RESUMO
Flow cytometric analysis of antigen-specific, idiotype-positive (id+), B cell development in transgenic mice expressing a rearranged M167-mu gene shows that large numbers of phosphocholine (PC)-specific, M167-id+ B cells develop in the spleen and bone marrow of these mice. Random rearrangement of endogenous V kappa genes, in the absence of a subsequent receptor-driven selection, should give rise to equal numbers of T15- and M167-id+ B cells. The observed 100-500-fold amplification of M167-id+ B cells expressing an endogenous encoded V kappa 24]kappa 5 light chain in association with the M167 VH1-id transgene product appears to be an antigen driven, receptor-mediated process, since no amplification of non-PC-binding M167 VH1/V kappa 22, T15-id+ B cells occurs in these mu-only transgenic mice. The selection and amplification of antigen-specific, M167-id+ B cells requires surface expression of the mu transgene product; thus, no enhancement of M167-id+ B cells occurs in the M167 mu delta mem-transgenic mice, which cannot insert the mu transgene product into the B cell membrane. Surprisingly, no selection of PC-specific B cells occurs in M167-kappa-transgenic mice although large numbers of B cells expressing a crossreactive M167-id are present in the spleen and bone marrow of these mice. The failure to develop detectable numbers of M167-id+, PC-specific B cells in M167-kappa-transgenic mice may be due to a very low frequency of M167-VH-region formation during endogenous rearrangement of VH1 to D-JH segments. The somatic generation of the M167 version of a rearranged VH1 gene may occur in less than one of every 10(5) bone marrow B cells, and a 500-fold amplification of this M167-Id+ B cell would not be detectable by flow cytometry even though the anti-PC antibody produced by these B cells is detectable in the serum of M167-kappa-transgenic mice after immunization with PC.
Assuntos
Linfócitos B/imunologia , Rearranjo Gênico , Genes de Imunoglobulinas , Idiótipos de Imunoglobulinas/análise , Cadeias mu de Imunoglobulina/genética , Fosforilcolina/farmacologia , Animais , Afinidade de Anticorpos , Sequência de Bases , Medula Óssea/imunologia , Feminino , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/imunologia , Cadeias mu de Imunoglobulina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , RNA Mensageiro/análiseRESUMO
Mouse spleen cells can be stimulated to proliferate in vitro by purified anti-mu or anti-gamma,kappa antibodies. These responses can be obtained in cell populations bearing membrane immunoglobulin (Ig), purified by the fluorescence activated cell sorter (FACS), but they are not observed in FACS-purified Ig- cell populations. Furthermore, treatment of spleen cell populations with anti-Thy 1.2 and complement does not impair the response, nor does addition of nylon wool-purified T lymphocytes enhance it. These results indicate that B lymphocytes respond to anti-Ig and that their response does not require T cells. On the other hand, cells from athymic nude (nu/nu) mice respond slightly less well to anti-mu than do cells from heterozygous littermate (nu/+) controls; nu/nu cells are almost unresponsive to anti-gamm,kappa and addition of nylon wool-purified T cells from nu/+ controls does not restore the response. This suggests that T lymphocytes or the thymus may control the appearance of cells responsive to anti-gamma,kappa. Responsiveness of normal mice to anti-mu does not appear until 4 wk of age and does not reach maximum levels until 8 wk of age. Acquisition of full responsiveness to anti-gamma,kappa is even more delayed. This, together with the failure of mice with the CBA/N B-cell defect to respond to anti-Ig, suggests that cells stimulated to proliferate by anti-Ig are a mature subset of B cells. Depletion of adherent cells by Sephadex G-10 treatment or by treatment with carbonyl iron and exposure to a magnetic field does not diminish anti-mu or anti-gamma,kappa responses, suggesting that the responsiveness does not require the presence of macrophages. Thus, activation of B-cell proliferation by anti-Ig appears to be a T-cell independent, macrophage-independent process in which membrane Ig plays a direct role in signal generation.
Assuntos
Anticorpos Anti-Idiotípicos , Linfócitos B/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos B , Animais , Reações Antígeno-Anticorpo , Linfócitos B/citologia , Cadeias gama de Imunoglobulina , Cadeias mu de Imunoglobulina , Cooperação Linfocítica , Tecido Linfoide/imunologia , Macrófagos/imunologia , Camundongos , Linfócitos T/imunologiaRESUMO
B lymphocyte-enriched cell populations cultured with mitogens in initial suspension cultures formed colonies in soft agar when the same mitogenic agent was present in the lower layer of a two-layer soft agar system. Colony formation depended upon the presence of T cells in the initial culture, and was optimal after an initial 72-h culture with phytohemagglutinin (PHA; 12.5 microliters/ml), pokeweed mitogen (PWM; 2.5 micrograms/ml), or protein A (10 micrograms/ml). The colonies could be picked from the agar and propagated by feeding every 3 d with medium supplemented with a growth factor-containing tissue culture supernate. The growth factor-containing supernate was prepared by stimulating pools of human peripheral blood mononuclear cells for 72 h with PHA or PWM. The lines propagated in this manner were membrane Ig+, lacked sheep erythrocyte rosette-forming ability, and did not ingest latex. They lacked the Epstein-Barr nuclear antigen (EBNA) and had 46 chromosomes. Such lines have been propagated for over 1 yr. One line (BL1) was subjected to limiting dilution cloning and a line, BL1.1, was prepared that contained 96% lambda-bearing cells and no kappa-bearing cells. This line was also EBNA negative. This procedure can thus be used to prepare and clone long-term lines of nontransformed human B lymphocytes.
Assuntos
Linfócitos B/citologia , Ativação Linfocitária , Linfócitos B/imunologia , Linhagem Celular , Células Cultivadas , Células Clonais/citologia , Células Clonais/imunologia , Substâncias de Crescimento/farmacologia , Humanos , Fito-Hemaglutininas/farmacologia , Mitógenos de Phytolacca americana/farmacologia , Receptores de Antígenos de Linfócitos B , Proteína Estafilocócica A/farmacologia , Linfócitos T/citologia , Fatores de TempoRESUMO
Spleen cell cultures from young adult mice of a variety of strains were stimulated to incorporate tritiated thymidine ([3H]TdR) by a goat anti-mouse IgM antiserum and by purified anti-mu antibodies prepared from this serum. This stimulation was shown to depend upon the anti-mu activity of the antiserum. In addition, ultracentrifuged anti-mu and F(ab')2 fragments of anti-mu were shown to be stimulatory. The anti-mu preparation lacked detectable endotoxin contamination and was also shown to stimulate response by two strains (C57BL/10ScCr and C3H/HeJ) which are unresponsive to the mitogenic effects of endotoxin, while it failed to stimulate a response by cells from a mouse strain (CBA/N) which responds to endotoxin. In addition purified goat anti-mouse gamma, kappa antibodies and rabbit anti-mouse kappa-antib odies stimulated uptake of [3H]TdR by mouse spleen cells, although to a lesser degree than the anti-mu preparation. The cell density, culture requirements, and kinetics of the response are presented.
Assuntos
Anticorpos Anti-Idiotípicos , Cadeias Pesadas de Imunoglobulinas , Cadeias mu de Imunoglobulina , Ativação Linfocitária , Receptores de Antígenos de Linfócitos B , Animais , Anticorpos Anti-Idiotípicos/isolamento & purificação , Cromatografia de Afinidade , Relação Dose-Resposta Imunológica , Fragmentos Fab das Imunoglobulinas , Cadeias kappa de Imunoglobulina , Cinética , Camundongos , Polissacarídeos Bacterianos/farmacologia , Relação Estrutura-AtividadeRESUMO
Host mutations in Escherichia coli K12 selected for the temperature-sensitive replication of the bacterial plasmid colicinogenic factor E(1) (ColE(1)) exhibit a pleiotropic effect with respect to the effect of the mutation on other extra-chromosomal elements. The mutations also vary with respect to the time of incubation of the cells at 43 degrees C required for complete cessation of ColE(1) DNA synthesis. While the synthesis of the bacterial chromosome appears unaffected, supercoiled ColE(1) DNA replication stops immediately in some mutants and gradually decreases during several generations of cell growth before stopping in others. Mutations isolated in the ColE(1) plasmid resulted in only a gradual cessation of ColE(1) DNA synthesis over several generations of cell growth at 43 degrees C. Conjugal transfer of the ColE(1) and ColV factors occurs normally in the host mutants when the transfer is carried out at the permissive temperature; however, the presence of a group I mutation in the donor cell prohibited conjugal transfer of either plasmid DNA at 43 degrees C to a normal recipient cell. Similarly, the presence of this mutation in the recipient prevented the establishment of ColE(1) or ColV in the mutant recipient cell upon conjugation with a normal donor at 43 degrees C. Various host ColE(1) replication mutants carrying either ColE(1) or ColE(2) were also defective in the mitomycin C-induced production of colicin E(1) or colicin E(2) at 43 degrees C. The majority of the host mutations examined exhibited a temperature sensitivity to growth in deoxycholate in addition to the inhibition of plasmid DNA replication, suggesting a membrane alteration in these mutants when grown at the restrictive temperature.
Assuntos
Replicação do DNA , Escherichia coli , Herança Extracromossômica , Genética Microbiana , Colífagos , DNA Viral , Ácido Desoxicólico/farmacologia , Escherichia coli/efeitos dos fármacos , Mitomicinas/farmacologia , Mutação , Temperatura , Fatores de TempoRESUMO
A method for the rapid separation of murine spleen cells into subpopulations on the basis of their size has been developed using counterflow centrifugation. Upon separation of normal spleen cells with a mean cell volume of 125.5 +/- 6.0, 5 fractions of cells were obtained with mean cell volumes which ranged from 107.8 +/- 3.2 microns3 in fraction 1 to 152.7 +/- 4.9 microns3 in fraction 5. The cells in these 5 fractions were characterized by analysis on a fluorescence activated cell sorter (FACS) after staining with fluorescein conjugated anti-mu, delta, Ia, or Thy 1.2 antibodies, and by assaying for the presence of non-specific esterase activity. Surface Ig+, Ia+ B lymphocytes and Thy 1.2 T lymphocytes were present in all 5 fractions. However, while these T and B lymphocytes accounted for virtually all of the cells in the first 3 fractions, non-T, non-B cells were found in fractions 4 and 5, and represented 30% of the total population in the 5th fraction. Comparison of the intensity of anti-mu, delta or Ia staining of the B cells in fractions 1-5 revealed differences which suggested that B cell size correlated with different activation states of these cells. Increases in the intensity of the staining of T lymphocytes by anti-theta antibodies were also noted in the various fractions. The capacity of the B and T cells in each fraction to proliferate to B or T cell mitogens, respectively, was proportional to their frequency within the fractions. By contrast, the fractions containing larger cells were enriched in cells which proliferated in vitro in the absence of added mitogen. Furthermore, only fractions containing larger cells had the capacity to stimulate allogeneic T cell proliferation in a mixed lymphocyte reaction. Our data suggest that this technique provides a useful method for separating splenocytes on the basis of cell size. Use of this methodology provides a way to correlate cell size with phenotypic surface markers and functional abilities.
Assuntos
Separação Celular/métodos , Centrifugação/métodos , Linfócitos/citologia , Macrófagos/citologia , Baço/citologia , Animais , Antígenos de Superfície/análise , Antígenos de Histocompatibilidade Classe II/análise , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Linfócitos/imunologia , Camundongos , Receptores de Antígenos de Linfócitos B/análise , Antígenos Thy-1RESUMO
Severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood lymphocytes (hu-PBL) were evaluated as an animal model for demonstrating dengue (DEN) viral infection. Reconstituted mice (hu-PBL-SCID) that demonstrated successful engraftment by the presence of serum titers of human immunoglobulin (Ig) were inoculated intraperitoneally with DEN virus serotype 1 (DEN-1). Serial blood samples were taken postinoculation and assayed for virus in C6/36 cells. The identity of all viral isolates was confirmed by an immunofluorescence antibody assay using DEN-1 monoclonal antibody. A total of six experiments were performed using different procedures of reconstitution and infection, and in three of these experiments, DEN-1 virus was recovered from the hu-PBL-SCID mice. In the first successful experiment, DEN-1 virus was recovered on postinoculation day (PID) 24 from blood, spleen, thymus, and lung tissues of one of eight hu-PBL-SCID mice. A second group of eight hu-PBL-SCID mice were inoculated with human monocytes infected in vitro with DEN-1 virus. Virus was recovered from the blood of mice between PID 15 and 23, and from lung tissue of one of these mice. In a third experiment, seven SCID mice were treated initially with anti-asialo GM1 antibody to eliminate natural killer cells, and then were injected simultaneously with a mixture of hu-PBL and DEN-1 virus. Virus was demonstrated in the blood of one mouse on PID 38, and in another mouse on PID 8, 12, 20, 24, and 36.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Dengue/imunologia , Modelos Animais de Doenças , Camundongos SCID , Animais , Vírus da Dengue/imunologia , Vírus da Dengue/isolamento & purificação , Estudos de Avaliação como Assunto , Feminino , Citometria de Fluxo , Humanos , Imunoglobulina G/sangue , Células Matadoras Naturais/imunologia , Linfócitos/imunologia , Masculino , Camundongos , Monócitos/imunologia , Viremia/imunologiaRESUMO
An IgG1 mouse monoclonal antibody (MAb) specific for a mouse IgM allotypic determinant in the a, c, f, g, h, and j haplotypes was derived from a fusion of SP2/O-Ag14 mouse myeloma cells with C57BL/6 mouse spleen cells (Igh-Cb) immune to TC31, a MAb of the IgMa allotype. MAb from one hybridoma derived from this fusion (designated DS1) was demonstrated to bind in an ELISA to immunoglobulin bearing the IgMa allotype (TC31, MOPC104E), but not to immunoglobulin bearing the IgMb allotype (C.BPC112). Fluorescein-conjugated DS1 was shown to bind to the surface of BALB/cByJ splenic B cells, but was shown to have negligible binding on C57BL/6J cells. Similarly, DS1-conjugated Sepharose beads were able to stimulate in vitro proliferation of BALB/c, but not C57BL/6 splenic B cells. DS1 was unable to bind to spleen cells from BALB/c allotype congenic strains, BAB/14 (Igh-Cb) and C.AL-20 (Igh-Co), demonstrating that DS1 recognizes a determinant under the control of a gene linked to the Igh-C gene complex. Using sera from recombinant inbred lines, the determinant defined by DS1 was shown to be linked to the Igh-1 locus. Furthermore, the determinant was localized to the CH1 domain of the mu heavy chain. Sera from BALB/cByJ, NMRI, CBA/J, SEA/GnJ, RIIIs/J, and CE/J mouse strains were shown to bind to DS1 in an ELISA, while sera from A/J, SJL/J, NZB/B1NJ, AKR/J, C57BL/6J, and C57BL/10SnJ mouse strains did not bind to DS1. From these data we propose that DS1 is reactive with specificity Igh-6.1, which was originally defined by an allotypic antiserum developed by Black et al. (Immunogenetics 7:213, 1978).
Assuntos
Anticorpos Monoclonais , Imunoglobulina M , Alelos , Animais , Anticorpos Anti-Idiotípicos , Especificidade de Anticorpos , Linfócitos B/imunologia , Epitopos/genética , Genes de Imunoglobulinas , Hibridomas/imunologia , Alótipos de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulina M/genética , Ativação Linfocitária , Camundongos , Camundongos EndogâmicosRESUMO
Four rat x mouse hybridomas secreting monoclonal anti-idiotypic (anti-Id) antibodies (MAb) specific for the transgene-encoded antibody of the 207-4 transgenic mouse line, which carries the VH1/V kappa 24 gene segments of the IgA, phosphocholine-(PC) specific MOPC167 myeloma, were developed from a fusion of Ag8-X63.653 mouse cells with spleen cells from a rat immunized with MOPC167 and HPCM27 anti-PC antibodies. The anti-Id MAb were shown by ELISA to be specific for PC-binding proteins of VH1/V kappa 24 H and L chains of various isotypes. They did not bind VH1/V kappa 22, VH1/V kappa 8, or VH1/V kappa 1 PC-binding proteins or other IgA or IgM myeloma proteins. Analysis by flow cytometry demonstrated that these MAb bind to the transgene-encoded membrane immunoglobulin (sIgM) as expressed on > 95% of the B220 positive 207-4 spleen cells. All four MAb were able to inhibit the binding of MOPC167 to PC conjugated to bovine serum albumin. Differences in fine specificity of binding were demonstrated by differential staining of spleen cells of the 216-7 mu kappa delta Mem MOPC167 transgenic mice. In these mice endogenous H chains associate with the transgene encoded L chain to form MOPC167 crossreactive idiotopes. Two of the MAb, 28-4-3 and 28-6-20, stained significant numbers of cells, while MAb 28-5-15 did not bind to 216-7 cells. Three of the MAb, 28-5-15, 28-6-20, and 28-4-3, when conjugated to Sepharose beads, were able to induce DNA synthesis in cultures of 207-4 transgenic spleen cells. None of the MAb were able to induce an antibody response in vivo. These MAb should prove useful in staining PC-transgenic B cells for flow cytometry studies and in defining early cellular events in the activation of idiotype positive B cells by anti-Id antibodies.
Assuntos
Anticorpos Monoclonais/imunologia , Idiótipos de Imunoglobulinas/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Proteínas do Mieloma/imunologia , Fosforilcolina/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Linfócitos B/imunologia , Sítios de Ligação , Bovinos , Imunoglobulina A/metabolismo , Idiótipos de Imunoglobulinas/metabolismo , Imunoglobulina M/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Ratos , Ratos Sprague-DawleyAssuntos
Linfócitos B/imunologia , Imunoglobulina D/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Anticorpos Anti-Idiotípicos , Anticorpos Monoclonais , Linfócitos B/citologia , Células Cultivadas , Imunoglobulina M/imunologia , Ativação Linfocitária , Macrófagos/imunologia , Camundongos , Baço/imunologiaRESUMO
Germ-free mice were intentionally associated with drug-resistant donor strains of Escherichia coli known to carry R factors and with drug-sensitive recipient strains. In vivo transfer of R factors was observed in all experiments, involving five different donor-recipient combinations. The number of converted recipients varied, depending upon the donor-recipient combination, but in all cases it was restricted by limiting numbers of either recipient or donor strains in the digestive tract of the microbially defined mice. Converted recipients were detected in fecal material as early as 5.5 hr after mice were associated with donor and recipient bacteria. Donors, recipients, and converted recipients were detectable in the stomach, small intestine, cecum, and large intestine of the microbially defined mice and their suckling young.
Assuntos
Bactérias , Resistência Microbiana a Medicamentos , Genética Microbiana , Animais , Ceco/microbiologia , Escherichia coli , Fezes/microbiologia , Vida Livre de Germes , Intestino Grosso/microbiologia , Intestino Delgado/microbiologia , Camundongos , Estômago/microbiologiaRESUMO
Fifteen sulfonamide-resistant cultures isolated from urinary tract infections in eastern Nebraska were screened for transferable drug resistance by three methods. Seven of the 15 resistant cultures could transfer resistance of varying levels to two or more chemotherapeutic agents. Transfer of drug resistance occurred without accompanying transfer of chromosomal traits and required cell to cell contact. In mixed culture, the number of drug-resistant recipients increased exponentially, reaching a plateau 2 hr after mixing. Spontaneous or artificial elimination of resistance was found to be a rare event. In addition, several drug-sensitive isolates from urinary tract infections were shown to be competent recipients of drug resistance determinants. From these data, it appears that the transferable drug resistance observed was mediated by R factors.
Assuntos
Resistência Microbiana a Medicamentos , Enterobacteriaceae/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Genética Microbiana , Infecções Urinárias/microbiologia , Cloranfenicol/farmacologia , Conjugação Genética , Humanos , Klebsiella/efeitos dos fármacos , Ácido Nalidíxico/farmacologia , Proteus/efeitos dos fármacos , Estreptomicina/farmacologia , Sulfisoxazol/farmacologia , Sulfonamidas/farmacologia , Tetraciclina/farmacologiaRESUMO
The ability of various cell populations to bind and present the thymus-independent antigen TNP-Ficoll to a responding cell population was assessed. The in vitro antibody response to TNP-Ficoll depends upon the presence of B lymphocytes and plastic-adherent accessory cells, but does not require T lymphocytes. Purified B cells were the most effective population in binding and presenting TNP-Ficoll, and adherent cells did not perform this function. Antigen binding and presentation was antigen specific and could be blocked with anti-mu antibody, but not by antibodies directed against other immunoglobulin classes. Spleen cells from mice genetically unresponsive to TNP-Ficoll (CBA/N X BALB/c F1 males) were equally effective as normal spleen cells in antigen binding and presentation. We conclude that the initial events in the induction of the antibody response involves antigen binding by B cells, and that subsequent activation of the subset of B cells that can respond to TNP-Ficoll proceeds either via B cell-B cell interaction or B cell-dependent transfer of antigen to macrophage-like cells.
Assuntos
Formação de Anticorpos , Antígenos , Imunidade Celular , Timo/imunologia , Animais , Linfócitos B/imunologia , Adesão Celular , Sobrevivência Celular , Feminino , Ficoll/imunologia , Imunoglobulina M , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , Coelhos , Linfócitos T/imunologia , Trinitrobenzenos/imunologiaRESUMO
Two monoclonal rat anti-mouse IgM antibodies, Bet 1 and Bet 2, are described in this paper. Bet 1 defines a new allotypic determinant, Igh-6.5, expressed on IgM molecules in serum and on B lymphocytes, whereas Bet 2 recognizes a determinant on IgM molecules of all mouse strains tested. Both reagents bind to the IgM myeloma protein MOPC104E, but not to IgG myeloma proteins, including FLOPC21, MOPC21, and UPC10. Using serum from various mouse strains to inhibit the binding of Bet 1 to MOPC104E, 3 distinct inhibition patterns were found. BALB/c, DBA/2, and CBA sera inhibited strongly, C56BL/6 (B6), SJL, AKR, and NZB sera inhibited weakly, and A and AL sera showed no inhibition of binding of BET 1 to MOPC104E. All sera tested were equivalent in their inhibition of the binding of Bet 2 to MOPC104E. When spleen cells from different mouse strains were reacted with fluorescein-conjugated Bet 1 (F-Bet 1) and subjected to flow microfluorometry analysis, 3 types of staining patterns, corresponding to those obtained with the serum inhibition assay, were also found. The determinant recognized by Bet 1 is controlled by a gene linked to the Igh-C gene complex. C.AL20 behaved like AL, and C.B20 and BAB/14 behaved like B6, both in the serum inhibition assay and in flow microfluorometry analysis of spleen cells stained with F-Bet 1. In addition, the capacity of serum from individual (BALB/c X B6) X B6 backcross progeny to inhibit the binding of Bet 1 to MOPC104E was linked to the expression of the Ig-1a marker.
Assuntos
Anticorpos , Epitopos , Alótipos de Imunoglobulina , Imunoglobulina M , Animais , Anticorpos Anti-Idiotípicos , Anticorpos Monoclonais , Ligação Competitiva , Imunofluorescência , Ligação Genética , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos AKR , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , Camundongos Endogâmicos NZB , Proteínas do Mieloma/imunologia , RatosRESUMO
Mice expressing the xid gene exhibit an altered immune response to phosphocholine (PC)-conjugated keyhole limpet hemocyanin (KLH). Less than 25% of their anti-PC-KLH response is PC specific, and most of these antibodies lack the normally predominant T15 idiotype. These findings suggested that immune defective mice might employ different variable region genes than normal mice in their anti-PC response. To examine this possibility, we characterized by Southern blot analysis the gene family encoding PC-VH regions and determined the amino acid sequence and fine specificity of binding of a T15-, IgG2, PC-specific hybridoma (1B8E5) produced by fusion of the SP2/O cell line and PC-KLH immune CBA/N spleen cells. Southern blot analysis of DNA from CBA/N mice by using a PC-VH probe (S107 VH) revealed a hybridization pattern virtually identical to that of DNA from normal CBA/J mice, indicating that CBA/N mice do not suffer from a gross deletion of PC-VH genes. Analysis of the 1B8E5 antibody reveals that both the binding specificity and relative affinity of this antibody are different from the anti-PC antibodies of the T15, M167-M511, and M603 families. The complete amino acid sequence of the heavy (H) chain variable region shows that 1B8E5 uses a VH segment identical to the allelic form of T15 (C3) but has a unique D region of three amino acids and use the JH1 joining segment. Both the DH and JH regions are unusual when compared to PC-specific antibodies from normal mice, which have a D region composed of five to eight amino acids and use the JH1 joining segment. The amino terminal sequence of the 1B8E5 light (L) chain demonstrates that this anti-PC antibody carries a Vk3 subgroup L chain. Chains from this subgroup have not previously been found in association with PC-binding antibodies. Thus, the Vk, DH, and JH segments expressed in 1B8E5 make this hybridoma unique in terms of the anti-PC antibodies studied to date, and suggests that additional PC-specific antibodies exist in inbred mice that employ "unusual" V gene segments.
Assuntos
Anticorpos Monoclonais/biossíntese , Sítios de Ligação de Anticorpos , Colina/análogos & derivados , Idiótipos de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Fosforilcolina/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Especificidade de Anticorpos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Hibridomas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Mitocôndrias Hepáticas/metabolismo , Fosforilcolina/imunologiaRESUMO
Mouse spleen cells were stimulated to proliferate in vitro by soluble affinity-purified heterologous antibodies to mouse delta. Antibodies from goat or rabbit antisera to TEPC 1017, a mouse IgD myeloma protein, were purified on an affinity column of TEPC 1033, a second mouse IgD myeloma protein. Maximum uptake of [3H]thymidine in the range of 60,000 cpm was obtained after 48 hr of culture with anti-delta at concentrations of 50 micrograms/ml. In contrast, the hybridoma 10-4.22 anti-delta was nonmitogenic at similar concentrations. The proliferative response was not impaired upon removal of T cells by treatment with an anti-thymocyte serum (ATS), nor by removal of adherent cells by passage of spleen cells over Sephadex G-10 columns and counter-flow centrifugation. Splenic lymphocytes isolated on the fluorescence activated cell sorter (FACS) with intermediate-to-high amounts of surface IgD (sIgD) were responsive to soluble anti-delta, while IgD-negative cells, or cells with low amounts of sIgD, were unresponsive. Spleen cells from mice less than 4 weeks of age, or from mice carrying the X-linked B cell defect (xid), were unresponsive to anti-delta. These results indicate that anti-delta acts similarly to anti-mu in stimulating a proliferative response by later maturing B cells, which are characterized by a high density of sIgD.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias delta de Imunoglobulina/imunologia , Ativação Linfocitária , Animais , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Divisão Celular , Cabras/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Cadeias mu de Imunoglobulina/imunologia , Cinética , Macrófagos/imunologia , Masculino , Camundongos , Coelhos/imunologia , Solubilidade , Baço/citologiaRESUMO
Transgenic mice carrying immunoglobulin genes coding for mu heavy chain and kappa light chain have been used to study the mechanisms involved in allelic and isotypic exclusion. We report here that individual cells from transgenic mice carrying a functionally rearranged mu heavy chain gene (capable of generating both membrane and secreted forms of IgM) can rearrange an endogenous mu heavy chain gene and simultaneously produce both transgenic and endogenous IgM. These "double-producing" cells express both endogenous and transgenic IgM in the cytoplasm (detected by immunohistology) and on the cell surface (detected by multiparameter fluorescence-activated cell sorter analysis). In addition, they secrete mixed IgM molecules containing both transgenic and endogenous mu heavy chains (detected in serum by radioimmune assay). The transgenic mice studied also have relatively large numbers of cells that produce endogenous immunoglobulin in the absence of detectable transgenic immunoglobulin ("endogenous-only cells"). The mechanisms that generate double-producing cells and endogenous-only cells appear to be under genetic control because the frequencies of these B-cell populations are characteristic for a given transgenic line. Thus, our findings indicate that more is involved in triggering allelic exclusion than the simple presence or absence of membrane mu heavy chains (as has been previously postulated).
Assuntos
Linfócitos B/imunologia , Genes de Imunoglobulinas , Imunoglobulina M/genética , Receptores de Antígenos de Linfócitos B/genética , Animais , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos , Camundongos TransgênicosRESUMO
In pursuing the mechanism of endotoxin action, we examined the effect of lipopolysaccharide (LPS) and its chemically defined components, lipid A and lipid X on cultured bovine endothelial cells. We report that LPS and lipid A caused detachment and altered morphology of endothelial cells while lipid X did not. Phorbol myristate acetate, a compound known to activate protein kinase C, also caused endothelial cell detachment. Morphologic changes were readily apparent in the endothelial cells after 6 hours of exposure to lipopolysaccharide (1 microgram/ml); at that time many of the cells had contracted and formed bleblike structures on the surface. Large vacuoles, dense bodies, and pyknotic nuclei were found in the detaching cells, indicating necrosis or cell death. Preceding the morphologic changes and actual detachment, endothelial cell DNA and RNA synthesis was impaired by LPS. The changes in DNA and RNA synthesis occurred within 4 hours of exposure to 1 microgram/ml of LPS when the cells were still able to maintain normal levels of ATP. In addition to the inhibition of nucleic acid synthesis, protein synthesis was inhibited after 6 and 8 hours of LPS exposure. DNA, RNA, and protein synthesis returned to control levels after 24 hours of exposure. Investigation on the cultured bovine endothelial cells as a model for LPS action was useful in that these cells are sensitive to relatively low levels of LPS and the endothelium may be an important target in sepsis.
Assuntos
Endotélio Vascular/citologia , Glicolipídeos/farmacologia , Lipídeo A/farmacologia , Lipopolissacarídeos/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Animais , Bovinos , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , DNA/biossíntese , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Biossíntese de Proteínas , RNA/biossínteseRESUMO
X-linked immune deficient (Xid) mice fail to produce anti-phosphocholine (PC) antibodies even after immunization with Streptococcus pneumoniae. Consequently, Xid mice are extremely susceptible to infection with S. pneumoniae, PC-specific B cells appear to undergo clonal deletion in Xid mice; however, a new thymus-dependent form of PC, 6-(O-phosphocholine)hydroxyhexanoate (EPC), can rescue PC-specific B cells from the bone marrow presumably by providing T cell help before clonal deletion. Analysis of PC-specific IgG hybridomas from Xid mice revealed utilization of several V-D junctional variants of the VH1 gene segment rearranged to different D and JH gene segments. The majority of Xid anti-PC antibodies exhibit an Asp-->Gly95H replacement at the V-D junction. These Gly95H VH1 variants associate with kappa 1C L chains to produce anti-PC antibodies that: (1) have low relative affinity for PC, (ii) are heteroclitic for nitrophenylphosphocholine and (iii) fall to bind to or provide protection against S. pneumoniae. Single prototypic V-D variants of the T15 idiotype (Asp95H), M603 idiotype (Asn95H) and M167 idiotype (Asp95H-Ala96H) were also induced in Xid mice. The M603-like and M167-like antibodies bound to and protected against S. pneumoniae even though they exhibited Kas for PC which were lower than T15 idiotype+ antibodies. These data demonstrate that small changes in the V-D junctional sequence of the T15 (VH1) heavy chain alter L chain usage and the structure of the PC binding site so that the PC expressed on S. pneumoniae is no longer recognized.