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D D Chaplin

Publications and source records attributed to D D Chaplin.

At least 37 records · Page 2Linked to original sources
PubMed1998

Targets in cytokine activation.

The first morning session of the Eighth International Conference of the Inflammation Research Association was titled 'Targets in Cytokine Activation'. It encompassed four areas of research that may be considered as either current or future targets. Probably the best established target of the four is interleukin-1 beta converting enzyme (ICE) and Winnie Wong from BASF Bioresearch Corporation presented an overview of work in this field. This was followed by a newly emerging target called TACE (TNF-alpha converting enzyme) in a presentation from Douglas Cerretti of Immunex. The final two presentations covered work with chemoattractant receptors (Craig Gerard, Harvard) and mice where the inducible NO synthase gene had been deleted (John Mudgett, Merck).

ADAM Proteins↗
PubMed1998

Cytokine regulation of secondary lymphoid organ development.

Lymphotoxin and tumor necrosis factor provide essential signals for the formation of secondary lymphoid tissue structures. Lymphotoxin in its membrane form (LT alpha 1 beta 2 heterotrimer) is required for the development of lymph nodes and Peyer's patches and supports the development of normal spleen structure. In the spleen, lymphotoxin acts during embryonic development to support the formation of distinct B and T cell zones. Lymphotoxin also acts in a tonic fashion-supporting the formation and maintenance of the follicular dendritic cell network and of primary B cell follicle structure. The cells that deliver the tonic lymphotoxin signal supporting follicular dendritic cell structure are B cells; thus, B cells participate fundamentally in the development of the lymphoid tissue structure in which they subsequently mature.

Animals↗
PubMed1997

Distinct roles of lymphotoxin alpha and the type I tumor necrosis factor (TNF) receptor in the establishment of follicular dendritic cells from non-bone marrow-derived cells.

In mice deficient in either lymphotoxin alpha (LT-alpha) or type I tumor necrosis factor receptor (TNFR-I), organized clusters of follicular dendritic cells (FDC) and germinal centers (GC) are absent from the spleen. We investigated the role of LT-alpha and TNFR-I in the establishment of spleen FDC and GC structure by using reciprocal bone marrow (BM) transfer. When LT-alpha-deficient mice were reconstituted with wild-type BM, FDC organization and the ability to form GC were restored, indicating that the LT-alpha-expressing cells required to establish organized FDC are derived from BM. The role of LT-alpha in establishing organized FDC structure was further investigated by the transfer of complement receptor 1 and 2 (CR1/2)-deficient BM cells into LT-alpha-deficient mice. Organized FDC were identified with both the FDC-M1 and anti-CR1 monoclonal antibodies in these BM-chimeric mice, indicating that these cells were derived from the LT-alpha-deficient recipient. Thus, expression of LT-alpha in the BM-derived cells, but not in the non-BM-derived cells, is required for the maturation of FDC from non-BM precursor cells. In contrast, when TNFR-I-deficient mice were reconstituted with wild-type BM, they showed no detectable FDC clusters or GC formation. This indicates that TNFR-I expression on non-BM-derived cellular components is necessary for the establishment of these lymphoid structures. TNFR-I-deficient BM was able to restore FDC organization and GC formation in LT-alpha-deficient mice, indicating that formation of these structures does not require TNFR-I expression on BM-derived cells. The data in this study demonstrate that FDC organization and GC formation are controlled by both LT-alpha-expressing BM-derived cells and by TNFR-I-expressing non-BM-derived cells.

Animals↗
PubMed1997

Abrogation of the alternative complement pathway by targeted deletion of murine factor B.

To investigate the role of complement protein factor B (Bf) and alternative pathway activity in vivo, and to test the hypothesized potential genetic lethal effect of Bf deficiency, the murine Bf gene was interrupted by exchange of exon 3 through exon 7 (including the factor D cleaving site) with the neor gene. Mice heterozygous for the targeted Bf allele were interbred, yielding Bf-deficient offspring after the F1 generation at a frequency suggesting that Bf deficiency alone has no major effect on fertility or fetal development. However, in the context of one or more genes derived from the 129 mouse strain, offspring homozygous for Bf deficiency were generated at less than expected numbers (P = 0.012). Bf-deficient mice showed no gross phenotypic difference from wild-type littermates. Sera from Bf-deficient mice lacked detectable alternative complement pathway activity; purified mouse Bf overcame the deficit. Classical pathway-dependent total hemolytic activity was lower in Bf-deficient than wild-type mice, possibly reflecting loss of the alternative pathway amplification loop. Lymphoid organ structure and IgG1 antibody response to a T-dependent antigen appeared normal in Bf-deficient mice. Sensitivity to lethal endotoxic shock was not significantly altered in Bf-deficient mice. Thus, deficiency of Bf and alternative complement activation pathway led to a less dramatic phenotype than expected. Nevertheless, these mice provide an excellent model for the assessment of the role of Bf and the alternative pathway in host defense and other functions in vivo.

Animals↗
PubMed1997

Lymphotoxin-alpha (LTalpha) supports development of splenic follicular structure that is required for IgG responses.

LTalpha-deficient (LTalpha-/-) mice show altered splenic microarchitecture. This includes loss of normal B cell-T cell compartmentalization, of follicular dendritic cell (FDC) clusters, and of ability to form germinal centers (GC). LTalpha-/- mice immunized with sheep red blood cells (SRBC) produced high levels of antigen-specific IgM but no IgG in either primary or secondary responses, demonstrating failure of Ig class switching. This inability to switch to IgG could have been due to the altered splenic microarchitecture in these mice. Alternatively, it could have been due directly to a requirement for LTalpha expression by lymphocytes cooperating in the antibody response. To investigate this, we performed reciprocal spleen cell transfers. When irradiated LTalpha-/- mice were reconstituted with wild-type splenocytes and immunized immediately with SRBC, splenic microarchitecture remained disturbed and there was no IgG response. In contrast, when irradiated wild-type animals received splenocytes from LTalpha-/- mice, follicle structure and a strong IgG response were retained. These data indicate that LTalpha-deficient B cells and T cells have no intrinsic defect in ability to generate an IgG response. Rather, the altered microenvironment characteristic of LTalpha-/- mice appears to result in impaired ability to switch to a productive IgG response. To investigate whether prolonged expression of LTalpha could alter the structure and function of spleen follicles, reciprocal bone marrow (BM) transplantation was performed. Six weeks after reconstitution of LTalpha-/- mice with wild-type BM, spleen follicle structure was partially restored, with return of FDC clusters and GC. B cell/T cell compartmentalization remained abnormal and white pulp zones were small. This was accompanied by restoration of IgG response to SRBC. Reconstitution of wild-type mice with LTalpha-/- BM resulted in loss of FDC clusters and GC, and loss of the IgG response, although compartmentalized B cell and T cell zones were largely retained. Thus, defective IgG production is not absolutely associated with abnormal B cell and T cell compartmentalization. Rather, expression of LTalpha supports the maturation of spleen follicle structure, including the development and maintenance of FDC clusters, which supports Ig class switching and an effective IgG response.

Animals↗
PubMed1997

Independent signals regulate development of primary and secondary follicle structure in spleen and mesenteric lymph node.

Lymphotoxin-alpha-deficient (LT-alpha-/-) mice manifest congenital absence of lymph nodes (LNs) and Peyer's patches and disturbed spleen follicle structure. The splenic white pulp areas show loss of discrete T and B lymphocyte zones, of follicular dendritic cell (FDC) clusters, and of germinal centers (GCs). Tumor necrosis factor receptor I-deficient (TNFR-I-/-) mice show similar absence of FDC clusters and GCs but retain segregation of T and B cell zones. Rarely are mesenteric LNs found in LT-alpha-/- mice. These mesenteric LNs show segregation of T and B cell zones similar to wild-type mice. In contrast, mesenteric LNs in TNFR-I-/- mice manifest grossly disturbed organization of T and B cells. Both LT-alpha-/- and TNFR-I-/- mice lacked FDC clusters in LNs and spleen. Interestingly, although both LT-alpha-/- and TNFR-I-/- mice that had been immunized with sheep red blood cells failed to form GCs in the spleen, they both developed GC-like clusters of peanut agglutinin-positive (PNA+) cells in their LNs. Furthermore, when lethally irradiated recombination activating gene (RAG)-1-deficient (RAG-1(-/-)) mice that had received spleen cells from LT-alpha-/- mice were immunized with sheep red blood cells, they failed to generate PNA+ clusters in the reconstituted spleen but showed robust PNA+ clusters in the reconstituted LNs. These data demonstrate that the signals that regulate the development of distinct T and B cell zones as well as the signals that regulate B cell activation to produce clusters of PNA+ cells differ between the spleen and LNs.

Animals↗
PubMed1997

Lymphotoxin-alpha-deficient and TNF receptor-I-deficient mice define developmental and functional characteristics of germinal centers.

Mice deficient in LT alpha (LT alpha-/-) lack lymph nodes and Peyer's patches. This action of LT alpha in lymph node organogenesis appears to be mediated by the membrane form of LT using a mechanism independent of TNF receptor I (TNFR-I) or II (TNFR-II). In contrast, normal Peyer's patch development appears to require both LT alpha and TNFR-I, with TNFR-I-/- mice showing hypoplastic Peyer's patch structures. LT alpha-/- mice also fail to support the normal segregation of T-cell and B-cell zones within the splenic white pulp. Again, this occurs via a mechanism independent of TNFR-I or TNFR-II. Additionally, follicular dendritic cell (FDC) clusters or germinal centers fail to develop in the spleen of LT alpha-/- animals. Mice deficient in either TNF alpha or TNFR-I also fail to develop splenic FDC clusters and germinal centers, indicating that signaling by both LT alpha and TNF alpha is required for development of these specialized lymphoid tissue structures. Finally, the splenic white pulp areas in LT alpha-/- mice lack the marginal zone of monoclonal antibody MOMA-1-staining metallophilic macrophages, whereas TNFR-I-deficient mice have preserved MOMA-1 staining. Thus, certain actions of LT alpha to regulate spleen white pulp architecture are mediated by receptors other than TNFR-I, most likely by the LT beta R or a closely related receptor. We tested whether germinal centers are essential for maturation of T-cell-dependent antibody responses. When LT alpha-/- mice were immunized with low doses of NP-ovalbumin (NP-OVA) adsorbed to alum, there was dramatically impaired production of high affinity anti NP IgG; however, after immunization with high doses of NP-OVA adsorbed to alum, LT alpha-/- mice mounted a high affinity NP-specific serum IgG response similar to wild-type mice, all in the absence of germinal centers or clustered FDC. Thus, although germinal centers enhance the processes required for maturation of the humoral immune response, the mechanisms responsible for affinity maturation are not absolutely dependent on the presence of germinal centers.

Animals↗
PubMed1996

Affinity maturation without germinal centres in lymphotoxin-alpha-deficient mice.

Affinity maturation by somatic hypermutation is thought to occur within germinal centres. Mice deficient in lymphotoxin-alpha (LT alpha-/- mice) have no lymph nodes or Peyer's patches, and fail to form germinal centres in the spleen. We tested whether germinal centres are essential for maturation of antibody responses to T-cell-dependent antigens. LT alpha-/- mice immunized with low doses of (4-hydroxy-3-nitrophenyl)acetyl-ovalbumin (NP-OVA) showed dramatically impaired production of high-affinity anti-NP IgG1. However, LT alpha-/- mice immunized with high doses of NP-OVA, even though they failed to produce germinal centres, manifested a high-affinity anti-NP IgG1 response similar to wild-type mice. Furthermore, when LT alpha-/- mice were multiply immunized with high doses of NP-OVA, the predominantly expressed anti-NP VH gene segment VH186.2 showed somatic mutations typical of affinity maturation. Thus, B-cell memory and affinity maturation are not absolutely dependent on the presence of germinal centres.

Amino Acid Sequence↗
PubMed1996

Markedly impaired humoral immune response in mice deficient in complement receptors 1 and 2.

Complement receptor 1 (CR1, CD35) and complement receptor 2 (CR2, CD21) have been implicated as regulators of B-cell activation. We explored the role of these receptors in the development of humoral immunity by generating CR1- and CR2-deficient mice using gene-targeting techniques. These mice have normal basal levels of IgM and of IgG isotypes. B- and T-cell development are overtly normal. Nevertheless, B-cell responses to low and high doses of a T-cell-dependent antigen are impaired with decreased titers of antigen-specific IgM and IgG isotypes. This defect is not complete because there is still partial activation of B lymphocytes during the primary immune response, with generation of splenic germinal centers and a detectable, although reduced, secondary antibody response. These data suggest that certain T-dependent antigens manifest an absolute dependence on complement receptors for the initiation of a normally robust immune response.

Animals↗
PubMed1996

Mice deficient in IL-1beta manifest impaired contact hypersensitivity to trinitrochlorobenzone.

Mice rendered deficient in IL-1 beta by gene targeting in embryonic stem cells develop and grow normally in a protected laboratory environment. Endotoxin-stimulated peritoneal macrophages from IL-1beta-deficient mice showed normal synthesis and cellular release of IL-1alpha after treatment with 5 mM ATP demonstrating that IL-1beta is not necessary for expression and release of the IL-1alpha isoform. Mice deficient in IL-1beta showed unaltered sensitivity to endotoxic shock, with or without pretreatment with D-galactosamine. In contrast, IL-1beta-deficient mice showed defective contact hypersensitivity responses to topically applied trinitrochlorobenzene (TNCB). This defect could be overcome either by application of very high doses of sensitizing antigen, or by local intradermal injection of recombinant IL-1beta immediately before antigen application. These data demonstrate an essential role for IL-1beta in contact hypersensitivity and suggest that IL-1beta acts early during the sensitization phase of response. They suggest an important role for IL-1beta in initiation of the host of response at the epidermal barrier.

Animals↗
PubMed1996

Role of lymphotoxin and the type I TNF receptor in the formation of germinal centers.

In mice deficient in either lymphotoxin-alpha (LT-alpha) or the type I tumor necrosis factor (TNF) receptor, but not the type II TNF receptor, germinal centers failed to develop in peripheral lymphoid organs. Germinal center formation was restored in LT-alpha-deficient mice by transplantation of normal bone marrow, indicating that the LT-alpha-expressing cells required to establish this lymphoid structure are derived from bone marrow.

Animals↗
PubMed1995

A novel Creb family gene telomeric of HLA-DRA in the HLA complex.

cDNA selection was used to identify genes encoded by a 440-kb yeast artificial chromosome (YAC) clone that spanned from HLA-DRA to CYP21 in the HLA complex. An initially selected short cDNA was used to isolate a 2639-nucleotide, apparently full-length cDNA from a human tonsil library. This cDNA contained one extended open reading frame that predicted a protein of 700 amino acids with a basic region and a leucine zipper that is highly similar to members of the Creb/ATF subfamily. High-stringency Southern blotting of total human genomic DNA using this cDNA as the probe showed only a single locus that mapped to the selecting YAC clone. This gene, designated Creb-related protein (Creb-rp), is expressed ubiquitously and is evolutionarily conserved in mammals. It is located in the HLA Class III region 6-10 kb centromeric of the XB gene, which encodes a tenascin-like extracellular matrix protein. Homologous sequences are located in the Class II-Class III interval of the mouse H-2 complex. The amino acid sequence homology and general structural features of the predicted protein indicate that this gene encodes a general transcription factor belonging to the Creb/ATF subfamily of the bZip super-family.

Amino Acid Sequence↗
PubMed1995

Feature mapping of the HLA class I region: localization of the POU5F1 and TCF19 genes.

The class I region of the human leukocyte antigen (HLA) complex located on chromosome 6p21.3 is gene dense. To define the gene content of the class I region, we are constructing genomic DNA feature maps. Here we report mapping of the POU5F1 and TCF19 genes to an approximately 0.2-Mb region between the HLA-C and the S genes. Localization of these genes was facilitated by subcloning genomic DNA fragments from the 0.2-Mb region into a transposon gamma delta-based vector, selecting transposon-mediated deletions in vivo in Escherichia coli, and sequencing a nested subset chosen for their uniform distribution of deletion endpoints. The POU5F1 and TCF19 genes are located approximately 130 kb telomeric of the HLA-C locus, approximately 0.6 kb apart from each other. Complete sequencing of a 5.5-kb EcoRI fragment containing the TCF19 gene revealed that it is composed of three exons, bounded by consensus splice signals. These experiments illustrate that the transposon-based nested deletion sequencing method provides an easy and efficient approach to feature mapping genomic fragments and to high-resolution analysis of selected subportions.

Base Sequence↗
PubMed1995

Restriction map of a 35-kb HLA fragment constructed by nested deletion 'drop-out' mapping.

An efficient method for generating detailed restriction maps of large cloned DNA segments is demonstrated. The mapping strategy entails comparing restriction fragments from a parent clone and from nested deletion derivatives of that clone. In a set of deletion plasmids of decreasing size, an individual fragment will be lost, or 'drop-out', according to its position in the cloned fragment. In this demonstration, nested deletions were generated in both directions in a 35-kb DNA segment from the human leukocyte antigen (HLA) region by intramolecular transposition of an engineered gamma delta (Tn1000) element present in a special 'deletion factory' cloning vector [Wang et al., Proc. Natl. Acad. Sci. USA 90 (1993) 7874-7878]. Fifteen plasmids with deletions extending in one direction and eleven plasmids with deletions extending in the opposite direction were digested singly by each of four restriction enzymes. A total of 36 cleavage sites were mapped in the 35-kb HLA fragment. This drop-out approach using nested deletions provides a simple and efficient means of mapping restriction sites, genes and other features of interest in cosmid-sized cloned DNA segments or DNAs.

Animals↗
PubMed1995

Construction of a genomic DNA 'feature map' by sequencing from nested deletions: application to the HLA class I region.

We are applying a transposon-based approach for detecting and mapping features of special interest to construct 'feature maps' of currently uncharacterized portions of the human leukocyte antigen (HLA) complex on chromosome 6. Such feature maps should facilitate identifying regions for high resolution analysis. Here we describe the feature mapping of a 35 kb DNA fragment located between the HLA-C and HLA-E loci. This fragment was cloned into a transposon gamma delta-based cosmid vector designed for generating nested deletions in vivo. Seventy informative nested deletions extending into the cloned fragment were isolated, and DNA adjacent to the deletion endpoints was sequenced by fluorescent automated technology. These islands of DNA sequences constituted the foundation of the feature map, and (i) identified putative exons, (ii) determined the positions of Alu elements, (iii) determined the span of the keratinocyte-specific S gene, and (iv) localized evolutionarily conserved sequences. The construction of feature maps using this in vivo nested deletion-sequencing approach provides a rapid and efficient means to identify DNA regions that merit more detailed analysis.

Base Sequence↗
PubMed1995

Macrophage apoptosis in the absence of active interleukin-1 beta-converting enzyme.

The interleukin-1 beta converting enzyme (ICE) is the cysteine proteinase responsible for cleaving the 31-kDa interleukin-1 beta (IL-1 beta) precursor to its active 17-kDa form. In lipopolysaccharide-stimulated cultured macrophages, induction of apoptosis but not necrosis effectively induces conversion of the IL-1 beta precursor to its mature form and results in the concomitant release of the mature cytokine from the cell. To determine whether ICE activity is required for macrophage apoptosis, we have exposed macrophages either to 5 mM ATP or to alloreactive cytolytic T lymphocytes (CTL) in the absence and presence of the ICE inhibitor peptide YVAD-chloromethylketone (YVAD-emk). Activated cells treated with YVAD-emk and ATP or CTL showed no mature IL-1 beta in either the cell lysates or the culture supernatants, indicating effective inhibition of ICE activity; however, the YVAD-treated macrophages showed no detectable change in 51Cr release or nuclear fragmentation, indicating failure to inhibit apoptotic cell death. Thus, in these cells, YVAD-emk uncouples IL-1 beta processing and apoptosis.

Adenosine Triphosphate↗