The T cell-dependent B cell immune response and germinal center reaction are intact in A-myb-deficient mice.

Expression of the protooncogene A-myb is restricted to the developing CNS, adult testes, breasts in late pregnancy, and germinal centers of secondary B cell follicles. The functional relevance of A-myb expression at three of these sites has been demonstrated previously via the generation and analysis of A-myb-deficient mice, which display behavioral abnormalities, male sterility, and perturbed breast development during pregnancy. In contrast, here we show that the germinal center response driven by T cell-dependent Ag immunization and the associated processes of Ab V gene somatic hypermutation, affinity maturation, and heavy chain class switching are overtly normal in A-myb-deficient mice. Nonetheless, these mice display mild splenic white pulp hypoplasia and blunted primary serum Ab responses, suggesting that although A-myb is not directly involved in the regulation of the memory B cell response, it may play a role in enhancing peripheral B cell survival or proliferative capacity.

Fc gamma receptor IIB on follicular dendritic cells regulates the B cell recall response.

Generation of the B cell recall response appears to involve interaction of Ag, in the form of an immune complex (IC) trapped on follicular dendritic cells (FDCs), with germinal center (GC) B cells. Thus, the expression of receptors on FDC and B cells that interact with ICs could be critical to the induction of an optimal recall response. FDCs in GCs, but not in primary follicles, express high levels of the IgG Fc receptor Fc gamma RIIB. This regulated expression of Fc gamma RIIB on FDC and its relation to recall Ab responses were examined both in vitro and in vivo. Trapping of IC in spleen and lymph nodes of Fc gamma RII-/- mice was significantly reduced compared with that in wild-type controls. Addition of ICs to cultures of Ag-specific T and B cells elicited pronounced Ab responses only in the presence of FDCs. However, FDCs derived from Fc gamma RIIB-/- mice supported only low level Ab production in this situation. Similarly, when Fc gamma RIIB-/- mice were transplanted with wild-type Ag-specific T and B cells and challenged with specific Ag, the recall responses were significantly depressed compared with those of controls with wild-type FDC. These results substantiate the hypothesis that FcgammaRIIB expression on FDCs in GCs is important for FDCs to retain ICs and to mediate the conversion of ICs to a highly immunogenic form and for the generation of strong recall responses.

Contrasting the in situ behavior of a memory B cell clone during primary and secondary immune responses.

Whether memory B cells possess altered differentiative potentials and respond in a qualitatively distinct fashion to extrinsic signals as compared with their naive precursors is a current subject of debate. We have investigated this issue by examining the participation of a predominant anti-arsonate clonotype in the primary and secondary responses in the spleens of A/J mice. While this clonotype gives rise to few Ab-forming cells (AFC) in the primary response, shortly after secondary immunization its memory cell progeny produce a massive splenic IgG AFC response, largely in the red pulp. Extensive clonal expansion and migration take place during the secondary AFC response but Ab V region somatic hypermutation is not reinduced. The primary and secondary germinal center (GC) responses of this clonotype are both characterized by ongoing V gene hypermutation and phenotypic selection, little or no inter-GC migration, and derivation of multiple, spatially distinct GCs from a single progenitor. However, the kinetics of these responses differ, with V genes containing a high frequency of total as well as affinity-enhancing mutations appearing rapidly in secondary GCs, suggesting either recruitment of memory cells into this response, or accelerated rates of hypermutation and selection. In contrast, the frequency of mutation observed per V gene does not increase monotonically during the primary GC response of this clonotype, suggesting ongoing emigration of B cells that have sustained affinity- and specificity-enhancing mutations.

Severe attenuation of the B cell immune response in Msh2-deficient mice.

Recently, results obtained from mice with targeted inactivations of postreplication DNA mismatch repair (MMR) genes have been interpreted to demonstrate a direct role for MMR in antibody variable (V) gene hypermutation. Here we show that mice that do not express the MMR factor Msh2 have wide-ranging defects in antigen-driven B cell responses. These include lack of progression of the germinal center (GC) reaction associated with increased intra-GC apoptosis, severely diminished antigen-specific immunoglobulin G responses, and near absence of anamnestic responses. Mice heterozygous for the Msh2 deficiency display an "intermediate" phenotype in these regards, suggesting that normal levels of Msh2 expression are critical for the B cell response. Interpretation of the impact of an MMR deficiency on the mechanism of V gene somatic hypermutation could be easily confounded by these perturbations.

The roles of antibody variable region hypermutation and selection in the development of the memory B-cell compartment.

Somatic hypermutation and selection of immunoglobulin (Ig) variable (V)-region genes, working in concert, appear to be essential for memory B-cell development in mammals. There has been substantial progress on the nature of the cis-acting DNA elements that regulate hypermutation. The data obtained suggest that the mechanisms of Ig gene hypermutation and transcription are intimately intertwined. While it has long been appreciated that stringent phenotypic selection forces are imposed on the somatically mutated Ig V regions generated during a T-cell dependent B-cell response, the mechanisms involved in this selection have remained enigmatic. Our studies have questioned the role of foreign antigen deposited on follicular dendritic cells in affinity-based positive selection of V regions, and have shown that this selection takes place in a "clone-autonomous" fashion. In addition, our data strongly suggest that affinity for antigen alone is not the driving force for selection of B-cell clones into the memory compartment. In contrast, we suggest that a combination of positive selection for increased foreign antigen binding, and negative selection of antibody V regions that are autoreactive at the onset of the response, or have acquired autoreactivity via hypermutation, results in the "specificity maturation" of the memory B-cell response.

A periarteriolar lymphoid sheath-associated B cell focus response is not observed during the development of the anti-arsonate germinal center reaction.

The behavior of p-azophenylarsonate (Ars)-specific B cell clones during the primary T cell-dependent splenic response of A/J mice was investigated using an immunohistochemical approach. The earliest Ars-specific B cells were observed as isolated cells in the red pulp by day 3 after immunization with Ars-keyhole limpet hemocyanin, (KLH) and at day 6, large clusters of Ars-specific B cells were first detected in germinal centers, which continued to be observed for an additional 8 to 15 days. Surprisingly, no Ars-specific B cell foci were observed in or near the CD4 T cell-rich periarteriolar lymphoid sheath (PALS) during the entire primary response. Nevertheless, A/J mice immunized with (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma globulin (NP-CGG) or Ars-CGG mounted robust splenic (4-hydroxy-3-nitrophenyl)acetyl or CGG-specific PALS-associated focus reactions, respectively. In contrast, no Ars-specific PALS B cell foci were detected in A/J mice immunized with Ars-CGG. These data add to a growing body of evidence indicating that B cell proliferation and differentiation in CD4 T cell-rich microenvironments are not prerequisites for the GC reaction. Taken together with previous results obtained using other model Ags, the data suggest that the specificity of the B cell Ag receptor may strongly influence the lymphoid microenvironment in which a B cell clone first undergoes Ag-driven clonal expansion and differentiation.

Amplified follicular immune complex deposition in mice lacking the Fc receptor gamma-chain does not alter maturation of the B cell response.

The role of Ag-Ab complexes (or immune complexes; ICs) in the regulation of the maturation of the B cell immune response was investigated in mice perturbed in the deposition and retention of such complexes. Loss of surface expression of Fc gammaRI and Fc gammaRIII due to targeted disruption of the common FcR gamma-chain gene results in dramatically increased deposition of ICs on follicular dendritic cells (FDCs) in germinal centers (GCs), attributed to altered clearance of circulating ICs. Despite these changes in the trapping of ICs by FDCs, serum Ab production, V gene hypermutation, isotype class switching and Ab affinity maturation are overtly unaltered. Thus, substantially augmenting B cell cognate Ag density on FDCs does not alter the outcome of the maturation of the B cell response. The significance of this finding in terms of the currently accepted model for the generation of B cell memory is discussed.

Evaluation of the role of the 3'alpha heavy chain enhancer [3'alpha E(hs1,2)] in Vh gene somatic hypermutation.

Previous work on the cis-acting elements that control heavy chain variable region (VH) gene somatic hypermutation has indicated the presence of an as yet unidentified element(s) 3' of the intron enhancer that is necessary for high rate mutation. Examination of cis-acting elements involved in kappa light chain V gene hypermutation has demonstrated a requirement for both the intronic and 3' kappa enhancers in this process. To examine whether the 3'alpha heavy chain enhancer [3'alpha E(hs1,2)] is required for somatic hypermutation of VH genes, we generated two types of transgenic mice. One type was generated using a construct containing a VH promoter, a rearranged VDJ, the heavy chain intronic enhancer, and the murine heavy chain 3'alpha E(hs1,2). The transgenes in the second lines were similar to the transgenes in the first with the addition of a second complete matrix attachment region (MAR) 3' of the heavy chain intronic enhancer, and splice acceptor and polyadenylation sites between the two enhancers. Analysis of both transgenes revealed levels of mutation at least 10-fold lower than endogenous VH genes. These data suggest that the 3'alpha E(hs1,2) does not play a role analogous to the 3' kappa enhancer in the regulation of the hypermutation process. Moreover, in one of the transgenes, the presence of the 3'alpha E(hs1,2) resulted in a lack of transcription in vivo, suggesting a negative regulatory role for this enhancer in certain contexts.

Characterization of the cis-acting elements required for somatic hypermutation of murine antibody V genes using conventional transgenic and transgene homologous recombination approaches.

We have used conventional transgenic technology and a novel transgene homologous recombination pathway to investigate the cis-acting elements necessary for murine antibody V-gene somatic hypermutation. These studies show that an undefined element 3' of the IgH intronic enhancer is required for VH hypermutation. This element appears not to be the 3' alpha IgH enhancer, at least in its "minimal' form. Elements 5' of the natural VH promoter are not necessary for hypermutation, and this promoter can be replaced by a non-Ig promoter, resulting in a reduction, but not ablation of the rate of hypermutation in the adjacent VH gene. Our analyses provide no support for "gene conversion' models of hypermutation, and support models that propose a role for transcription in hypermutation over purely DNA-based models. Analysis of a CD72/kappa chimeric transgene suggests that all of the factors that influence hypermutation of kappa transgenes have yet to be defined.

Altering the antibody repertoire via transgene homologous recombination: evidence for global and clone-autonomous regulation of antigen-driven B cell differentiation.

Antibody VH transgenes containing small amounts of natural 5' and 3' flanking DNA undergo nonreciprocal homologous recombination with the endogenous Igh locus in B cells. The resulting "hybrid" heavy chain loci are generated at a low frequency but are fully functional, undergoing somatic hypermutation and isotype class switching. We have used this recombination pathway to introduce a somatically mutated variable (V) region with an unusually high affinity for the hapten p-azophenylarsonate (Ars) into the preimmune antibody repertoire. The affinity of this V region for Ars is 100-fold higher than any unmutated anti-Ars antibody previously characterized. Expression of the transgene-encoded V region did not affect many aspects of antigen-driven B cell differentiation, including somatic hypermutation, in either Ars-specific transgene- or endogenous V gene-expressing clones. Thus, the regulation of these processes appears to operate in a "global" fashion, in that the mechanisms involved are imperceptive of the relative affinities for antigen of the antibodies expressed by B cell clones participating in the immune response. In contrast, the selection of V region mutants leading to affinity maturation and memory cell formation was found to be strongly influenced by the transgenic V region, but only in clones expressing this V region. Hybridomas derived from transgene- and endogenous V region-expressing memory cells were isolated at similar frequencies from individual transgenic mice. The V regions expressed by hybridomas in both of these groups had 2- to 30-fold greater affinity for Ars than their unmutated precursors, despite the fact that the transgene-encoded precursors had 100-fold higher affinity than their endogenous counterparts. These results show that the criterion for entry into the memory compartment is established not by the affinity of a B cell's V region relative to all other V regions expressed during the response, but by the affinity of this V region relative to its unmutated precursor. Thus, the development of B cell memory is regulated in a "clone-autonomous" fashion.

Expression of nitric oxide synthase in human peripheral blood mononuclear cells and neutrophils.

It has been clearly demonstrated in rodents that nitric oxide (NO) plays an important role in host defense and immunity. However, evidence that human leukocytes express inducible nitric oxide synthase (iNOS) or its products has been inconclusive and a source of controversy. We report that iNOS could not be detected in human monocytes, HL-60 cells, neutrophils, and T cells by Western blotting analysis (< or = 10 pg) or by radiolabeled L-arginine-to-L-citrulline conversion (< or = 20 pmol L-citrulline) under conditions sufficient to induce iNOS in the rodent system and in human hepatocytes, which include activation with cytokines, endotoxins, and/or chemoattractants. However, sensitive methods such as RT-PCR and Northern blot analysis show "constitutively expressed" iNOS mRNA from human monocytes, neutrophils, Jurkat cells, and HL-60 cells. This iNOS mRNA is 4.4 kb and is similar to that seen in human hepatocytes and rodent macrophages. In spite of the constitutive expression of mRNA in neutrophils and the lack of detectable NOS activity (based on Western blotting and L-arginine-to-L-citrulline conversion assay), stimulation of human neutrophils unit FMLP in vitro induced the ADP-ribosylation of an intracellular NO target, glyceraldehyde-3-PO4 dehydrogenase (GAPDH), in a NO-dependent manner. These studies indicate that low levels of NOS protein are expressed in neutrophils (and perhaps T cells and monocytes) and produce NO following stimulation. The data indicate that, in addition to its phagocytic and tumoricidal activity. NO may also function as an autacoid signaling molecule within the cells.

B cell differentiation: I. Development and functional analysis of murine B cells immortalized by a recombinant retrovirus.

B lymphoblast cells were immortalized by infection with a v-myc and v-raf/mil containing recombinant retrovirus. The immortalized B cells do not require exogenous growth factors or mitogens for growth. These cells express characteristic mature B cell phenotypic markers including IgM and IgD. Northern blot analysis detected mu and delta mRNA, and Southern blot analysis revealed rearrangements of Ig genes but not TCR genes in these cells. The immortalized B cells were found to respond to B cell mitogens by proliferation and IgM secretion, and to respond to IL-4 by an increase in la expression. Furthermore, they were found to differentiate to secrete IgM and to switch to IgG1 production following interaction with Staphylococcal enterotoxin B- activated Th2 cells. These cell lines are thus valuable tools for analyzing the molecular events involved in B lymphocyte growth and differentiation.

Metabolism of 3-chlorobenzoate by a Pseudomonas (diff) spp.

Pseudomonas (diff) spp. was isolated from a complex petrochemical sludge, using benzoate as the sole source of carbon. The organism could metabolize 3-chlorobenzoate, releasing approximately 30% of organically bound chloride. 3-Chlorodihydrodihydroxybenzoate and 3-chlorocatechol were confirmed as pathway intermediates by mass spectral and HPLC analysis. About 3-fold higher levels of catechol 1,2-oxygenase were detected in cells grown on 3-chlorobenzoate as compared to that of benzoate. 3-Chlorocatechol inhibited the catechol 1,2-oxygenase activity, when used as assay substrate. A 15-fold purified catechol 1,2-oxygenase had a Km of 0.37 mumole and Vmax of 2.3 with 3-chlorocatechol. Catechol gave Km of 0.2 mumole and Vmax of 40, suggesting that 3-chlorocatechol is not metabolised further and hence blocks the metabolic pathway for 3-chlorobenzoate degradation. In contrast catechol 1,2-oxygenase was not inhibited by 4-chlorocatechol and probably is an intermediate for the total/complete degradation of 3-chlorobenzoate (approx. 30%).