Defining a transcriptional fingerprint of murine splenic B-cell development.

B-cell development occurs in a stepwise fashion that can be followed by the expression of B cell-specific surface markers. In this study, we wished to identify proteins that could contribute to the changes in expression of such markers. By using RNA from freshly isolated B220+ cells, we hoped to reduce the effect of artifacts that occur during the isolation and amplification steps necessary to use flow cytometry analysis-sorted subsets in microarray experiments. Analyses comparing expression patterns from B220+ 2-week bone marrow (pro-B, pre-B, immature B cells), 2-week spleen (predominantly transitional cells) and 8-week spleen (mainly mature B cells) yielded hundreds of genes. We also examined the B cell-activating factor (BAFF)-dependent effects on immature splenic B cells by comparing expression patterns in the spleen between 2-week A/J vs 2-week A/WySnJ mice, which lack functional BAFF receptor signaling. Genes that showed the expression differences between spleen and bone marrow samples were then analyzed through quantitative PCR on B-cell subsets isolated using two different sorting protocols. A comparison of the results from our study with the results from other analyses showed not only some overlap of preferentially expressed genes but also an expansion of other genes potentially involved in B-cell development.

Expression of the mouse fragilis gene products in immune cells and association with receptor signaling complexes.

The mouse genome possesses five genes encoding proteins homologous to human Leu-13. The Leu-13 protein associates with immune cell receptor activation complexes: a monoclonal antibody against Leu-13 induces T and B cells to form homotypic aggregates, inhibits activation-induced proliferation and induces the shedding of L-selectin. The mouse fragilis proteins have not been previously analyzed as components of the immune response. Antibody and nucleic acid reagents were generated that are specific for each of the five fragilis gene products. Expression of some of these genes (fragilis and fragilis3) is wide spread in a variety of mouse immune (and nonimmune) tissues while others (fragilis5) appear to be much more restricted. These proteins have been predicted to span the membrane twice with both amino- and carboxyl-terminal sequences extracellular: we show that a highly conserved loop of the protein between the transmembrane domains is intracellular. The fragilis proteins are associated with tetraspanin proteins CD81 and CD9: B cell activation positions fragilis into lipid rafts along with the CD81, CD19, and CD21. The mouse functional equivalent to human Leu-13 may not be single gene product, but instead may require the contribution of multiple fragilis proteins.

Overexpression of RANKL implicates IFN-beta-mediated elimination of B-cell precursors in the osteopetrotic bone of microphthalmic mice.

The microphthalmic (mi) mouse possesses a dominant negative mutation in the microphthalmia-associated transcript factor (MITF) transcription factor. These animals are characterized by reduced numbers of peripheral mast and natural killer (NK) cells, are osteopetrotic because of osteoclast reduction and malfunction, lack functional melanocytes, and are deficient for maturing B-cells within the bone marrow. Granulocyte precursor cells, however, are functionally maintained within the mi bone marrow. A central question has been whether the B-cell deficiency of the mi mouse marrow is caused by the absence of an MITF-controlled gene product or because of the compromised, osteopetrotic environment. In this report, we examined mi marrow by performing transcriptional mapping analyses of candidate genes whose products are instrumental for functional osteoclast and B-cell development. Surprisingly, the expression of a subset of such genes including RANKL, stromal-derived factor (SDF-1), B-cell lymphotactin chemokine (BLC), and RANK was dramatically enhanced in the mi marrow. Normal and mutant marrow were also analyzed by subtractive transcript cloning, which identified a number of known and unknown genes with altered transcriptional activity. One such unknown mouse gene possesses a human counterpart that is interferon-beta (IFN-beta) inducible, suggesting the osteopetrotic marrow is enriched for IFN-beta, a cytokine that is known to eliminate B-cell precursors. A model is proposed suggesting excess RANKL sets off a cascade of cytokine production including IFN-beta that leads to the preferential elimination of B-cell precursors in the marrow of osteopetrotic marrow.

Functional characterization of Pactolus, a beta-integrin-like protein preferentially expressed by neutrophils.

Murine Pactolus is a beta-integrin-like molecule expressed exclusively on the surface of granulocytes. Cell surface expression of Pactolus is dramatically increased following activation of bone marrow neutrophils with known agonists, and cross-linking of cell surface Pactolus, suggesting the bulk of the protein is in intracellular stores. The mature protein is found in two forms depending upon the extent of N-linked glycosylation. There is no evidence to suggest that Pactolus requires an associated alpha chain for expression. In some mouse strains, a truncated form of the protein is predicted based upon alternative splicing: this form, however, is unstable and rapidly degraded after synthesis. Differences in the quantities of these Pactolus mRNA isoforms have defined two alleles. BALB/c and C3H/HeJ mice possess allele B and preferentially express the truncated, unstable product, whereas C57BL/6 mice possess allele A and only produce the membrane-bound form. Sequence analysis has shown the difference between these two alleles is due to a single base pair difference at the splice acceptor site for the truncated product. The increased expression of the membrane form of Pactolus by granulocytes of C57BL/6 mice suggests a compensatory adhesion function that is reduced in cells from the low producing strains.

Cell-specific regulation of the CD21 gene.

The Complement Receptor Type 2 (Cr2-145,CR2, CD21) is an important receptor in the innate and acquired immune response. CD21 is produced by B cells and follicular dendritic cells, where it binds cleavage products of the C3 complement protein. CD21 facilitates internalization of immune complexes by B cells to enhance antigen presentation. CD21, in association with CD19/CD81, also serves as a coaccessory activation complex with the B-cell antigen receptor, permitting a lower antigen concentration to achieve maximal B-cell activation. CD21 traps immune complexes on the surface of follicular dendritic cells and displays them to activated B cells in germinal centers. Much work has been conducted to determine the transcriptional control mechanisms dictating CD21 expression. Appropriate transcriptional control of the CD21 gene evidently requires the CD21 promoter, as well as intronic sequences with enhancer and suppressor functions. Chromatin structure has been implicated in regulating the coordination of CD21 promoter and intronic control sequences by regulating access to them by putative transcription factors. This review assesses the past and current research into CD21 transcriptional regulation and offers insight into future experimental directions.

Amplification of cosmid and plasmid libraries.

The purpose of amplification is to provide a reagent library that can be used many times. In this protocol, bacteria containing the recombinant clones are grown on agar plates, washed off the plates, and stored in glycerol. An amplified cosmid or plasmid library will contain one library's equivalence in 10 to 100 microl. By producing 100 microl of such a library frozen in 100 1-ml tubes, over 1000 platings can be expected.

Size fractionation using sucrose gradients.

DNA fragments migrate through a linear sucrose gradient at a rate that is dependent on their size. This procedure described in this unit provides good resolution for DNA fragments 5 to 60 kb in size, and sucrose gradients are also useful for purification of bacteriophage l vector arms. Partially digested genomic DNA can be fractionated for the production of cosmid or bacteriophage libraries and completely digested DNA can be fractionated for subgenomic DNA libraries. Protocols are provided in this unit for both partial and complete enzyme digestion of genomic DNA.

Plating and transferring cosmid and plasmid libraries.

There are two commonly used protocols for the screening of recombinant bacteria with hybridization probes. One method involves the spreading of bacteria on the surface of agar using a sterile spreader. A nitrocellulose membrane filter is then placed on top of the colonies and most of each colony is transferred to the filter. This method works well when relatively small numbers of positive colonies are being selected (up to several thousand). A second method, described in this unit, employs a matrix of some type (here nitrocellulose filters are used) upon which bacteria can be plated and grown when the filter is placed on top of a nutrient agar surface. Once the plated bacteria have grown into visible colonies, the filters can be used for replica plating and in situ hybridization analysis.

Purification of cosmid and plasmid clones.

Cosmid- or plasmid-bearing colonies that are identified by hybridization are purified by spreading the cosmids or plasmids on an agar plate and repeating the colony hybridization.

Cell-specific expression of the murine CD21 gene depends on accessibility of promoter and intronic elements.

The murine complement receptor type 2 gene (Cr2/CD21) is transcriptionally active in murine B and follicular dendritic cells, but not in murine T cells. We have previously shown that altering chromatin structure via histone deacetylase inhibitors results in CD21 expression in murine T cells, and that the minimal CD21 promoter provided appropriate cell-specific expression of luciferase reporter constructs only in the presence of the first third of intron 1, fragment A. We extend this work by showing that replacing the CD21 gene promoter with the SV40 promoter resulted in the loss of this cell-specific control. Further delineation of intronic regulatory elements by fragmentation also resulted in the loss of cell-specific gene expression, suggesting that multiple CD21 promoter and intronic elements interact for appropriate CD21 gene expression. To assess this model, we performed EMSAs to define protein binding sites within promoter and intronic regions and DNase I hypersensitivity assays to determine chromatin accessibility. Multiple DNA binding factors were shown to be present in B and T cell extracts; a minority demonstrated B cell specificity. However, the DNase I sensitivity of T cell CD21 regulatory elements was not comparable to that of B cells until the histone acetylation status of the gene was altered. Taken together, these data suggest that chromatin remodeling facilitates cell-specific CD21 gene expression by modulating access of transcription factors to regulatory elements in the promoter and intron.

Interleukin-4 (IL-4) and IL-13 signaling pathways do not regulate Borrelia burgdorferi-induced arthritis in mice: IgG1 is not required for host control of tissue spirochetes.

Previous studies have suggested that interleukin-4 (IL-4) has a protective effect in host defense to Borrelia burgdorferi infection, both in limiting the severity of arthritis and in controlling spirochete numbers in tissues, and a mapping study revealed suggestive linkage to a cluster of genes on mouse chromosome 11, including the genes for IL-4 and IL-13. In contrast, other studies have questioned the importance of IL-4. In this study the involvement of IL-4 in murine Lyme disease was examined in C57BL/6J and BALB/cJ mice with targeted disruptions in the IL-4 gene, the IL-4Ralpha chain gene, or both. A spectrum of arthritis severity was seen in BALB/cJ mice, and ablation of IL-4, IL-4Ralpha, or both had no effect on the overall severity of arthritis as determined by joint swelling and histopathology. Wild-type C57BL/6J mice exhibited mild to moderate arthritis, and ablation of IL-4 again had no effect on arthritis severity. IL-4- and IL-4Ralpha-deficient mice produced extremely low levels of immunoglobulin G1 (IgG1) and showed increased production of IgG2b. This shift in immunoglobulin isotype had no effect on the host's ability to control spirochete growth in either strain of mouse, as determined by PCR detection of B. burgdorferi DNA from heart and ankle tissues. In summary, the IL-4-IL-4Ralpha pathway, including IL-13 signaling, neither limits arthritis severity nor is required for control of spirochete growth during B. burgdorferi infection of mice. Furthermore, the IgG1 isotype is not required to control B. burgdorferi cell numbers in tissues. These findings suggest the host defense against B. burgdorferi infection is not dependent on the Th1-Th2 paradigm of T-cell responses.

Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine toll-like receptor 2.

Toll-like receptor (TLR) 2 has recently been associated with cellular responses to numerous microbial products, including LPS and bacterial lipoproteins. However, many preparations of LPS contain low concentrations of highly bioactive contaminants described previously as "endotoxin protein," suggesting that these contaminants could be responsible for the TLR2-mediated signaling observed upon LPS stimulation. To test this hypothesis, commercial preparations of LPS were subjected to a modified phenol re-extraction protocol to eliminate endotoxin protein. While it did not influence the ability to stimulate cells from wild-type mice, repurification eliminated the ability of LPS to activate cells from C3H/HeJ (Lpsd) mice. Additionally, only cell lines transfected with human TLR4, but not human or murine TLR2, acquired responsiveness to both re-extracted LPS and to a protein-free, synthetic preparation of lipid A. These results suggest that neither human nor murine TLR2 plays a role in LPS signaling in the absence of contaminating endotoxin protein.

Microphthalmic mice display a B cell deficiency similar to that seen for mast and NK cells.

The microphthalmic mouse (mi) possesses a 3-bp deletion of the Mi gene that alters the DNA binding site of the transcription factor gene product. This animal has diminished numbers of NK and mast cells (MC) and is osteopetrotic due to a lack of the normal complement of functional osteoclasts. The reduction of MC has been proposed to be due to the lack of adequate c-Kit expression that is required for MC differentiation. However, data from other labs has questioned this interpretation. In this report, we present data suggesting bone marrow-derived deficiencies of the mi mouse are not due to a lack of c-Kit expression and function, but instead due to an inhospitable environment within the bone marrow itself. Specifically, we have found that such animals also lack virtually all B cell precursors within the marrow and rely upon other lymphatic sites, such as the spleen, for B cell development and maturation. Although the animal has depressed numbers of NK cells, B cells, and MC, it still possesses a normal thymus and peripheral T cells. Therefore, the block in cellular differentiation must be within the marrow environment, which is essential for maturing B cells, NK cells, and MC but not T cells.

Genomic organization, chromosomal localization, and transcriptional variants of the murine Pactolus gene.

The Pactolus gene encodes a cell surface protein similar to that of the beta integrin subunit family. The murine Pactolus gene is comprised of 16 exons that encompass 24 kb of the genome. The genomic organization of the Pactolus gene is very similar to that described for the human beta2 integrin gene (and deduced for murine beta2 integrin), including a separate exon containing only 5' untranslated sequences. The Pactolus gene was mapped to a terminal region of murine Chromosome (Chr) 16, distinct from the previously mapped site of the beta2 integrin gene on murine Chr 10. The Pactolus gene encodes three distinct transcripts via alternative splicing. The Pactolus A transcript encodes the full-length protein including transmembrane and cytoplasmic domains, while the Pactolus B transcript truncates translation before reaching these membrane-anchoring sequences. A newly discovered form, Pactolus C, is found in neonatal samples (along with Pactolus A) and would also encode a prematurely terminated protein. This form is derived from an alternative splicing event that skips part of exon 11, deletes exon 12, and uses an alternative acceptor site upstream of exon 13. The formation of the Pactolus B and C forms is thus governed by a complex alternative splicing mechanism that is affected by the developmental status of the animal.

Cutting edge: inflammatory signaling by Borrelia burgdorferi lipoproteins is mediated by toll-like receptor 2.

The agent of Lyme disease, Borrelia burgdorferi, produces membrane lipoproteins possessing potent inflammatory properties linked to disease pathology. The recent association of toll-like receptors (TLR) 2 and 4 with LPS responses prompted the examination of TLR involvement in lipoprotein signaling. The ability of human cell lines to respond to lipoproteins was correlated with the expression of TLR2. Transfection of TLR2 into cell lines conferred responsiveness to lipoproteins, lipopeptides, and sonicated B. burgdorferi, as measured by nuclear translocation of NF-kappaB and cytokine production. The physiological importance of this interaction was demonstrated by the 10-fold greater sensitivity of TLR2-transfected cells to lipoproteins than LPS. Futhermore, TLR2-dependent signaling by lipoproteins was facilitated by CD14. These data indicate that TLR2 facilitates the inflammatory events associated with Lyme arthritis. In addition, the widespread expression of lipoproteins by other bacterial species suggests that this interaction may have broad implications in microbial inflammation and pathogenesis.

Lymphoid transcription of the murine CD21 gene is positively regulated by histone acetylation.

Intronic transcriptional control sequences influence the cell- and tissue-specific expression of the CD21 gene. The interactions of such intronic control sequences, which are physically separated from the gene's promoters, suggest that factors that alter chromatin structure might be influential in this process. Accordingly, we analyzed the effect of histone acetylation on the expression of CD21 in nonexpressing T and B lymphocytes, respectively. The acetylase inhibitors sodium butyrate and trichostatin A were used to create hyperacetylated histones. The CD21 gene was specifically activated in the previously transcriptionally quiescent cells in a time- and dose-dependent fashion: the expression of a number of other genes was not influenced. These data suggest a model of cell-type-specific deacetylase activity that serves to repress gene transcription when present and active.

Rapid and sensitive quantification of Borrelia burgdorferi-infected mouse tissues by continuous fluorescent monitoring of PCR.

The quantity of Borrelia burgdorferi organisms in tissue samples is an important determinant for infection studies in the mouse model of Lyme disease. This report presents the development of a rapid and sensitive external-standard-based PCR assay for the absolute quantification of B. burgdorferi in mouse tissue samples. The assay uses a double-stranded DNA dye to continuously monitor product formation and in less than an hour was able to quantify samples ranging up to 6 log units in concentration. The PCR efficiencies of the sample and the standard were matched by using a standard composed of purified B. burgdorferi chromosome mixed with tissue-matched mouse genome lacking bacterial DNA. Normalization of B. burgdorferi quantities to the mouse nidogen gene allowed comparison of B. burgdorferi numbers in samples isolated from different tissues and strains. PCR analysis of the chromosomal gene recA in cultured B. burgdorferi was consistent with a single recA per bacterium. The parameters defined in this assay should be applicable to quantification of other organisms, even infectious agents for which no ready source of DNA standard is available. In summary, this report presents a rapid external-standard-based PCR method for the quantification of B. burgdorferi in mouse DNA samples.

Identification of quantitative trait loci governing arthritis severity and humoral responses in the murine model of Lyme disease.

A spectrum of disease severity has been observed in patients with Lyme disease, with approximately 60% of untreated individuals developing arthritis. The murine model of Lyme disease has provided strong evidence that the genetic composition of the host influences the severity of arthritis following infection with Borrelia burgdorferi: infected C3H mice develop severe arthritis while infected C57BL/6N mice develop mild arthritis. Regions of the mouse genome controlling arthritis severity and humoral responses during B. burgdorferi infection were identified in the F2 intercross generation of C3H/HeNCr and C57BL/6NCr mice. Rear ankle swelling measurements identified quantitative trait loci (QTL) on chromosomes 4 and 5, while histopathological scoring identified QTL on a unique region of chromosome 5 and on chromosome 11. The identification of QTL unique for ankle swelling or histopathological severity suggests that processes under distinct genetic control are responsible for these two manifestations of Lyme arthritis. Additional QTL that control the levels of circulating Igs induced by B. burgdorferi infection were identified on chromosomes 6, 9, 11, 12, and 17. Interestingly, the magnitude of the humoral response was not correlated with the severity of arthritis in infected F2 mice. This work defines several genetic loci that regulate either the severity of arthritis or the magnitude of humoral responses to B. burgdorferi infection in mice, with implications toward understanding the host-pathogen interactions involved in disease development.

E and P selectins are not required for resistance to severe murine lyme arthritis.

Borrelia burgdorferi-induced arthritis in mice is characterized by tendonitis, synovitis, and inflammatory-cell infiltrate, predominantly of neutrophils. Because genetic deficiency in E and P selectins results in delayed recruitment of neutrophils to sites of inflammation, mice with this deficiency were tested for their response to infection with B. burgdorferi. E and P selectins were not required for the control of B. burgdorferi numbers, nor did deficiency in E and P selectins result in alteration of arthritis severity.

The role of CD14 in signaling mediated by outer membrane lipoproteins of Borrelia burgdorferi.

Borrelia burgdorferi possesses membrane lipoproteins that exhibit stimulatory properties and, consequently, have been implicated in the pathology related to Lyme disease. As CD14 has been shown to mediate signaling by a number of lipid-modified bacterial products, the involvement of CD14 in signaling mediated by two B. burgdorferi lipoproteins, outer surface protein A (OspA) and OspC, was determined. Lipoprotein-mediated induction of nuclear factor-kappaB nuclear translocation and production of IL-8 and IL-6 in HUVEC was enhanced in the presence of serum or soluble rCD14. CD14-specific Abs that block LPS-mediated signaling also inhibited lipoprotein-dependent signaling in HUVEC and neutrophils. The formation of stable complexes between OspA and CD14 was demonstrated by native gel electrophoresis. LPS was found to compete with OspA for binding with CD14, suggesting that LPS and OspA bind similar regions on CD14. The similarity in binding was further supported by the finding that a mutant soluble CD14, lacking the LPS binding site, did not facilitate lipoprotein signaling, nor did it form a complex with OspA. Binding of OspA to CD14 was dependent on the lipid modification, as unlipidated OspA did not form a complex with CD14 or stimulate cells. In contrast, the lipopeptide remaining after proteinase K digestion both formed a complex with CD14 and retained stimulatory properties. These findings indicate that CD14 facilitates bacterial lipoprotein signaling in mammalian cells.