Trypanosoma cruzi infection induces up-regulation of cardiac muscarinic acetylcholine receptors in vivo and in vitro.

The pathogenesis of chagasic cardiomyopathy is not completely understood, but it has been correlated with parasympathetic denervation (neurogenic theory) and inflammatory activity (immunogenic theory) that could affect heart muscarinic acetylcholine receptor (mAChR) expression. In order to further understand whether neurogenic and/or immunogenic alterations are related to changes in mAChR expression, we studied two models of Trypanosoma cruzi infection: 1) in 3-week-old male Sprague Dawley rats chronically infected with T. cruzi and 2) isolated primary cardiomyocytes co-cultured with T. cruzi and peripheral blood mononuclear cells (PBMC). Using [3H]-quinuclidinylbenzilate ([3H]-QNB) binding assays, we evaluated mAChR expression in homogenates from selected cardiac regions, PBMC, and cultured cardiomyocytes. We also determined in vitro protein expression and pro-inflammatory cytokine expression in serum and cell culture medium by ELISA. Our results showed that: 1) mAChR were significantly (P < 0.05) up-regulated in right ventricular myocardium (means +/- SEM; control: 58.69 +/- 5.54, N = 29; Chagas: 72.29 +/- 5.79 fmol/mg, N = 34) and PBMC (control: 12.88 +/- 2.45, N = 18; Chagas: 20.22 +/- 1.82 fmol/mg, N = 19), as well as in cardiomyocyte transmembranes cultured with either PBMC/T. cruzi co-cultures (control: 24.33 +/- 3.83; Chagas: 43.62 +/- 5.08 fmol/mg, N = 7 for both) or their conditioned medium (control: 37.84 +/- 3.84, N = 4; Chagas: 54.38 +/- 6.28 fmol/mg, N = 20); 2) [(3)H]-leucine uptake was increased in cardiomyocytes co-cultured with PBMC/T. cruzi-conditioned medium (Chagas: 21,030 +/- 2321; control 10,940 +/- 2385 dpm, N = 7 for both; P < 0.05); 3) plasma IL-6 was increased in chagasic rats, IL-1beta, was increased in both plasma of chagasic rats and in the culture medium, and TNF-alpha level was decreased in the culture medium. In conclusion, our results suggest that cytokines are involved in the up-regulation of mAChR in chronic Chagas disease.

Trypanosoma cruzi infection induces up-regulation of cardiac muscarinic acetylcholine receptors in vivo and in vitro

The pathogenesis of chagasic cardiomyopathy is not completely understood, but it has been correlated with parasympathetic denervation (neurogenic theory) and inflammatory activity (immunogenic theory) that could affect heart muscarinic acetylcholine receptor (mAChR) expression. In order to further understand whether neurogenic and/or immunogenic alterations are related to changes in mAChR expression, we studied two models of Trypanosoma cruzi infection: 1) in 3-week-old male Sprague Dawley rats chronically infected with T. cruzi and 2) isolated primary cardiomyocytes co-cultured with T. cruzi and peripheral blood mononuclear cells (PBMC). Using [³H]-quinuclidinylbenzilate ([³H]-QNB) binding assays, we evaluated mAChR expression in homogenates from selected cardiac regions, PBMC, and cultured cardiomyocytes. We also determined in vitro protein expression and pro-inflammatory cytokine expression in serum and cell culture medium by ELISA. Our results showed that: 1) mAChR were significantly (P < 0.05) up-regulated in right ventricular myocardium (means ± SEM; control: 58.69 ± 5.54, N = 29; Chagas: 72.29 ± 5.79 fmol/mg, N = 34) and PBMC (control: 12.88 ± 2.45, N = 18; Chagas: 20.22 ± 1.82 fmol/mg, N = 19), as well as in cardiomyocyte transmembranes cultured with either PBMC/T. cruzi co-cultures (control: 24.33 ± 3.83; Chagas: 43.62 ± 5.08 fmol/mg, N = 7 for both) or their conditioned medium (control: 37.84 ± 3.84, N = 4; Chagas: 54.38 ± 6.28 fmol/mg, N = 20); 2) [³H]-leucine uptake was increased in cardiomyocytes co-cultured with PBMC/T. cruzi-conditioned medium (Chagas: 21,030 ± 2321; control 10,940 ± 2385 dpm, N = 7 for both; P < 0.05); 3) plasma IL-6 was increased in chagasic rats, IL-1â, was increased in both plasma of chagasic rats and in the culture medium, and TNF-á level was decreased in the culture medium. In conclusion, our results suggest that cytokines are involved in the up-regulation of mAChR in chronic Chagas disease.

Tissue inhibitor of metalloproteinase-1 alters the tumorigenicity of Burkitt's lymphoma via divergent effects on tumor growth and angiogenesis.

Epstein-Barr virus (EBV)-positive Burkitt's lymphoma cells and EBV-infected B cells elicit humoral factors that inhibit tumor-induced angiogenesis, resulting in tumor necrosis and regression. Of the chemokine factors identified in association with this growth behavior, none have induced complete tumor regression. We have previously identified tissue inhibitors of metalloproteinase (TIMP)-1 in various B cell lymphoma cell lines. Here we show that induction of TIMP-1 expression in an EBV-negative Burkitt's lymphoma cell line results in a biphasic, in vivo tumor growth pattern in the nude mouse that is essentially identical to EBV-positive Burkitt's lymphoma cell lines. The initial effect of TIMP-1 is to enhance tumor growth, consistent with the reported anti-apoptotic effect of TIMP-1 on B cell growth. Tumor necrosis and regression then follow the initial period of rapid, increased tumor growth. Only microscopic foci of residual, proliferating tumor cells are observed on biopsy of the tumor site. This latter effect is mediated by TIMP-1 inhibition of an angiogenic response within the developing tumor mass, as demonstrated by immunostaining and microvessel counts. These findings suggest that TIMP-1 is an important mediator of the in vivo growth properties of EBV-positive Burkitt's lymphoma.

Tissue inhibitor of metalloproteinases 1 regulation of interleukin-10 in B-cell differentiation and lymphomagenesis.

Tissue inhibitors of metalloproteinases (TIMPs), first described as specific inhibitors of matrix metalloproteinases, have recently been shown to exert growth factor activities. It was previously demonstrated that TIMP-1 inhibits apoptosis in germinal center B cells and induces further differentiation. Interleukin-10 (IL-10) is reported as a vital factor for the differentiation and survival of germinal center B cells and is also a negative prognostic factor in non-Hodgkin lymphoma (NHL). However, the mechanism of IL-10 activity in B cells and the regulation of its expression are not well understood. IL-10 has been shown to up-regulate TIMP-1 in tissue macrophages, monocytes, and prostate cancer cell lines, but IL-10 modulation of TIMP-1 in B cells and the effect of TIMP-1 on IL-10 expression has not been previously studied. It was found that TIMP-1 expression regulates IL-10 levels in B cells and that TIMP-1 mediates specific B-cell differentiation steps. TIMP-1 inhibition of apoptosis is not IL-10 dependent. TIMP-1 expression in B-cell NHL correlates closely with IL-10 expression and with high histologic grade. Thus, TIMP-1 regulates IL-10 expression in B-cell NHL and, through the inhibition of apoptosis, appears responsible for the negative prognosis associated with IL-10 expression in these tumors.

In vitro suppression of programmed cell death of B cells by tissue inhibitor of metalloproteinases-1.

Cellular pathways for induction of programmed cell death (PCD) have been identified, but little is known about specific extracellular matrix processes that may affect apoptosis along those pathways. In this study, a series of Burkitt's lymphoma (BL) cell lines were assayed for their expression of tissue inhibitor of metalloproteinases (TIMP)-1. Results indicate that TIMP-1-positive BL lines show resistance to cold-shock-induced apoptosis. Furthermore, recombinant TIMP-1, but not TIMP-2 or a synthetic metalloproteinase inhibitor (BB-94), confers resistance to apoptosis induced by both CD95-dependent and -independent (cold shock, serum deprivation, and gamma-radiation) pathways in TIMP-1-negative BL lines. TIMP-1 suppression of PCD is not due to metalloproteinase inhibition, as reduction and alkylation of the TIMP-1 did not abolish this activity. Retroviral induction of TIMP-1 not only resulted in cell survival but also in continued DNA synthesis for up to 5 d in the absence of serum, while controls underwent apoptosis. This resistance to apoptosis is reversed by anti-TIMP-1 antibodies, demonstrating that secreted TIMP-1 is active in blocking apoptosis. Furthermore, TIMP-1 upregulation induced expression of Bcl-XL but not Bcl-2 as well as decreased NF-kappaB activity as compared with controls. These results demonstrate that TIMP-1 suppresses apoptosis in B cells and suggests a novel activity for TIMP-1 in tissue homeostasis.

Tissue inhibitor of metalloproteinase (TIMP)-1 induces differentiation and an antiapoptotic phenotype in germinal center B cells.

Tissue inhibitors of metalloproteinases (TIMPs) have been shown to be multifunctional factors. Contrasting with their enzyme-inhibitory activity, TIMPs also promote cell growth. Previously, we have reported an enhanced expression of TIMP-1 by normal reactive B cells and high-grade lymphomas. In the present study, a series of Burkitt's lymphoma (BL) cell lines were analyzed for their expression of TIMP-1. TIMP-1 expression correlates with upregulation of activation and survival markers. TIMP-1-negative cells express the phenotype associated with group I BL lines and Epstein-Barr virus (EBV)-negative, nonendemic BLs (CD10+, CD38+, sIg+, and CD77+). However, TIMP-1+ BL lines showed group II/III BL phenotype, downregulation of the above markers, and upregulation and secretion of the activation marker CD23. Also, TIMP-1+ cells have high levels of CD40 expression. To determine whether TIMP-1 is directly involved in the BL phenotype, an EBV-negative BL line JD38 was infected with timp-1-expressing retrovirus and analyzed. In the absence of EBV, upregulation of TIMP-1 is sufficient to induce the same phenotype seen in TIMP-1+, EBV+ BL lines (CD10-, CD38-, sIg-, CD77-, CD23+, CD40 bright). This study not only suggests a role for TIMP-1 in BLs, but also supports its value as a prognostic factor. This is a US government work. There are no restrictions on its use.

Bleomycin-induced differentiation of bcl-2-transfected U937 leukemia cells.

In this study, the effect of bleomycin on myeloid leukemic U937 cells transfected with murine bcl-2 or vector alone (vector containing neomycin-resistant gene only; MNC) was investigated. Sublethal concentrations of bleomycin (1 microgram/ml) induced a decrease in cell growth in both vector-only and bcl-2-transfected U937 cells. In MNC-transfected U937 cells, loss of viable cells and colony-forming cells was observed following 4 days of bleomycin treatment. This was accompanied by accumulation of cells in the G0-G1 phase of the cell cycle and morphological changes as well as induced expression of markers associated with myeloid differentiation (i.e., increased granularity and CD11b expression). In contrast, bcl-2-transfected U937 cells maintained viable cell numbers and colony forming cells for up to 2 weeks in the presence of bleomycin. These cells did not show cell cycle accumulation in G0-G1 and in addition, displayed delayed expression of differentiation markers when compared with bleomycin-treated, vector-only transfected U937 cells. One day following a 5-day exposure to 1 microgram/ml bleomycin, a loss of differentiated cells by apoptosis, as demonstrated by dUTP and analyzed by flow cytometry, was observed in the MNC-transfected U937 cell population. In contrast, differentiated bcl-2-transfected U937 cells remained viable for 2 weeks following bleomycin treatment. The results of this study suggest that up-regulated Bcl-2 not only blocks apoptosis in proliferating myeloid cells but also delays or prevents apoptosis in differentiated myeloid cells.

Quantitation of resistance to cytosine arabinoside by myeloid leukemic cells expressing bcl-2.

The presence of bcl-2 in myeloid leukemias has been associated with a decrease in therapy-induced apoptosis, reduced patient survival and in vitro autonomous growth of leukemic cells. The present study focuses on the quantitation of resistance to increasing doses of 1-beta-d-arabinofuranosylcytosine (Ara-C) by using hematological tumors expressing different levels of bcl-2. Scanning densitometry of Western blots demonstrated that the myeloid U-937 cells express low levels of bcl-2 (RD = 0.008), whereas the follicular lymphoma RL-7 expressed very high levels (RD = 3.084). Colony formation was also examined following incubation with Ara-C and RL-7 cells demonstrated a higher clonogenic survival (LD50 = 0.5 microns) when compared with U-937 cells (LD50 = 0.005 microM). Similarly, the level of bcl-2 expression in each cell line was also related to apoptosis with U-937 cells demonstrating increased DNA fragmentation when compared with RL-7 cells. To further evaluate the effect of upregulated bcl-2 on Ara-C treatment, U-937 cells were transfected with a retroviral vector carrying the murine bcl-2 or vector alone. Upregulation of bcl-2 by myeloid leukemic cells increased the resistance by 3 logs to Ara-C when comparing LD50 values from clonogenic assays, and decreased apoptosis by at least 3 logs when measuring dUTP positive cells by flow cytometry.

The role of metalloproteinases and their inhibitors in hematological disorders.

The balance between the activities of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) is an important control point during the turnover of the extracellular matrix. Previous studies have demonstrated that an uncontrolled rate of production of these proteins leads to various pathological conditions. Overexpression of MMPs by malignant cells can increase the invasive behavior of solid tumors and their metastatic potential. In an increasing number of reports, it has become evident that expression of MMPs and TIMPs is not restricted to solid tumors. Normal and malignant hematological cells also express these proteins and play a pivotal role in the hematological-cell physiology. In addition to modifying the extracellular matrix, MMPs and TIMPs exert other functions in hematological cells, including growth-factor activity, removal of cell-surface receptors, and autoimmunity. The goal of this review is to gather recent studies concerning the properties of MMPs and TIMPs in hematological cells, their regulation of gene expression, their cellular distribution, and their potential role in the pathogenesis of hematological disorders such as inflammation and neoplasia.

IL-7 induces human lymphokine-activated killer cell activity and is regulated by IL-4.

Induction of lymphokine-activated killer (LAK) activity by IL-2 has been described and characterized as broadly cytolytic activity against both fresh and cultured tumors. rIL-7 in the absence of IL-2 also induces LAK activity in human cells. This activity is unique for IL-7, because it is not shared by other cytokines including IL-1, IL-4, IL-6, and TNF-alpha. IL-7 also induces either de novo or increased expression of the surface markers CD25 (Tac, IL-2R alpha-chain), CD54 (ICAM-1), Mic beta 1 (IL-2R beta-chain) and CD69 (early T cell activation Ag). IL-7-induced LAK activity is independent of IL-2 secretion, because it is not abrogated by IL-2 antisera. The LAK precursor responding to IL-7 stimulation is enriched in the null cell fraction as has been demonstrated for IL-2-induced LAK cells. TGF-beta and IL-4 interfere with generation of LAK activity by IL-7. Anti-IL-4 antiserum enhances IL-7-induced LAK activity and augments induction of surface marker expression by IL-7. This may be indirect evidence that IL-7 stimulation leads to induction of IL-4 activity. Our results describe the activation of mature lymphoid cells by IL-7. This and the previously described role of IL-7 in lymphohemopoiesis makes it a cytokine of potential therapeutic value for treatment of immunodeficiency states and possibly the immunotherapy of cancer.