Coinduction of granulocyte-macrophage colony-stimulating factor release and lymphokine-activated killer cell susceptibility in monocytes by interleukin-2 via interleukin-2 receptor beta

Human monocytes express interleukin-2 receptor beta (IL-2R beta) constitutively; however, the function of these receptors has not been fully delineated. We discovered that IL-2R beta directs two biologic activities in human monocytes, the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and increased susceptibility to lysis by lymphokine-activated killer cells (LAK) cells. Human monocytes were purified from peripheral blood mononuclear cells by plastic adherence and anti-CD2 plus complement lysis. By a 5-hour 51Cr-release assay, monocytes cultured in IL-2 were found to gain increasing susceptibility to LAK cells with time and this effect was dose dependent. Maximal susceptibility was obtained with a 4-day culture in 1,000 U/mL of IL-2. Monocytes were also found to release GM-CSF in response to IL-2 using a CSF-dependent cell line, Mo7e. Because IL-2- induced GM-CSF release coincides with LAK lysis of IL-2-cultured monocytes, we treated monocytes with anti-GM-CSF and anti-IL-2R beta to determine whether GM-CSF release and LAK susceptibility were dependent or independent events. We found that both phenomena were inhibited by either antibody. Therefore, we conclude that IL-2-induced release of GM- CSF is mediated by IL-2R beta, which then acts to modulate the susceptibility of monocytes to lysis by LAK cells.     

Pancreatitis-associated ascitic fluid induces hepatocyte death independent of local cytokines

INTRODUCTION: Kupffer-cell-derived cytokines mediate liver injury, yet macrophage pacification does not abolish hepatocyte injury. We undertook this study to examine the role of pancreatitis-associated ascitic fluid (PAAF) in liver injury. METHODS: Pathogen-free PAAF was perfused into healthy rat livers in situ for 60 min (n = 5, sham = 5, LPS = 5). AST, ALT, LDH, and TNF were measured in the effluent. Primary cultures of rat Kupffer cells or hepatocytes were treated with PAAF; AST, ALT, LDH, and TNF were measured and cell proliferation was determined by MTT assay. A hepatocyte human cell line (CCL-13) was treated with PAAF and apoptosis was measured by flow cytometry. RESULTS: Liver perfusion with PAAF induced a >15-fold increase in AST/ALT/LDH (P < 0.001 PAAF vs sham), but not in TNF. In vitro, Kupffer cell viability was sharply reduced by PAAF in a dose-dependent manner; however, 5% PAAF (50% viability) did not induce TNF production from Kupffer cells. PAAF induced a multifold increase in AST/ALT/LDH from fresh hepatocytes (P < 0.001 vs control), which was not attenuated by a protease inhibitor. The CCL-13 cell population was reduced to 15 +/- 2% of baseline by PAAF (P < 0.001 vs control), whereas elastase, trypsin, or TNF had no effect. PAAF increased the percentage of nonviable CCL-13 cells (78 +/- 4% vs 28 +/- 1%, P < 0.001 vs control). Neither protease inhibitor nor heat inactivation of PAAF altered this pattern of hepatocyte death. CONCLUSION: PAAF induces direct hepatocyte injury and death by heat-stable factors other than pancreatic enzymes but not via local production of Kupffer-cell-derived cytokines.     

Kupffer cell-derived fas ligand plays a role in liver injury and hepatocyte death

Liver injury is an important prognostic indicator during acute pancreatitis. The aim of this study was to determine the role of Fas ligand (FasL) in hepatocyte injury. Liver parenchymal enzymes were measured in cocultures of hepatocytes and Kupffer cells treated with elastase. FasL and FasL mRNA were measured in elastase-treated Kupffer cells. Hepatocytes were treated with FasL and their viability was assessed by monotetrazolium (MTT), apoptosis by flow cytometry, as well as caspase-3 and p38-mitogen-activated protein kinase (MAPK) by immunoblotting. Elastase increased aspartate aminotransferase and lactate dehydrogenase in cocultures of hepatocyte and Kupffer cells (P < 0.040). Elastase increased FasL production from Kupffer cells (P = 0.02) and upregulated FasL mRNA (FasL/beta-2 microglobulin (BMG): 0.23 ± 0.03 vs. 0.11 ± 0.003; P = 0.04). FasL increased alanine aminotransferase and lactate dehydrogenase (P < 0.03) and reduced hepatocyte viability by 45% (P = 0.01). FasL increased the number of dually labeled cells with AnnexinV/7AAD (P = 0.03) while upregulating cleavage of caspase-3 and the phosphorylation of p38-MAPK. FasL antibody attenuated the FasL-related increase in dually labeled cells (P = 0.02), the cleavage of caspase-3, and phosphorylation of p38-MAPK. Pancreatic elastase upregulates FasL within Kupffer cells. FasL induces hepatocyte injury and death and upregulates p38-MAPK and caspase-3 within hepatocytes. The ability to manipulate interactions between Kupffer cells and hepatocytes may have important therapeutic implications. Presented at the Forty-Fourth Annual Meeting of The Society for Surgery of the Alimentary Tract, Orlando, Florida, May 18–21, 2003 (poster presentation) and published as an abstract in Gastroenterology 2003. Supported by SSAT Career Development Award (M.M.), Dr. Bob Haines Pancreatitis Research Fund (M.M.), VA Merit Award (J.N.) and a VA Merit Award (P.K.E.-B.).     

The prognostic value of circulating myeloblasts in patients with myelodysplastic syndromes

The prognostic value of peripheral blasts (PB) is not well-studied in patients with myelodysplastic syndromes (MDS). We evaluated the impact of PB on overall survival (OS) and transformation to acute myeloid leukemia (AML) in a large cohort. The MDS database at the Moffitt Cancer Center was retrospectively reviewed to identify patients with ≥?1% PB (PB-MDS) and those without PB (BM-MDS). We also assessed the correlation between PB and gene mutations. One thousand seven hundred fifty-eight patients were identified, among whom 13% had PB near the time of diagnosis. PB-MDS patients were more likely to be younger with trilineage cytopenia, complex karyotype, higher-risk disease, transfusion dependence, and therapy-related MDS. The rate of AML transformation was 49 vs. 26% (p?<?0.005) and median OS was 16.5 vs. 45.8 months (p?<?0.005) in the PB-MDS and BM-MDS groups, respectively. In Cox regression analysis, the presence of PB was an independent prognostic covariate for OS, HR 1.57 (95% CI 1.2–2). Among 51 patients with an available gene panel, the rate of ≥?1 gene mutation in the PB-MDS group (n?=?4) was 100% compared to 81% in the BM-MDS group (n?=?47). The presence of PB in MDS is an adverse independent prognostic variable that refines prognostic discrimination.     

Chronic neutropenia mediated by fas ligand

Chronic neutropenia, often associated with rheumatoid arthritis, is a characteristic finding in large granular lymphocyte (LGL) leukemia. The mechanism of neutropenia is not known. Normal neutrophil survival is regulated by the Fas-Fas ligand apoptotic system. We hypothesized that neutropenia in LGL leukemia is mediated by dysregulated expression of Fas ligand. Levels of Fas ligand in serum samples from patients with LGL leukemia were measured with a Fas ligand enzyme-linked immunosorbent assay. The effects of serum from patients with LGL leukemia on apoptosis of normal neutrophils were determined by flow cytometry and morphologic assessment. High levels of circulating Fas ligand were detected in 39 of 44 serum samples from patients with LGL leukemia. In contrast, Fas ligand was undetectable in 10 samples from healthy donors. Serum from the patients triggered apoptosis of normal neutrophils that depended partly on the Fas pathway. Resolution of neutropenia was associated with disappearance or marked reduction in Fas ligand levels in 10 of 11 treated patients. These data suggest that high levels of Fas ligand are a pathogenetic mechanism in human disease. (Blood. 2000;95:3219-3222)     

Induction of myelodysplasia by myeloid-derived suppressor cells

Myelodysplastic syndromes (MDS) are age-dependent stem cell malignancies that share biological features of activated adaptive immune response and ineffective hematopoiesis. Here we report that myeloid-derived suppressor cells (MDSC), which are classically linked to immunosuppression, inflammation, and cancer, were markedly expanded in the bone marrow of MDS patients and played a pathogenetic role in the development of ineffective hematopoiesis. These clonally distinct MDSC overproduce hematopoietic suppressive cytokines and function as potent apoptotic effectors targeting autologous hematopoietic progenitors. Using multiple transfected cell models, we found that MDSC expansion is driven by the interaction of the proinflammatory molecule S100A9 with CD33. These 2 proteins formed a functional ligand/receptor pair that recruited components to CD33’s immunoreceptor tyrosine-based inhibition motif (ITIM), inducing secretion of the suppressive cytokines IL-10 and TGF-β by immature myeloid cells. S100A9 transgenic mice displayed bone marrow accumulation of MDSC accompanied by development of progressive multilineage cytopenias and cytological dysplasia. Importantly, early forced maturation of MDSC by either all-trans-retinoic acid treatment or active immunoreceptor tyrosine-based activation motif–bearing (ITAM-bearing) adapter protein (DAP12) interruption of CD33 signaling rescued the hematologic phenotype. These findings indicate that primary bone marrow expansion of MDSC driven by the S100A9/CD33 pathway perturbs hematopoiesis and contributes to the development of MDS.     

Cross-sectional associations between cutaneous viral infections and regulatory T lymphocytes in circulation

Approximately three million non-melanoma skin cancers (NMSCs) are diagnosed worldwide each year, although this number is likely an underestimate given that these cancers are not always recorded in cancer registries. Studies have suggested that skin (cutaneous) infections with human papillomaviruses (HPV) and polyomaviruses (HPyV) may play a role in the development of some NMSC types. Suppression of the immune system is also a risk factor for NMSC. This study, from the U.S.A, aimed to understand the relationship between T-regulatory (Treg) cells, cells which suppress immune response, and cutaneous viral infections. Blood, skin swabs and eyebrow hairs were collected from 352 patients who underwent skin cancer screening and did not have cancer detected. The researchers examined whether the skin swabs (SSW) and eyebrow hairs (EBH) contained genetic material (DNA) corresponding to 98 cutaneous HPV types (including beta HPV and gamma HPV) and 5 HPyV types. The blood samples were analyzed to determine proportions of different types of Treg cell populations in circulation (in the blood). The researchers found no association between total percent of circulating Treg cells and beta HPV or HPyV infection. However, two types of Treg cells were found at lower levels in those that had gamma HPV infection in their EBH and/or SSW. Those two types were CLA⁺ Treg cells (known to travel to the skin) and effector CD27^-CD45RA^-FOXP3⁺CD4⁺ Treg cells (known to become active when exposed to a foreign viral infection). The study results suggest that gamma HPV infection may stimulate Treg cells to move from circulation into the skin tissues.     

Acquired amegakaryocytic thrombocytopenia and pure red cell aplasia associated with an occult large granular lymphocyte leukemia

Acquired amegakaryocytic thrombocytopenia and pure red cell aplasia rarely occur concurrently. We report a case in which these disorders were associated with an occult large granular lymphocyte leukemia. The peripheral blood cytopenias improved after glucocorticoids and intravenous immunoglobulin were administered, and response was maintained with cyclosporine. Large granular lymphocyte leukemia should be suspected in the setting of unexplained bone marrow failure.     

ERK couples chronic survival of NK cells to constitutively activated Ras in lymphoproliferative disease of granular lymphocytes (LDGL)

Chronic NK lymphoproliferative disease of large granular lymphocytes (LDGL) is characterized by the expansion of activated CD3-, CD16+ or CD56+ lymphocytes. The mechanism of survival of NK cells from LDGL patients is unknown but may be related to antigenic stimulation. There is currently no standard effective therapy for LDGL, and the disease is characteristically resistant to standard forms of chemotherapy. We found evidence of constitutive activation of extracellular-regulated kinase (ERK) in NK cells from 13/13 patients with NK-LDGL (one patient with aggressive and 12 patients with chronic disease). Ablation of ERK activity by inhibitors or a dominant-negative form of MEK, the upstream activator of ERK, reduced the survival of patient NK cells. Ras was also constitutively active in patient NK cells, and exposure of cells to the Ras inhibitor FTI2153 or to dominant-negative-Ras resulted not only in ERK inhibition but also in enhanced apoptosis in both the presence and absence of anti-Fas. Therefore, we conclude that a constitutively active Ras/MEK/ERK pathway contributes to the accumulation of NK cells in patients with NK-LDGL. These findings suggest that strategies to inhibit this signaling pathway may be useful for the treatment of the NK type of LDGL.