Robert C. Habbersett

Influence of vesicle size on complement-dependent immune damage to liposomes

Complement-dependent antibody-mediated damage to multilamellar lipid vesicles (MLVs) normally results in a maximum release of 50-60% of trapped aqueous marker. The most widely accepted explanation for this is that only the outermost lamellae of MLVs are attacked by complement. To test this hypothesis, complement damage to two different types of large unilamellar vesicles (LUVs), large unilamellar vesicles prepared by the reverse-phase evaporation procedure (REVs) and large unilamellar vesicles prepared by extrusion techniques (LUVETs), were determined. In the presence of excess antibody and complement the LUVs released a maximum of only approx. 25 to 40% of trapped aqueous marker, instead of close to 100% that would be expected. Since small unilamellar vesicles apparently differ from LUVs in that they can release 100% of trapped aqueous marker it appeared that the size of the vesicles was an important factor. Because of these observations the influence of MLV size on marker release was examined. Three populations of MLVs of different sizes were separated by a fluorescence activated cell sorter. Assays of the separated MLV populations showed that the degree of complement-dependent marker release was inversely related to MLV size. No detectable glucose was taken up by MLVs when glucose was present only outside the liposomes during complement lysis. Our results can all be explained by the closing, or loss, of complement channels. We conclude that complement channels are only transiently open in liposomes, and that loss of channel patency may be due to either channel closing or to loss of channels.

High level expression of the plasma cell antigen PC.1 on the T-cell subset expanding in MRL/MpJ-lpr/lpr mice: detection with a xenogeneic monoclonal antibody and alloantisera.

MRL/MpJ-lpr/lpr (MRL-lpr/lpr) mice spontaneously develop a systemic lupus erythematosus-like syndrome associated with the expansion of a T-cell subset exerting helper activity for autoantibody production. Several studies have demonstrated that these T cells have unusual phenotypic characteristics including the expression of the B220 B-cell marker. To further characterize the antigenic profiles of these T cells, we have generated monoclonal antibodies (MAb) by immunizing rats with MRL-lpr/lpr T cells. Using flow cytofluorometry analysis, one of these MAb (4G6), described here, was found to react strongly with T cells of MRL-lpr/lpr mice but weakly with T cells of congenic mice lacking the lpr mutation (MRL/MpJ-+/+ mice). This MAb also stained brightly T cells from C3H/Hej-lpr/lpr mice and dimly those from normal C3H/Hej mice. However, it failed to react with T cells from C57Bl/6-lpr/lpr mice or normal C57Bl/6 (B6) mice. Analysis of 4G6 reactivity (weak vs negative) of T cells in a series of inbred strains demonstrated a correlation with the Pca-1a genotype known to result in expression of the PC.1 antigen on plasma cells. Immunoprecipitation studies revealed that the 4G6 antigen has a mean apparent molecular weight of 115,000, under reducing conditions, similar to that of PC.1. Moreover, high expression of 4G6 was found on plasmacytoma lines and B blasts but not on T blasts. Identity of the 4G6 antigen with PC.1 was confirmed by the finding that conventional anti-PC.1 alloantisera could block the cell surface binding of the 4G6 MAb. Therefore, T cells from MRL-lpr/lpr (and C3H-lpr/lpr) mice aberrantly carry high levels of a plasma cell antigen, detected by the 4G6 MAb, which substantiates further that these T cells represent a unique subset with some surface properties of the B-cell lineage.

Alterations of the T-cell population in BXSB mice: Early imbalance of 9F3-defined Lyt-2+ subsets occurs in the males with rapid onset lupic syndrome

BXSB mice represent a model for systemic lupus erythematosus (SLE). Due to a Y chromosome-linked genetic defect, males of this strain suffer from SLE much earlier in life than do the females. Comparative study of male and female BXSB mice therefore provides a way to identify abnormalities of the immune system leading to accelerated SLE development. The present work is part of an effort to examine whether T-cell alterations accompany such immune abnormalities. We focused on the evolution of Lyt-2+ T-cell subsets as defined by the 9F3 monoclonal antibody (MAb). By means of two-color flow cytofluorometry analysis, 9F3+ Lyt-2+ and 9F3- Lyt-2+ cell subsets could be clearly distinguished in the lymph nodes (LN) of BXSB mice. At as early as 2 months of age, BXSB males showed an increase of 9F3+ Lyt-2+ cell frequency compared to the females. This sex-related difference became more pronounced upon further aging. In 9- to 11-month-old mice, 9F3+ cells accounted for 80-85% of the LN Lyt-2+ population in the males versus 40-45% in the females. This difference reflected the selective expansion of 9F3+ Lyt-2+ cells in the males. It was also observed at a younger age in autoimmune (NZW X BXSB)F1 hybrids but not in old nonautoimmune C57Bl/6 or (CBA/N X BXSB)F1 mice. Moreover, adult thymectomy of BXSB mice was found to hasten the shift of 9F3-defined Lyt-2+ subset proportions. We postulate that the early imbalance of 9F3-defined Lyt-2+ subsets seen spontaneously in BXSB males may result from some thymus dysfunction and may be related to the development of autoimmunity.

Thymic abnormalities induced by castration in aged (NZB × SJL)F1 male mice: Distinct effects of early and late castration

Female but not male (NZB X SJL)F1 (NS) hybrid mice develop thymic abnormalities during aging. To determine the possible participation of androgens in this sex-related difference, we investigated the effect of androgen deprivation, as can be achieved by orchidectomy, on the cellular composition of the thymus of old NS males. Mice were orchidectomized at ages ranging from 3 weeks to 9 months and their thymuses were studied at the ages of 12 or 18 months. Phenotypic characterization of the intrathymic lymphocyte population was carried out using flow cytofluorometry analysis. Orchidectomy performed early in life (3 weeks-3 months) resulted, at 12 months of age, in thymic alterations (expansion of dull Thy-1+ cells, emergence of surface immunoglobulin-bearing cells) resembling those occurring spontaneously in old NS females. In contrast, when orchidectomy was performed later in life (6-9 months), there was a numerical increase of all thymocyte subsets but no major qualitative abnormality at either 12 or 18 months of age. Therefore, the absence of thymic disease in intact NS males may reflect primarily a suppressive effect of androgens that can be reversed by early but not by late orchidectomy.

Alterations of the intrathymic lymphocyte population in aged (NZB X SJL)F1 female mice as revealed by the differential effects of hydrocortisone and cyclophosphamide treatments.

Abstract The thymus of NZB x SJL female (NSF) mice becomes enlarged during aging. The purpose of this study was to define the alterations of the intrathymic lymphocyte population underlying this thymic hypertrophy. Thymocyte subsets were delineated by their differential sensitivity to in vivo administration of hydrocortisone (HC) or Cyclophosphamide (CY) and were further characterized by their physical properties and their surface phenotype. The subset of relatively HC-resistant thymocytes (high-electrophoretic mobility (EPM), low Thy-1+, Lyt-1+,2±, and low PNA+) was found expanded by four- to fivefold in 12-month-old NSF mice as compared to 3-month-old NSF mice. These cells were also resistant to CY. In contrast, the HC-sensitive thymocytes (low EPM, high Thy-1+, Lyt-1+,2+, and high PNA+), a subset of which was CY-resistant, were depleted by five- to sixfold in aged mice. Additionally, two subsets of HC-resistant, CY-sensitive cells were found to emerge in the thymus of 12-month-old NSF mice. The first of these subsets, with low EPM, was devoid of T-cell markers but expressed large amounts of surface immunoglobulins and thus probably corresponds to B cells. The other subset, which was more HC-resistant and possessed a higher EPM and larger volume than the former one, has been previously identified as plasma cells. These data indicate that the thymic hypertrophy of aging NSF mice reflects the intrathymic accumulation of HC-resistant, CY-resistant T cells and of HC-resistant, CY-sensitive cells of the B lineage.