3 clones from another tumour; these expressed PGK-1 A, B and AB respectively. Again, there was no evidence of immunological cross reactivity between the A and B clones, but there was some cross reactivity between the A clone and AB clone. These results, coupled with previous observations of changes in the clonal composition of pleoclonal murine fibrosarcomas in culture and on transplantation, suggest that the antigenic specificity of these tumours is less stable than is commonly supposed.

Right angle light scatter: a necessary parameter in flow cytofluorimetric analysis of human peripheral blood mononuclear cells.

The techniques of flow cytometry are being increasingly used to classify lymphocytes according to their surface antigens. Accurate analyses depend on the exclusion of non-lymphocytic contaminant cells. Using a fluorescence-activated cell sorter (FACS IV), virtually complete separation of human lymphocytes from monocytes and granulocytes can be achieved by means of wide-angle (90 degrees) light scatter. The technique described is simple and suitable for routine use. Used in conjunction with fluorescein-labelled monoclonal antibodies and logarithmic amplification of the fluorescence signals, it improves the accuracy of lymphocyte subset analysis. Its use is particularly necessary in the study of subjects such as renal allograft recipients, who have a high or variable contaminant cell:lymphocyte ratio.

Circadian variation of lymphocyte subpopulations: a study with monoclonal antibodies.

Use of monoclonal antibodies to identify subpopulations of circulating lymphocytes in healthy adults showed pronounced circadian variations in total T cells, the two major T cell subsets, and HLA-DR+ lymphocytes. When the results for the T cell subsets were expressed as a ratio (helper:suppressor) no significant rhythmic variation was observed. Lymphocytes bearing a surface antigen identified by the HNK-1 antibody (a population containing the natural killer and antibody dependent killer activity) did not show significant rhythmic variation. There was an inverse relation between plasma cortisol concentration and numbers of T and B cells. These observations have therapeutic implications and should be considered in the course of immunological monitoring.