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R A Kroczek

Publications and source records attributed to R A Kroczek.

At least 37 records · Page 2Linked to original sources
PubMed1996

Induction, regulation, and function of soluble TRAP (CD40 ligand) during interaction of primary CD4+ CD45RA+ T cells with dendritic cells.

To assess the induction, regulation, and the relative roles of cell surface tumor necrosis factor-related activation protein (TRAP; CD40 ligand) and the soluble form of TRAP (sTRAP) in the initial phase of T cell activation, primary CD4+ CD45RA+ (naive) T cells were co-cultured with mature Langerhans' cells (mLC) in the presence of superantigen. In this cell system, TRAP was very efficiently induced in T cells at both the mRNA and protein levels. After appearing on the cell surface, TRAP was rapidly down-regulated by a mechanism triggered through interaction of TRAP with CD40 on mLC. Co-culture of T cells with mLC led to the release of sTRAP, an 18-kDa protein capable of binding to CD40. Experimental data strongly suggest that sTRAP is not released by proteolytic cleavage of TRAP on the cell surface, but is generated in an intracellular compartment. Release of sTRAP and induction of TRAP cell surface expression were found to be regulated independently. In terms of function, sTRAP cannot compete with cell surface TRAP for ligation of CD40 on mLC, indicating that sTRAP release is not a mechanism for termination of the TRAP/CD40 interaction. However, sTRAP on its own rapidly down-regulates CD40 expression on mLC and has long-lasting anti-apoptotic effects on dendritic cells. Thus, we infer from our results obtained in vitro that primary activation of CD4+ T cells by dendritic cells in the lymphoid tissues leads to release of sTRAP, which may act on CD40+ bystander cells in a cytokine-like fashion.

Adult↗
PubMed1996

Ontogeny of CD40L [corrected] expression by activated peripheral blood lymphocytes in humans.

The CD40 ligand (CD40L) is a molecule expressed by activated T cells which plays a critical role in the regulation of B-cell responses, including differentiation into Ig-producing cells. Using the specific monoclonal antibody TRAP1 we have evaluated the ontogeny of CD40L expression in 97 normal individuals between birth and 50 years of age. The expression of CD40L is a function of age; it is severely reduced at birth, progressively increases during the first months of life, and reaches a plateau in the second decade. This progressive attainment of the ability to express CD40L is due to a process of maturation of the CD4 + subset, being significantly correlated with the expression of the CD45RO antigen.

Adolescent↗
PubMed1996

[CD40 as a mediator of proliferation in normal ans neoplastic thymic epithelium].

AIMS AND METHODS: While CD40 is a costimulatory molecule of utmost importance for B-cell proliferation and Ig class switch its role has not been elucidated in the human thymus and in thymic epithelial tumors. To investigate, whether CD40 is functionally active there, we investigated the proliferative response by 3H-Thymidin incorporation of primary normal and neoplastic thymic epithelial cell cultures to CD40 triggering by soluble CD40 ligand (CD40L). RESULTS: Normal thymic epithelial cells exhibited proliferation in response to CD40 ligand in a dose dependent manner, while a CD40 ligand specific monoclonal antibody prevented this effect. This response was not significantly different from the response of neoplastic thymic epithelial cells derived from myasthenia gravis-associated thymomas. CONCLUSION: Our data demonstrate that CD40 is expressed on the surface of normal and neoplastic epithelial cells of the thymus and is a functional molecule in terms of epithelial cell proliferation in vitro. Given the differential expression of CD40 in the various histological thymoma subtypes in vivo, the finding suggests a role of CD40 in the different mechanisms of tolerance breakdown in thymomas.

Antigens, CD↗
PubMed1995

Undetectable CD40 ligand expression on T cells and low B cell responses to CD40 binding agonists in human newborns.

IgG, IgA, and IgE production by newborn B cells is limited both in vivo and in vitro in various activation conditions, whereas IgM production is readily detectable. It has been suggested that the Ig heavy chain switch inability could be the consequence of T and B cell immaturity. As the interaction between CD40 (expressed on B cells) and its ligand CD40-L (expressed on activated T cells) triggers a key signal required for isotype switching, we studied the expression and function of these two components in normal fetuses, newborns, and infants, compared with adults. CD40-L expression was not inducible in 28 of 30 specimens of newborn cord-blood T cells following incubation with PMA and ionomycin, whereas activation markers such as CD69 were inducible. CD40-L expression was triggered by activation of T cells from infants > 3 wk of age. Surprisingly, T cells from 19- to 28-wk-old fetuses also expressed CD40-L following activation. CD40-L expression on newborn T lymphocytes was induced on T cell lines generated in the presence of PHA and maintained with IL-2 following further stimulation with PMA and ionomycin. CD40-L mRNA transcripts and intracytoplasmic protein expression following activation of newborn T cells were strongly decreased, leading to undetectable protein membrane detection. These results point to a possible transcriptional down-regulation of CD40-L expression by neonatal T lymphocytes. In addition, fetal and cord-blood B cells were poorly able to switch to IgG or IgA by stimulation with CD40 agonists (Ab or soluble CD40-L) in the presence of IL-4 or IL-10 as also detected with surface IgD+ adult B cells. Both phenomena could contribute to the neonatal Ig switch inability, although distinct underlying regulatory mechanisms are probably involved, as suggested by different in vivo time courses.

Adult↗
PubMed1995

Cloning of ATAC, an activation-induced, chemokine-related molecule exclusively expressed in CD8+ T lymphocytes.

A cDNA clone, designated ATAC, was isolated from a collection of human T cell activation genes. Analysis of tissue distribution determined that ATAC mRNA of approximately 0.9 kb is exclusively expressed in activated CD8+ T cells. Induction of the ATAC gene requires stimulation by both phorbol 12-myristate 13-acetate and Ca2+ ionophore A23187 ("two-signal gene") and is fully abrogated by the immunosuppressive agent cyclosporin A. Upon stimulation, ATAC mRNA is detectable within 30 min, maximal expression is seen after 4 h. The polypeptide encoded by the open reading frame of ATAC mRNA is 114 amino acids long with a calculated M(r) of 12.52 kDa. The structural features predict the cleavage and secretion of a mature ATAC protein of approximately 10 kDa from the 12.52-kDa precursor. ATAC is highly similar to a very recently identified murine molecule designated lymphotactin both at the cDNA (73.8% identity) and the protein (61.4% identity) levels, and related to members of the C-C and C-X-C chemokine families. Two variants of the ATAC protein were expressed and tested for chemotaxis and Ca2+ release on a variety of target cells. The ATAC gene was located to chromosome 1q23.

Amino Acid Sequence↗
PubMed1995

A soluble form of TRAP (CD40 ligand) is rapidly released after T cell activation.

TRAP is a tumor necrosis factor (TNF)-related, 33-kDa type II transmembrane protein almost exclusively expressed on the surface of activated CD4+ T lymphocytes. Interaction of TRAP with CD40 on B cells is of paramount importance for immunoglobulin class switching and subsequent synthesis of IgG, IgA or IgE in vivo. We now provide evidence that activated T cells not only express cell membrane-associated TRAP but also a soluble form of TRAP (sTRAP). After generating monoclonal antibodies against TRAP and establishing a TRAP-specific enzyme-linked immunosorbent assay we were able to detect substantial amounts of sTRAP in the supernatants of activated T cells. The onset and rate of sTRAP release was found to parallel the expression of TRAP on the cell surface. sTRAP, an 18-kDa protein, is generated by proteolytic processing of full-length TRAP in an intracellular compartment. Starting with methionine 113 of full-length TRAP, sTRAP lacks the transmembrane region and a part of the extracellular domain but contains the entire TNF-alpha homology region and can, therefore, bind to CD40. Like other members of the TNF superfamily (e.g. TNF-alpha, Fas/APO-1 ligand), TRAP thus has the potential to be biologically active not only in a transmembrane form but also as a soluble molecule.

Amino Acid Sequence↗
PubMed1995

Spontaneous apoptosis of dendritic cells is efficiently inhibited by TRAP (CD40-ligand) and TNF-alpha, but strongly enhanced by interleukin-10.

In the lymphoid tissues, adaptive immune responses are initiated by the interaction of interdigitating dendritic cells (IDC) with naive T cells. To understand this interplay better, we used mature Langerhans cells (mLC), migrating from human epidermis, as the correlate of IDC ex vivo to evaluate the different effects of tumor necrosis factor (TNF)-alpha. TNF-related activation protein (TRAP; CD40-ligand) and interleukin-10 (IL-10) on induction or prevention of apoptotic cell death in these cells. Spontaneous decrease of mLC viability in culture was due to apoptosis, as determined by the appearance of typical morphological changes such as dilatation of the endoplasmic reticulum (ER), chromatin condensation and membrane blebbing. IL-10 strongly reduced mLC viability, whereas TRAP and TNF-alpha facilitated the survival of mLC. Spontaneous DNA fragmentation was detectable after 24 h in culture. IL-10 led to an earlier onset of DNA fragmentation, whereas TRAP and TNF-alpha delayed internucleosomal DNA cleavage. We found that IL-10-treated mLC were readily ingested and removed by macrophages. TNF-alpha and TRAP, in contrast, reduced engulfment of mLC by macrophages. Interestingly, IL-10, even at low concentrations, reverted the effects of TNF-alpha and TRAP in inhibiting mLC apoptosis. Furthermore, IL-10 led to the down-regulation of various surface antigens, especially of CD86 and CD54, whereas TNF-alpha and TRAP enhanced the expression of MHC class I and II antigens and of the accessory molecules CD40, CD54, CD80 and CD86. Taken together, these results show that mLC spontaneously undergo apoptosis in culture and that the progression of mLC to apoptosis is inhibited by TRAP and TNF-alpha, but accelerated by IL-10.

Apoptosis↗
PubMed1995

Synovial fibroblast-like cells strongly express jun-B and C-fos proto-oncogenes in rheumatoid- and osteoarthritis.

To identify recently activated cells in the synovial membrane (SM) of patients with rheumatoid arthritis (RA), the in situ expression of the proto-oncogenes jun-B, c-jun, jun-D, and c-fos was assessed by means of immunohistochemistry and in situ hybridization techniques. SM from patients with osteoarthritis (OA) or joint trauma (JT), as well as from normals (No) were used as controls. Numerous cells expressing high levels of jun-B and c-fos were found within lining layer and diffuse infiltrates in the vicinity of inflammatory cells, but only a few in lymphoid follicles and endothelia. The positive cells were spindle-shaped, CD14- and CD3-negative and, in addition, expressed mRNA for collagen alpha 2 (I) and alpha 1 (III), indicating that they were fibroblasts. In control OA, JT, and even No SM, individual fibroblast-like cells stained as strongly as in RA; however, the density of positive cells was substantially lower. In RA SM, fibroblasts, but not lymphocytes or macrophages, appear to undergo in situ activation. Quantitative differences among RA, OA, and JT may be related to different degrees of inflammatory infiltration.

Arthritis, Rheumatoid↗
PubMed1994

CD40 ligand (CD40L) expression and B cell function in agammaglobulinemia with normal or elevated levels of IgM (HIM). Comparison of X-linked, autosomal recessive, and non-X-linked forms of the disease, and obligate carriers.

Hyper-IgM syndrome is a rare immunodeficiency characterized by low or absent IgG, IgA, and IgE with normal or elevated levels of IgM. It can occur as an acquired or familial disorder with either X-linked or autosomal modes of inheritance. The X-linked form (HIGM1) is a result of mutations in the CD40 ligand (CD40L) gene, but the defect in non-X-linked forms of the disease (HIM) has not been determined. We show here that CD40L expression on activated T cells from non-X-linked patients can be detected by CD40Fc, 5c8 Mab, and anti-TRAP, whereas activated T cells from HIGM1 patients either had no detectable CD40L (Type I), or stained with anti-TRAP but not CD40Fc or 5c8 (Type II). Activated T cells from obligate carriers varied from low to normal expression of CD40L. B cells from HIGM1 and non-X-linked HIM patients proliferated in response to CD40L. Costimulation of B cells from HIGM1, from sporadic HIM, or from non-X-linked HIM patients with CD40L plus IL-2 resulted in some IgM production, but no significant IgG or IgA. Costimulation with CD40L plus IL-10 resulted in significant IgG and/or IgA secretion by B cells from some HIGM1 patients, but consistently failed to stimulate IgG or IgA secretion by B cells from non-X-linked patients. In addition, costimulation with CD40L and IL-4 failed to induce IgE secretion by B cells from one non-X-linked HIM patient, and induced a weak response in another. These results suggest that patients with non-X-linked forms of HIM may have an intrinsic B cell defect preventing heavy chain switching, which is not related to expression of CD40L.

Adolescent↗
PubMed1994

Leishmania major parasites share an epitope with the murine CD3-T cell receptor complex.

After immunization of BALB/c mice with a low molecular mass fraction (FrD; < or = 31 kDa) isolated from a soluble extract of Leishmania major promastigotes, a panel of monoclonal antibodies (mAb) was obtained. One of these antibodies (mAb 9C) recognized a cytosol-associated antigen from L. major of approximately 21 kDa as shown by Western blot and immunoprecipitation. In addition, mAb 9C reacted with surface structures of murine splenic T cells and T cell clones. Reactivity was confined to murine cells, but was not strain restricted. Immunoprecipitation studies and surface-labeling experiments with CD4+ T cell clones and the T cell receptor (TCR)-CD3-T cell line TG40 transfected with V alpha/beta chains from human TCR and concomitant co-expression of murine CD3 suggested that mAb 9C binds to an epitope located within the murine CD3-TCR complex. In addition, mAb 9C induced strong T cell proliferation. We conclude that L. major parasites share an epitope with the murine CD3-TCR complex which is functionally important for T cell activation.

Animals↗
PubMed1994

Defective expression of CD40 ligand on T cells causes "X-linked immunodeficiency with hyper-IgM (HIGM1)".

X-linked immunodeficiency with hyper-IgM (HIGM1) is a rare disorder, characterized by recurrent infections associated with very low or absent IgG and IgA, and normal to increased IgM serum levels. The disease has been earlier mapped to the q26-27 region of the X-chromosome. We have identified a novel molecule expressed on the surface of activated T cells, which was designated TRAP (Tumor necrosis factor Related Activation Protein), and could demonstrate that TRAP is a ligand for the CD40 receptor expressed on B cells. Our mapping of the TRAP gene to the Xq26.3-27.1 region suggested a causal relationship to HIGM1. Further work revealed that various mutations of the TRAP/CD40 ligand (CD40L) gene may lead to a defective expression of the TRAP/CD40L molecule on the T-cell surface in HIGM1 patients. A combination of structural and functional analyses finally demonstrated that the failure of TRAP/CD40L on T cells to interact with CD40 on B cells is responsible for the inefficient T-cell help for B cells observed in HIGM1. The observations made in HIGM1 allowed us to conclude that TRAP/CD40L is not required for IgM synthesis. In contrast, functional expression of TRAP is a prerequisite for effective immunoglobulin isotype switching and subsequent production of IgG, IgA and IgE by B cells in vivo. The interaction of TRAP/CD40L with CD40 thus provides a very critical link between the cellular and the humoral part of the immune system. The knowledge of TRAP/CD40L cDNA sequence, the availability of various reagents for the testing of expression and function of TRAP/CD40L, and our recent elucidation of the exon-intron structure of the TRAP/CD40L gene now provide all necessary tools for early diagnosis of affected patients and the detection of female carriers of HIGM1. The available information will also provide a basis for future attempts at gene therapy in this disease.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1994

NOT, a human immediate-early response gene closely related to the steroid/thyroid hormone receptor NAK1/TR3.

By analyzing the early genetic response of human T cells following mitogenic activation we have identified NOT, a member of the steroid/thyroid hormone family of receptors. NOT has all structural features of steroid/thyroid hormone receptors (C2C2 zinc-finger domain, ligand binding domain), but is rapidly and only very transiently expressed after cell activation, which is clearly at variance with classical steroid receptors such as glucocorticoid or estrogen receptors. NOT gene induction is independent of de novo protein synthesis, defining NOT as an immediate-early response gene. Short-lived NOT mRNA (4.2 kilobases) expression could be observed in vitro in a greater number of tissue types following activation by a variety of distinct stimuli. In vivo, NOT mRNA expression was detected exclusively in the brain, where a very strong signal was observed. By immunoblot analysis of human T cell lysates with NOT specific antisera two activation-dependent protein bands (66 and 59 kilodaltons) could be detected. NOT gene was localized to human chromosome 2q22-q23. Sequence comparison revealed that NOT is the human homolog of the murine NURR1 and rat RNR-1. Moreover NOT is closely related to NAK1/TR3, a previously identified human orphan steroid receptor. Several lines of evidence indicate that NOT and NAK1/TR3 form a distinct and exclusive subgroup of orphan steroid receptors, whose expression characteristics in vitro and in vivo resemble the expression of nonsteroid immediate-early transcription factors such as jun and fos. NOT and NAK1/TR3 thus may function as general coactivators of gene transcription rather than participate in the induction of specific target genes, as is the case with classical steroid receptors.

Amino Acid Sequence↗
PubMed1993

Southern and northern analysis.

This review on Southern and Northern analysis, rather than providing step-by-step protocols, focuses on a critical evaluation of the existing experimental methods and modifications thereof. Principal parameters influencing electrophoresis of DNA/RNA in agarose gels are outlined and at the same time alterations in these parameters for optimal resolution of DNA of varying length are discussed. Further, methods for evaluating the quality of DNA/RNA size separation in agarose gels are described. Since efficient transfer of DNA/RNA from the gel onto a membrane support is critical in both methods, several experimental approaches for transfer are compared. Also discussed in this review are alternative methods for radioactive and non-radioactive labeling of DNA probes. Finally, detailed protocols are provided for an effective hybridization of Southern and Northern blots.

Animals↗
PubMed1993

Defective expression of T-cell CD40 ligand causes X-linked immunodeficiency with hyper-IgM.

X chromosome-linked immunodeficiency with hyper-IgM (HIGM1, MIM number 308230) is a rare disorder characterized by recurrent bacterial infections, very low or absent IgG, IgA and IgE, and normal to increased IgM and IgD serum levels. HIGM1 has been suggested to result from ineffective T-cell help for B cells. We and others have identified a novel, TNF-related activation protein (TRAP) that is exclusively expressed on the surface of stimulated T cells. TRAP, a type II transmembrane protein of M(r) 33,000, is the physiological ligand for CD40 (refs 5-8). Crosslinking of CD40 on B cells induces, in the presence of lymphokines, immunoglobulin class switching from IgM to IgG, IgA or IgE. Mapping of the TRAP gene to the X-chromosomal location q26.3-q27.1 (ref. 6) suggested a causal relationship to HIGM1, which had previously been assigned to Xq26 (refs 12-14). Here we present evidence that point mutations in the TRAP gene give rise to nonfunctional or defective expression of TRAP on the surface of T cells in patients with HIGM1. The resultant failure of TRAP to interact with CD40 on functionally intact B cells is responsible for the observed immunoglobulin isotype defect in HIGM1.

Antigens, CD↗
PubMed1993

Biased T cell receptor V gene usage in rheumatoid arthritis. Oligoclonal expansion of T cells expressing V alpha 2 genes in synovial fluid but not in peripheral blood.

OBJECTIVE: To analyze the T cell receptor (TCR) variable (V) region gene usage in the rheumatoid joint. METHODS: Monoclonal antibodies (MAb) were used to determine the prevalence of selected V elements on T cells in synovial fluid (SF) from rheumatoid arthritis (RA) patients and in peripheral blood (PB) from RA patients and normal controls. V alpha 2-positive PB and SF T cells from 1 patient were cloned by immediate limiting-dilution and analyzed by restriction mapping. RESULTS: In 9 of 14 RA patients, SF was enriched in at least 1 of the selected V elements, compared with PB. TCR genes of the V alpha 2 family were the most frequently overrepresented in the SF (4 patients). The expanded V alpha 2-positive cells were oligoclonal in SF but heterogeneic in PB. CONCLUSION: Our data showing biased and clonally restricted TCR elements in the rheumatoid joint indicate major histocompatibility complex-restricted antigen recognition, rather than a "superantigen," in the pathogenesis of RA.

Adult↗
PubMed1993

Expression of PILOT, a putative transcription factor, requires two signals and is cyclosporin A sensitive in T cells.

Few known genes (IL-2, members of the IL-8 family, interferon-gamma) are induced in T cells only through the combined effect of phorbol myristic acetate (PMA) and a Ca(2+)-ionophore, and expression of only these genes can be fully suppressed by Cyclosporin A (CyA). We have identified a putative transcription factor, designated PILOT, with an identical dual signal requirement for expression. Induction of the PILOT gene is detectable in human T cells 20 min following activation in the presence of cycloheximide and is fully suppressed by CyA. The PILOT protein has a calculated M(r) of 42.6 kDa and contains three zinc fingers of the C2H2-type at the carboxyl-terminus which are highly homologous to the zinc finger regions of the transcription factors EGR1, EGR2, and pAT 133. In contrast to T cells, in fibroblasts PILOT gene expression requires only one signal (PMA) and is not affected by CyA. This observation directly demonstrates the existence of a Ca2+ signal-dependent regulatory element obligatory for expression of some genes in T cells but not in fibroblasts. This differential expression model will be valuable in the dissection of the dual signal pathway in T cells and the effects of CyA upon it.

Amino Acid Sequence↗
PubMed1992

Cloning of TRAP, a ligand for CD40 on human T cells.

A cDNA clone, designated TRAP (TNF-related activation protein) was isolated from a collection of T cell activation genes. The polypeptide encoded by a mRNA of approx. 2.3 kb is 261 amino acids long with a calculated M(r) of 29.3 kDa. The structural features predict a type II transmembrane protein, but are also compatible with a secreted form. TRAP is highly similar to an identified murine CD40 ligand both at the cDNA (82.8% identity) and the protein (77.4% identity) levels, and related to tumor necrosis factor/lymphotoxin. Expressed in a murine myeloma, TRAP was identified as a ligand for CD40 by binding to a soluble CD40 construct. TRAP mRNA is expressed in a T cell-specific fashion with a maximum at 8 h after stimulation. The TRAP gene is located in the q26.3-q27.1 region of the X chromosome.

Amino Acid Sequence↗