==================================BSR18================================== 18. Characteristics of NZB (Mouse) B-lymphocytes: especially cell surface phenotype (cell surface markers, antigens) and response to LPS (Lipopolysaccharide). 1 UI - 86305831 AU - Huston DP ; Tavana G ; Rich RR ; Gressens SE TI - Regulation of primary cytotoxic T lymphocyte responses generated during mixed leukocyte culture with H-2d identical Qa-1-disparate cells. AB - Cytotoxic lymphocyte (CTL) responses are not usually generated during primary mixed leukocyte culture (MLC) with H-2 identical cells. Thus NZB mice are unusual in that their spleen cells do mount CTL responses during primary MLC with H-2d identical stimulator cells; the predominant target antigen for these NZB responses is Qa-1b. Considering the numerous immunoregulatory defects in NZB mice, we postulated that these NZB anti-Qa-1 primary CTL responses were due to an abnormality in T suppressor cell activity. Cellular interactions capable of suppressing NZB anti-Qa-1 primary CTL responses were investigated by using one-way and two-way MLC with spleen cells from NZB mice and other H-2d strains. Although H-2d identical one-way MLC with the use of NZB responders resulted in substantial CTL responses, only minimal CTL responses were detected from two-way MLC with the use of NZB spleen cells plus nonirradiated spleen cells from other H-2d mice. Thus the presence of non-NZB spleen cells in the two-way H-2d identical MLC prevented the generation of NZB CTL. Noncytotoxic mechanisms were implicated in the suppression of the NZB CTL responses during two-way MLC, because only minimal CTL activity was generated when NZB spleen cells were cultured with semiallogeneic, H-2d identical (e.g., NZB X BALB) F1 spleen cells. The observed suppression could be abrogated with as little as 100 rad gamma-irradiation to the non-NZB spleen cells. The phenotype of these highly radiosensitive spleen cells was Thy-1+, Lyt-1+, Lyt-2-, L3T4+. The functional presence of these cells in the spleens of semiallogeneic, H-2d identical F1 mice indicated that their deficiency in NZB mice was a recessive trait. These data suggest that NZB mice lack an L3T4+ cell present in the spleens of normal mice that is capable of suppressing primary anti-Qa-1 CTL responses. This model system should facilitate additional investigations of the cellular interactions and immunoregulatory mechanisms responsible for controlling primary CTL responses against non-H-2K/D class I alloantigens. The model may also provide insight into the immunoregulatory defects of autoimmune NZB mice. MH - Animal ; Antigens, Surface/ANALYSIS/*IMMUNOLOGY ; Cells, Cultured ; *Cytotoxicity, Immunologic/RADIATION EFFECTS ; H-2 Antigens/*IMMUNOLOGY ; Leukocyte Culture Test, Mixed ; Mice ; Mice, Inbred NZB/*IMMUNOLOGY ; Mice, Inbred Strains ; Spleen/IMMUNOLOGY ; Support, U.S. Gov't, P.H.S. ; Suppressor Cells/*IMMUNOLOGY ; T Lymphocytes, Cytotoxic/CLASSIFICATION/ *IMMUNOLOGY SO - J Immunol 1986 Sep 15;137(6):1776-81 2 UI - 86225625 AU - Caughman SW ; Breathnach SM ; Sharrow SO ; Stephany DA ; Katz SI TI - Culture and characterization of murine dendritic Thy-1+ epidermal cells. AB - Although numerous advances have been made in characterizing the phenotype, ontogeny, ultrastructure, and cytochemistry of the murine Thy-1+ dendritic epidermal cell (Thy-1+ EC), elucidation of its functional qualities has been hampered by the difficulty in preparing pure populations of these cells. We therefore sought to obtain expanded, purified populations of Thy-1+ EC using culture techniques. Since Thy-1+ EC are bone marrow-derived, density gradient enriched populations of freshly harvested epidermal cells (FH-EC) were placed in culture under conditions known or suspected to promote mitogenesis among leukocyte subsets. FH-EC prepared from truncal skin of C3H/HeN mice (Thy-1.2+) were cultured at 37 degrees C in 5% CO2 in complete medium (CM) of Eagle's Hanks' amino acid with 10% fetal calf serum, nutrients, and antibiotics at 10(6) FH-EC/well in 24-well culture plates. CM was supplemented with one or more of the following: concanavalin A (Con-A), interleukin-1/epidermal cell-derived thymocyte-activating factor (IL-1/ETAF), IL-2, IL-3, gamma interferon, indomethacin (IM), and anti-Thy-1.2 antibody. Media with appropriate supplements were changed every 2-3 days. Freshly isolated, enriched FH-EC contained 7-20% Thy-1+ EC (defined as brightly fluorescing cells readily distinguishable from weakly fluorescing keratinocytes), which also stained with antibodies directed against asialo GM1, Ly 5.1, and vimentin but did not stain with antibodies to other T cell-, B cell- or macrophage phenotypic markers. Analysis of 10 separate cultures revealed a 3- to 10-fold expansion of nonkeratinocyte Thy-1+ cells after 21 +/- 4 days in culture in CM supplemented with Con-A and IM, and 70-100% of viable cells after expansion were Thy-1+. Phenotypic analysis of expanded cells revealed the emergence in 10 separate cultures of one of two mutually exclusive distinct populations: one Thy-1+, asialo GM1+, L3T4- (natural killer phenotype) and the other Thy-1+, asialo GM1-, L3T4+ (T helper phenotype). Experiments designed to explain the emergence of an L3T4+ population suggest that phenotypic modulation occurred in vitro. MH - Animal ; Antigens, Ly/*IMMUNOLOGY ; Antigens, Surface/GENETICS/*ISOLATION & PURIFICATION ; Cells, Cultured ; Concanavalin A/PHARMACODYNAMICS ; Female ; Indomethacin/PHARMACODYNAMICS ; Lymphoid Tissue/*CYTOLOGY/ IMMUNOLOGY ; Male ; Mice ; Mice, Inbred NZB ; Mice, Inbred Strains ; Microscopy, Electron ; Phenotype ; Skin/*CYTOLOGY ; T Lymphocytes/ IMMUNOLOGY ; Trypsin/PHARMACODYNAMICS SO - J Invest Dermatol 1986 Jun;86(6):615-24 3 UI - 86197780 AU - Cowdery JS ; McKiernan FE TI - Analysis of T cell and B cell function in Peyer's patch and lamina propria of New Zealand Black and DBA/2 mice. AB - We have analyzed gastrointestinal immune function in both DBA/2 and spontaneously autoimmune New Zealand Black (NZB) mice. We have studied both in vitro proliferation and differentiation of Peyer's patch cells and have measured immunoglobulin (Ig) secretion by cultured jejunal segments. Peyer's patch B cells and T cells from both DBA/2 and NZB mice showed similar proliferative responses to Con A and lipopolysaccharide (LPS), respectively. Unlike NZB splenic B cells, isolated Peyer's patch B cells from NZB mice did not spontaneously secrete Ig of any isotype. Seven-day cultures of equal numbers of Peyer's patch T cells and B cells resulted in similar patterns of secretion of IgA, IgG, and IgM in both strains. The addition of Con A to cultures of DBA/2 Peyer's patch cells consistently resulted in a onefold to threefold increase in IgA secretion after 7 days. Con A stimulation of NZB Peyer's patch cells did not produce any increment in IgA secretion. LPS stimulation of Peyer's patch cells from either strain resulted in a similar increase in IgG secretion with little effect on IgA secretion. The in vivo correlate of this finding was seen in the IgA to IgG ratio of Ig secreted by cultured jejunal fragments. In DBA/2 mice the rates of IgA/IgG varied from 2.36 to 4.85, whereas in NZB mice the ratio never exceeded 0.5. These experiments show that defects on the T cell compartment of NZB mice encompass gut-associated lymphoid tissue. The possible relationship of these findings and previously observed defects in oral tolerance is discussed. MH - Animal ; B Lymphocytes/*IMMUNOLOGY/METABOLISM ; Concanavalin A/ PHARMACODYNAMICS ; Female ; IgA/BIOSYNTHESIS ; IgG/BIOSYNTHESIS ; Intestinal Mucosa/*IMMUNOLOGY/METABOLISM ; Jejunum/IMMUNOLOGY/METABOLISM ; Lipopolysaccharides/PHARMACODYNAMICS ; Lymphocyte Transformation ; Mice ; Mice, Inbred DBA ; Mice, Inbred NZB ; Peyer's Patches/*IMMUNOLOGY/ METABOLISM ; Species Specificity ; Support, U.S. Gov't, P.H.S. ; T Lymphocytes/*IMMUNOLOGY/METABOLISM ; Tissue Culture SO - J Immunol 1986 Jun 1;136(11):4070-4 4 UI - 86169676 AU - Kotzin BL ; Arndt R ; Okada S ; Ward R ; Thach AB ; Strober S TI - Treatment of NZB/NZW mice with total lymphoid irradiation: long-lasting suppression of disease without generalized immune suppression. AB - We used total lymphoid irradiation (TLI; total dose = 3400 rad) to treat the lupus-like renal disease of 6-mo-old female NZB/NZW mice. Similar to our past studies, this treatment resulted in a marked prolongation of survival, decrease in proteinuria, and decrease in serum anti-DNA antibodies compared with untreated littermate controls. Although there was no evidence of disease recurrence in TLI-treated mice until after 12 mo of age, the in vitro proliferative response to phytohemagglutinin by NZB/NZW spleen cells recovered within 6 wk such that responses were greater than control NZB/NZW animals. A similar recovery and overshoot after TLI were evident in the primary antibody response to the T cell-dependent antigen sheep red blood cells (SRBC). Both the total and IgG anti-SRBC antibody responses after TLI were greater than those of untreated NZB/NZW controls, and were comparable with those of untreated non-autoimmune mice. Despite this increased response to mitogens and antigens after TLI, we noted a decrease in spontaneous splenic IgG-secreting cells and a decrease in IgG but not IgM antinuclear antibody production. Nonspecific suppressor cells of the mixed leukocyte response were detectable in the spleens of NZB/NZW mice early after TLI. However, the disappearance of suppressor cells was not associated with recrudescence of disease activity. Furthermore, transfer of large numbers of spleen cells from TLI-treated NZB/NZW mice did not result in disease suppression in untreated age-matched recipients. In summary, treatment of NZB/NZW mice with TLI results in a prolonged remission in autoimmune disease, which is achieved in the absence of generalized immunosuppression. MH - Animal ; Antibody-Producing Cells/METABOLISM ; Autoimmune Diseases/ *IMMUNOLOGY/PHYSIOPATHOLOGY ; B Lymphocytes ; Female ; Immunoglobulin Allotypes/BIOSYNTHESIS ; *Immunosuppression/METHODS ; Leukocyte Count/ RADIATION EFFECTS ; Leukocyte Culture Test, Mixed ; Lipopolysaccharides/ PHARMACODYNAMICS ; Lymphocyte Transformation ; Mice ; Mice, Inbred BALB C ; Mice, Inbred NZB ; Phytohemagglutinins/PHARMACODYNAMICS ; Spleen ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, Non-P.H.S. ; T Lymphocytes ; Time Factors ; *Whole Body Irradiation SO - J Immunol 1986 May 1;136(9):3259-65 5 UI - 86169604 AU - Rabinovitch PS ; Torres RM ; Engel D TI - Simultaneous cell cycle analysis and two-color surface immunofluorescence using 7-amino-actinomycin D and single laser excitation: applications to study of cell activation and the cell cycle of murine Ly-1 B cells. AB - The DNA-binding, fluorescent dye 7-amino-actinomycin D (7AAD) is efficiently excited by the 488 nm laser line commonly used in flow cytometry, but yields fluorescence emission further into the red spectrum than alternative DNA-specific fluorochromes. In this report, we show that the spectral properties of 7AAD allow single-laser analysis of DNA content and cell cycle simultaneously with two cell surface markers labeled with fluorescein (green)-and phycoerythrin (orange)-conjugated antibodies. The use of 7AAD makes three-color analysis practical and feasible, using the most widely available flow cytometric instruments. The power of this technique was demonstrated in two systems. Staining of human peripheral blood lymphocytes (PBL) with 7AAD was demonstrated to be dependent on cell activation and chromatin conformation; PHA-stimulated cells which have become activated and express IL 2 receptors had greater 7AAD fluorescence than nonactivated, IL 2 receptor-negative cells. Cell cycle analysis of mouse splenocytes stained with fluorescent antibodies to IgM and to Ly-1 demonstrated that the proportion of S and G2 phase cells in native spleen varies strongly among the subsets of cells identified with these markers. Of particular interest was the striking finding that the Ly-1+/IgM+ subset (Ly-1 B cells) is greatly enriched for cells in the S phase fraction. This is important because Ly-1 B cells have been associated with the production of autoantibodies, and is consistent with reports that these cells have a lymphoblastoid or a plasmablast morphology. We hypothesize that Ly-1 B cells may belong to a subset of in vivo activated cells which are either rapidly proliferating or are arrested in S phase. MH - Animal ; Antigens, Ly ; B Lymphocytes/CLASSIFICATION/*CYTOLOGY/IMMUNOLOGY ; *Cell Cycle ; Dactinomycin/*ANALOGS & DERIVATIVES ; Female ; *Flow Cytometry/METHODS ; Fluorescent Antibody Technic ; *Fluorescent Dyes ; Human ; Interphase ; Lasers ; *Lymphocyte Transformation ; Mice ; Mice, Inbred BALB C ; Mice, Inbred NZB ; Spectrometry, Fluorescence ; Stains and Staining ; Support, U.S. Gov't, P.H.S. SO - J Immunol 1986 Apr 15;136(8):2769-75 6 UI - 86113401 AU - Sekigawa I ; Ishida Y ; Hirose S ; Sato H ; Shirai T TI - Cellular basis of in vitro anti-DNA antibody production: evidence for T cell dependence of IgG-class anti-DNA antibody synthesis in the (NZB X NZW)F1 hybrid. AB - An in vitro system was designed to measure anti-DNA antibody synthesis, and the cellular basis of this autoantibody production in NZB X NZW (B/W)F1 (B/W F1) mice was analyzed. The spleen cells from old B/W F1 mice contained a number of B cells that spontaneously produced anti-DNA antibodies of both IgM and IgG classes in the absence of stimulants, thereby demonstrating that these B cells had been activated in vivo. These activated B cells could be removed by Sephadex G-10 column (G-10) filtration. Such G-10-passed, homogeneously small B cells were activated by the stimulant lipopolysaccharide (LPS) and produced both IgM and IgG class anti-DNA antibodies. The G-10-passed cells contained both B and T cells, and the cytotoxic treatment of the cells with monoclonal antibodies to T cells, anti-Thy-1 and anti-L3T4, abolished the LPS-induced IgG class, but not IgM class, anti-DNA antibody syntheses. Thus, the LPS-induced production of IgG class anti-DNA antibodies in B/W F1 mice is regulated by T cells. Reconstitution experiments revealed the requirement of T-B cell contact but not of the proliferative response of T cells. Moreover, there was no apparent adherent cell requirement. Such IgG class anti-DNA antibodies were produced only by spleen cells from old B/W F1 mice, but not from young B/W F1, NZB, NZW, and C57BL/6 mice. Like IgM class anti-DNA antibodies, LPS-induced synthesis of polyclonal IgM was T cell-independent. Only a slight reduction in the polyclonal IgG synthesis was observed after the G-10-passed cells had been treated with anti-Thy-1 antibody plus complement. This study should facilitate investigation of cell to cell interactions in the formation of autoantibodies and their correlations to immunologic abnormalities in autoimmune disease. MH - Animal ; Antinuclear Factors/*BIOSYNTHESIS/CLASSIFICATION ; Autoimmune Diseases/IMMUNOLOGY ; Cell Separation ; Chromatography, Gel ; Crosses, Genetic ; DNA/*IMMUNOLOGY ; Female ; IgG/*BIOSYNTHESIS ; Lipopolysaccharides/PHARMACODYNAMICS ; Lymphocyte Transformation ; Mice ; Mice, Inbred C57BL ; Mice, Inbred NZB ; Spleen/CYTOLOGY ; Support, Non-U.S. Gov't ; T Lymphocytes/IMMUNOLOGY/*METABOLISM SO - J Immunol 1986 Feb 15;136(4):1247-52 7 UI - 86113392 AU - Smith HR ; Yaffe LJ ; Kastner DL ; Steinberg AD TI - Evidence that Lyb-5 is a differentiation antigen in normal and xid mice. AB - These studies addressed the question of whether the Lyb-5 determinant on mouse B cells might represent a marker for a unique lineage of B cells as has been thought, or rather, a differentiation antigen. Previous studies have addressed this question by treating splenic B cells with anti-Lyb-5 + complement and then culturing them with various antigens. In this study, we first immunized in vivo or in vitro and then determined susceptibility to anti-Lyb-5. These studies demonstrate that a substantial proportion of antibody-forming cells produced in response to immunization with sheep red blood cells, trinitrophenyl (TNP)-keyhole limpet hemocyanin, TNP-Brucella abortus, TNP-lipopolysaccharide, as well as TNP-Ficoll, are eliminated by anti-Lyb-5 + complement treatment at the end of culture. Some generation of Lyb-5+ antibody-forming cells occurred in the last 24 hr of culture. These studies suggest that the Lyb-5 marker represents a differentiation antigen on relatively mature B cells. MH - Animal ; Antigens, Ly/ANALYSIS/*IMMUNOLOGY ; Antigens, Surface/ANALYSIS/ *IMMUNOLOGY ; B Lymphocytes/*IMMUNOLOGY/METABOLISM ; Brucella Abortus/ IMMUNOLOGY ; Complement/PHYSIOLOGY ; Cytotoxicity, Immunologic ; Female ; Guanosine/ANALOGS & DERIVATIVES/PHARMACODYNAMICS ; Hemolytic Plaque Technic ; IgG/BIOSYNTHESIS ; Isoantibodies/PHYSIOLOGY ; Lipopolysaccharides/IMMUNOLOGY ; *Lymphocyte Transformation/DRUG EFFECTS ; Male ; Mice ; Mice, Inbred DBA ; Mice, Inbred NZB ; Spleen/CYTOLOGY ; Time Factors ; Trinitrobenzenes/IMMUNOLOGY ; X Chromosome/IMMUNOLOGY SO - J Immunol 1986 Feb 15;136(4):1194-200