Tissue-specific function of lymph node fibroblastic reticulum cells.

OBJECTIVE: We present the first characterization of the cytokine expression pattern of lymph node fibroblastic reticulum cells (FRC), which are the stromal cells responsible for maintaining the highly structured nodal reticular fiber framework. METHODS: Microarray expression profiles of cultured nodal FRC and dermal fibroblasts (DF) were compared as well as their response to TNF, IL-4, IL-6 and IL-13, cytokines responsible for intranodal stromal activation. RESULTS: Hierarchical clustering of FRC and DF short-term culture samples revealed genes that were differentially expressed in FRC and DF. Identified differently regulated genes were confirmed by RNase protection analysis, PCR or immunohistochemistry. At earlier culture time points, FRC showed higher levels of several chemokines, including CCL2/MCP-1, and cytokines, e.g. IL-6, whereas several genes related to the production of extracellular matrix and angiogenesis were preferentially expressed in early DF cultures. By 60 days in culture, FRC and DF showed similar expression patterns consistent with homogenization of specialized stromal subsets. FRC and DF showed nearly identical transcriptional responses to exogenous TNF stimulation. CONCLUSIONS: Cultured FRC showed an overall transcriptional profile similar to cultured DF, including parallel responsiveness to TNF, but with differences in the expression of chemotactic chemokines, which reflect their biological roles.

Constitutive activation of JAK3/STAT3 in colon carcinoma tumors and cell lines: inhibition of JAK3/STAT3 signaling induces apoptosis and cell cycle arrest of colon carcinoma cells.

Signal transducer and activator of transcription 3 (STAT3) has oncogenic potential. The biological effects of STAT3 have not been studied extensively in the pathogenesis of colon cancer, nor has the role of Janus kinase 3 (JAK3), the physiological activator of STAT3, been evaluated. Here, we demonstrate that activated STAT3 (pSTAT3) and activated JAK3 (pJAK3) are expressed constitutively in two colon cancer cell lines, SW480 and HT29. To evaluate the significance of JAK3/STAT3 signaling, we inhibited JAK3 with AG490 and STAT3 with a dominant-negative construct. Inhibition of JAK3 down-regulated pSTAT3. The blockade of JAK3/STAT3 signaling significantly decreased viability of colon cancer cells due to apoptosis and cell-cycle arrest through down-regulation of Bcl-2, Bcl-X(L), Mcl-1, and cyclin D2 and up-regulation of p21(waf1/cip1) and p27(kip1). We also examined histological sections from 22 tumors from patients with stage II or stage IV colon cancer and found STAT3, JAK3, and their activated forms to be frequently expressed. Furthermore, quantitative reverse transcriptase-polymerase chain reaction identified JAK3 mRNA in colon cancer cell lines and primary tumors. Our findings illustrate the biological importance of JAK3/STAT3 activation in the oncogenesis of colon cancer and provide novel evidence that JAK3 is expressed and contributes to STAT3 activation in this malignant neoplasm.

EBV-associated B- and T-cell posttransplant lymphoproliferative disorders following primary EBV infection in a kidney transplant recipient.

Posttransplant lymphoproliferative disorders (PTLDs) usually are of B-cell lineage and associated with Epstein-Barr virus (EBV). PTLDs of T-cell lineage are much less common and infrequently associated with EBV. We report a rare case of a girl in whom B-cell and T-cell PTLDs developed following 2 EBV-negative kidney transplants. Within 2 years of the second transplantation, the originally EBV-negative patient developed both an EBV-associated clonal B-cell PTLD involving lymph nodes and an EBV-positive T-cell PTLD involving bone marrow and liver. These proliferations occurred concurrently with evidence of primary EBV infection and high plasma viral load. The patient eventually died of multiorgan failure 5 years after the initial transplant (3 years after the second transplant). To our knowledge, only 4 cases of both B-cell and T-cell PTLDs have been reported. Only 2 cases have been proven to be monoclonal and EBV-associated, as in this case, the first following kidney transplantation.

Transient down-modulation of CD20 by rituximab in patients with chronic lymphocytic leukemia.

Lymphoid cells in most patients with chronic lymphocytic leukemia (CLL), when treated with rituximab, become CD20-. This is thought to be due to masking of CD20 by rituximab. We used specific antimouse immunoglobulin antibodies to detect rituximab on the surface of CLL lymphocytes and we demonstrate that rituximab is rarely detectable after therapy. Only 3 of 65 patients with CLL had rituximab detectable on their lymphocytes after rituximab therapy despite the fact that most had no detectable CD20 expression. In vitro mixing of CLL or Raji cells with rituximab demonstrated that rituximab was detectable on the surface of cells due to its binding to CD20. However, the addition of plasma led to the down-modulation of CD20 expression, and the rituximab became undetectable. This down-modulation of CD20 protein expression was associated with a down-modulation of CD20 mRNA. CLL cells that lost their CD20 expression regained CD20 expression after 24 hours in culture. These data suggest that rituximab therapy leads to a substantial but transient down-modulation of CD20 expression and that negativity for CD20 in cells from patients treated with rituximab is not necessarily due to CD20 masking. The importance of this down-modulation in the efficacy of current therapy with rituximab needs further investigation.

Phenotypic modulation of the stromal reticular network in normal and neoplastic lymph nodes: tissue transglutaminase reveals coordinate regulation of multiple cell types.

The lymph node paracortex is composed of a network of fibroblastic reticular cells (FRC) and reticular fibers linking sinuses to blood vessels. Using immunostaining for the inducible enzyme/adhesion molecule tissue transglutaminase (TG), we demonstrate coordinate regulation of multiple stromal cell types of this reticular network including FRC, endothelial cells and sinus lining cells. Tissue transglutaminase is expressed at low levels in the paracortex around primary follicles but is markedly up-regulated in stromal cells around hyperplastic germinal centers and then down-regulated around regressing follicles. In cultured FRC, TG is induced 5- to 20-fold and becomes colocalized with matrix proteins by treatment with interleukin-4, but not by other cytokines that are commonly increased in lymph upon immune activation. Coordinate TG up-regulation is observed surrounding nodules of follicular lymphoma (14 of 15 cases) and in the FRC and endothelium of classical Hodgkin's disease, two tumor types that also showed an activated paracortical phenotype. Small lymphocytic lymphoma showed minimal TG staining, with other lymphoma types showing patterns suggesting differential TG regulation. Using TG as a stromal marker, we have identified differential modulation of the phenotype of the lymph node reticular network that parallels change in the B-cell compartment.

Determination of cyclin D1 and CD20 mRNA levels by real-time quantitative RT-PCR from archival tissue sections of mantle cell lymphoma and other non-Hodgkin's lymphomas.

Cyclin D1 overexpression is a valuable marker for the diagnosis of mantle cell lymphoma (MCL). We used a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) method to quantify levels of cyclin D1, CD20, and cyclophilin A mRNA in manually microdissected, paraffin-embedded tissue sections using an ABI 7700 qRT-PCR system. The study group included 21 cases of MCL and 37 cases of other types of B-cell non-Hodgkin's lymphoma. Cyclin D1 mRNA copy number was normalized to CD20 and cyclophilin A mRNA and evaluated statistically by analysis of variance. The relative cyclin D1 levels were similar whether normalized to CD20 or cyclophilin A, indicating that CD20 levels are stable and can be used as a B-cell-specific normalizer. Statistically significant differences were found in the median levels of cyclin D1 mRNA (expressed as % CD20 mRNA) among cases of MCL (87.6), small lymphocytic lymphoma (9.9), follicular lymphoma (2.4), diffuse large B-cell lymphoma (5.9), marginal zone B-cell lymphoma (39.8), and Burkitt lymphoma (7.1) (P < 0.05). We conclude that qRT-PCR can be used to quantify cyclin D1 mRNA levels in archival tissue sections. Normalization of cyclin D1 to a B-cell-specific marker more accurately reflects overexpression by MCL than other methods that normalize using constitutively expressed mRNA species.

Real-time RT-PCR assay for quantifying cyclin D1 mRNA in B-cell non-Hodgkin's lymphomas.

Mantle cell lymphoma (MCL) is a distinct type of non-Hodgkin's lymphoma (NHL) characterized by the t(11;14)(q13;q32), in which the ccnd1 gene is juxtaposed with the immunoglobulin heavy chain gene, resulting in up-regulation of cyclin D1. Cyclin D1 overexpression is a useful finding that supports the diagnosis of MCL. In this study, we used a 5' --> 3' exonuclease-based real-time reverse-transcriptase polymerase chain reaction (RT-PCR) method to quantify cyclin D1 mRNA in 108 B-cell NHL and nonneoplastic specimens, including 25 cases of MCL. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also quantified to normalize cyclin D1 mRNA levels, and the data were expressed as a cyclin D1 to GAPDH ratio. At each anatomic site, MCL cases had higher cyclin D1 levels than other types of NHL or nonneoplastic specimens, without overlap. For example, in lymph node specimens, the median cyclin D1/GAPDH ratio was 147 (range, 94-160) in MCL, compared with 8.6 (range, 4-18) in chronic lymphocytic leukemia/small lymphocytic lymphoma; 5.8 (range, 1.8-24) in follicular lymphoma; 4.8 in one case of marginal zone lymphoma; and 20.2 (range, 5.8-44) in reactive specimens. Statistical analysis using one-way analysis of variance (ANOVA) showed that MCL cases had significantly higher cyclin D1 levels than other groups (P <.05). In peripheral blood specimens involved by MCL, cyclin D1 levels correlated with extent of involvement. We conclude that this real-time RT-PCR method to quantify cyclin D1 expression is helpful in distinguishing MCL from other types of B-cell NHL and from nonneoplastic specimens. This method is rapid, can be applied to the analysis of fluid specimens, and obviates the need for time-consuming and laborious detection methods that are required by traditional semi-quantitative RT-PCR methods.