nitrophenols and Immunologic-Deficiency-Syndromes

Mice with a disrupted C4 locus (C4(-/-)) have an impaired immune response to thymus-dependent Ags. To test the role of bone marrow-derived C4 in humoral immunity, we reconstituted deficient animals with wild-type bone marrow or an enriched fraction of bone marrow-derived macrophages. C4 chimeras were immunized with 4-hydroxy-3-nitrophenyl(5) conjugated to keyhole limpet hemocyanin (NP(5)- KLH) or infected with HSV-1, and the Ab response was evaluated. Wild-type bone marrow rescued the humoral immune response to both Ags, i.e., the soluble Ag and HSV-1, demonstrating that local C4 production is sufficient for humoral responses. Although the C4 chimeric animals lacked detectable C4 in their sera, C4 mRNA was identified in splenic sections by in situ hybridization, and C4 protein deposits were identified in the germinal center areas of splenic follicles by immunofluorescence staining. Macrophages derived from bone marrow produced sufficient C4 protein to restore the humoral response to NP(5)-KLH in C4-deficient animals when administered along with Ag. Cell-sorting experiments, followed by C4-specific RT-PCR, identified splenic macrophages (CD11b(+), CD11c(-)) as a cellular source for C4 synthesis within the spleen.

Topics: Adoptive Transfer; Animals; Antibody Formation; Bone Marrow Transplantation; Cells, Cultured; Complement C4; Haptens; Hemocyanins; Immunoglobulin G; Immunologic Deficiency Syndromes; Injections, Intravenous; Macrophages; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Nitrophenols; Phenylacetates; Radiation Chimera; Spleen

X-linked immunodeficient (Xid) mice carry a Bruton's tyrosine kinase (Btk) mutation and exhibit a selective failure to produce antibodies against bacterial capsular polysaccharides. Studies in vitro point to a fundamental survival defect of Xid B cells after receptor cross-linking by thymus-independent type-2 (TI-2) antigen because B cells undergo apoptosis without proliferating. We describe results from a novel model, which we have used to investigate the impact of the Xid mutation on migration, proliferation and differentiation of B cells after polysaccharide immunization in vivo. Immunoglobulin knock-in mice, in which a large proportion of B cells express transgene-encoded receptors specific for (4-hydroxy-3-nitrophenyl)-acetyl (NP), were crossed with CBA/N mice. The male progeny contain NP-specific Xid B cells, while the female progeny contain NP-specific B cells with normal Btk. After immunization with the TI-2 antigen NP-Ficoll, NP-specific Xid B cells migrate to the T zones and proliferate. Despite transient up-regulation of blimp-1 and survival beyond the time when terminal differentiation is normally underway, Btk-defective B cells fail to differentiate to plasmablasts or germinal center cells. CD40 ligation partially restores their ability to form plasma cells in response to TI-2 antigen.

Topics: Agammaglobulinaemia Tyrosine Kinase; Animals; Antibodies; Antigens, T-Independent; Apoptosis; B-Lymphocytes; CD40 Antigens; Cell Differentiation; Cell Division; Cells, Cultured; Chemotaxis, Leukocyte; Female; Gene Deletion; Genetic Linkage; Immunologic Deficiency Syndromes; Lymphocyte Activation; Male; Mice; Mice, Transgenic; Nitrophenols; Phenotype; Positive Regulatory Domain I-Binding Factor 1; Protein-Tyrosine Kinases; Repressor Proteins; RNA, Messenger; Transcription Factors; Up-Regulation; X Chromosome

Cr2-/- mice have an impairment in humoral immunity, as shown by the decrease in the Ab titers against T cell-dependent Ags and abnormalities in germinal center formation. Germinal centers are present, but they are decreased in size and number, indicating problems in their development. In this study, we investigated whether this abnormality in germinal center development is associated with problems in the establishment of optimal affinity maturation and the generation of memory B cells, processes closely related to the germinal center reaction. We immunized the Cr2-/- animals with different Ags with or without adjuvants. We showed that, when immunized without adjuvants, complement receptors are absolutely required for optimal affinity maturation. Although limited affinity maturation is elicited in the Cr2-/- Ab response, it is decreased as compared with normal animals. Memory B cell generation is also impaired. In the presence of adjuvants, germinal center development in the Cr2-/- mice is still abnormal, as demonstrated by their decreased size and number. Surprisingly, adjuvants establish optimal affinity maturation and partially restore the amount of Ab produced during the primary response and memory B cell generation. However, adjuvants cannot improve the ability of follicular dendritic cells to retain Ags in the form of immune complexes. These observations indicate that immunization with inflammatory Ags offset some of the immunological abnormalities found in the Cr2-/- mice and show that optimal affinity maturation in the Cr2-/- mice can be achieved in the absence of normal germinal centers.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibody Affinity; Antigens; B-Lymphocyte Subsets; Base Sequence; Cell Differentiation; Dendritic Cells, Follicular; Germinal Center; Haptens; Hemocyanins; Immunoglobulin G; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Immunologic Deficiency Syndromes; Immunologic Memory; Injections, Intraperitoneal; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates; Receptors, Complement 3d; Spleen

In this study, we examine the relationship between primary and secondary T cell-dependent immune responses using the response of xid mice to the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) as an experimental model. The reduced serologic primary immune response of xid mice was demonstrated to be caused by a substantially decreased Ab-forming cell (AFC) generation. Furthermore, the germinal center reaction in the primary xid immune response was diminished and the frequency of NP-specific memory B cells prior to secondary immunization was reduced 10-fold. Despite the poor primary response of xid mice, secondary exposure to Ag generated a response that was qualitatively and quantitatively equal to that of wt mice. The number of IgG1 AFCs in spleen and bone marrow increased equally in both groups, as did the proportion of AFCs secreting high affinity Ab in both locations. The extent and distribution of somatic mutations in the V(H) genes of xid secondary response B cells was also found to be normal, indicating that the xid gene product is not critical for the processes that result in affinity maturation. Thus, although xid mice generate memory B cells of normal phenotype but at a substantially lower frequency, this does not limit the magnitude of the secondary response. Therefore, our results imply that the reduced memory B cell frequency in xid mice is still above some threshold value necessary for a normal secondary immune response.

Topics: Animals; Antibody-Producing Cells; Antigens, CD; B-Lymphocytes; B7-2 Antigen; CD40 Ligand; Female; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Genetic Linkage; Haptens; Hematopoietic Stem Cells; Hemocyanins; Immunization; Immunization, Secondary; Immunoglobulin G; Immunologic Deficiency Syndromes; Immunologic Memory; Lymphocyte Activation; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Mutant Strains; Mutation; Nitrophenols; Phenylacetates; Transfection; X Chromosome