Mechanism of CD11b down-regulation from phorbol myristate acetate stimulated polymorphonuclear neutrophils.

OBJECTIVE: To investigate the mechanism of CD11b down-regulation in phorbol myristate acetate (PMA) stimulated polymorphonuclear leukocytes (PMN). METHODS: Purified PMN were stimulated with PMA in the presence, or absence, of various enzyme inhibitors. Following stimulation, PMN CD11b expression was examined by flow cytometry and Western-blotting. The entire work was carried out at Liverpool University between the period of 1998 and 2001. RESULTS: Stimulation of PMN with PMA induced the down-regulation of CD11b by a mechanism involving a combination of an oxidant and a serine protease; most likely the primary granule derived elastase. CONCLUSION: The present study shows for the first time the cooperation of both oxidants and enzymes in the down-regulation of PMN adhesion receptors.

Serological diagnosis of celiac disease.

Celiac disease CD is an inflammatory disease of the small intestine brought about by exposure to gluten in genetically predisposed individuals. Celiac disease most often presents with non specific, or extra-intestinal, manifestations and, consequently, the disease remains under diagnosed. Untreated CD is associated with high morbidity and, therefore, early diagnosis is essential. The availability of non-invasive and relatively cheap serological tests has made it possible to screen large numbers of patients and resulted in increased, and earlier, diagnosis of patients with CD. However, these tests have varying degrees of sensitivities and specificities and the results generated can lead to a lot of confusion with regards to the diagnosis, or exclusion, of CD. In the present review, we discuss in detail these tests and suggest how they can be used in screening patients for CD with the hope that such information will help clinicians to select the right tests and interpret the generated results more effectively, and thus lead to improved identification and treatment of patients with CD.

An acquired form of Bernard Soulier syndrome associated with acute myeloid leukemia.

OBJECTIVE: To investigate glycoprotein-1b (GP-1b) expression on platelets from patients with acute myeloid leukemia (AML). METHODS: Purified platelets, obtained from AML-patients and normal control subjects, were examined for surface membrane GP1b-expression by flow cytometry and GP1b-mediated aggregation responses by aggregometry. The level of elastase in plasma from patients and controls was measured by enzymed-linked immunosorbent assay. The whole of this work was carried out at the University of Liverpool, Liverpool, United Kingdom during the period of 1994-2001. RESULTS: Platelets from the majority of AML-patients showed reduced GP1b-expression and reduced GP1b-mediated aggregation responses. Reduction in platelet GP1b-expression was associated with increased plasma elastase levels. CONCLUSION: The present study suggests that elastase, released from leukemic blasts, degrades platelet GP1b, resulting in dysfunctional circulating platelets in AML-patients. These results could explain the bleeding disorders observed in these patients.

Ineffectiveness of rat liver tissues in the screening of connective tissue disease.

OBJECTIVE: To assess the effectiveness of using rat liver tissues (RLT) for the screening of connective tissue disease (CTD). METHODS: Results of patient samples, submitted to the Clinical Immunology Laboratory, Birmingham Heartlands Hospital, Birmingham, United Kingdom for investigation of CTD between 2001 and 2002, were analyzed. Positive results for anti-double stranded DNA (dsDNA) antibodies and anti-extractable nuclear antigen (ENA) antibodies were correlated with the results of the corresponding anti-nuclear antibodies (ANA), obtained by indirect immunofluorescence (IIF) using RLT. In the second part of the study samples that were previously tested positive for anti-ENA or anti-dsDNA antibodies, were investigated prospectively for ANA using both RLT and human epithelial (Hep-2) cell line. RESULTS: The IIF method employing RLT for screening of CTD, failed to detect ANA patterns from 45% and 25% of patient samples know to contain antibodies to dsDNA and ENA. The anti-dsDNA antibodies that failed to be detected by the RLT were of low avidity and their clinical significance is unknown. In contrast, the antibodies to ENA were mostly directed against the Ro antigen and a clear marker of CTD. Hep-2 cell line enhanced the detection rate of anti-ENA antibodies, particularly those against the Ro antigen. In contrast, and like RLT, Hep-2 cell line failed to detect the low avidity anti-dsDNA antibodies. CONCLUSION: The present study has clearly shown that RLT are ineffective for screening of CTD. It is recommended that laboratories, which are still using these tissues, should consider replacing them with the Hep-2 cell line.

The role of the clinical immunology laboratory in the diagnosis and monitoring of connective tissue diseases.

Connective tissue diseases (CTD) are a group of autoimmune systemic diseases that can affect any organ-system in the body. The initial clinical presentations of these diseases overlap, not only with each other, but also with a wide range of other rheumatological and non-rheumatological disorders. Due to these reasons, clinicians depend heavily on the use of the clinical immunology laboratory for the diagnosis of CTD. A large number of tests exist in the laboratory for the investigation of CTD and each test can be performed by a number of different methods, each with its own limitations. Consequently, the significance of the results generated not only has to be interpreted in relation to the clinical picture, but also to the method used to generate the results. Moreover, within the laboratory, there is a hierarchical testing system for the investigation of CTD and if this system is used appropriately, in conjunction with the clinical picture, can result in the diagnosis/exclusion of CTD more efficiently and economically. In contrast, random use of the laboratory tests, combined with limited knowledge of the methods used to carry out these tests, can lead to delay or even misdiagnosis, as well as can lead to wastage of resources. In the following review, we have discussed the various tests that are used in the investigation of CTD, as well as the different methods used to carry out these tests, with the hope that such knowledge would lead to a more efficient and economical use of the clinical immunology laboratory in the investigation of CTD.

CD44 mediates polymorphonuclear leukocyte motility on hyaluronan.

OBJECTIVE: To investigate the behavior of polymorphonuclear (PMN) leukocytes on the extracellular matrix carbohydrate component, hyaluronan (HA), in the presence and absence of the chemokine, interleukin-8 (IL-8). METHODS: The present study was conducted at the Department of Hematology, University of Liverpool, United Kingdom, between the period 2000 to 2001. Polymorphonuclear cells were isolated from whole venous blood using Mono-Poly-Resolving Medium. Purified PMN were added alone or with IL-8 to HA-coated plates and the behavior of these cells monitored by time-lapse video microscopy over a period of 40 minutes. For the identification of surface receptor(s) mediating PMN migration on HA, PMN were incubated with blocking and non-blocking antibodies against cluster of differentiation 44 (CD44) and Receptor for Hyaluronan Mediated Motility (RHAMM) prior to addition to HA-coated surfaces. RESULTS: Approximately 55% of PMN were found to interact and migrate on HA-coated plates with a mean speed of 6.4 +/- 0.7 mm/min. Addition of IL-8 reduced both the percentage moving cells (7.5%) and the average speed of the remaining moving cells (2.0 +/- 0.3 mm/min). The inhibitory effect of IL-8 on PMN migration was associated with reorganization of the cytoplasmic fibrillar form of actin. Anti-CD44 blocking antibody substantially reduced the speed of PMN (2.5 +/-0.9 mm/min), while non-blocking anti-CD44 and anti-RHAMM antibodies had no effect. CONCLUSION: The present study demonstrates for the first time that PMN are able to interact and migrate on the widely distributed extracellular matrix component, HA, using the cell surface receptor, CD44. Such interaction is modified by the chemokine, IL-8, in a way that optimizes the host defense against invading pathogens.

Elucidation of the mechanisms of hairy-cell localization in tissues and the process of the bone marrow fibrosis in hairy-cell leukemia.

Hairy cell leukemia is a chronic B cell leukemia with a number of distinctive features including the unusual tissue distribution of the leukemic cells, hairy cells, and the bone marrow fibrosis. We have been working, for a number of years, on the potential mechanisms behind hairy-cell localization in tissues. In this review, it is summarized how our work has shed very important information regarding these mechanisms and led, eventually, to the full elucidation of the process of the bone marrow fibrosis in hairy cell leukemia.

The role of autocrine FGF-2 in the distinctive bone marrow fibrosis of hairy-cell leukemia (HCL).

Bone marrow (BM) fibrosis is a central diagnostic and pathogenetic feature of hairy-cell leukemia (HCL). It is known that fibronectin (FN) produced and assembled by the malignant hairy cells (HCs) themselves is a major component of this fibrosis. It is also known that FN production is greatly enhanced by adhesion of HCs to hyaluronan (HA) via CD44. The aim of the present study was to establish the roles of fibrogenic autocrine cytokines (fibroblast growth factor-2 [FGF-2] and transforming growth factor beta [TGFbeta]) and of different isoforms of CD44 in this FN production. We show that HC adhesion to HA stimulates FGF-2, but not TGFbeta, production and that HCs possess FGF-2 receptor. In a range of experiments, FN production was greatly reduced by blocking FGF-2 but not TGFbeta. Moreover FN, but not FGF-2, secretion was blocked by down-regulation of the v3 isoform of CD44 and by addition of heparitinase. These results show that autocrine FGF-2 secreted by HCs is the principal cytokine responsible for FN production by these cells when cultured on HA. The central role of FGF-2 in the pathogenesis of the BM fibrosis of HCL was supported by our immunohistochemical demonstration of large amounts of this cytokine in fibrotic BM but not in HCL spleen where there is no fibrosis. As regards CD44 isoforms, the present work demonstrates that CD44v3 is essential for providing the heparan sulfate necessary for HC stimulation by FGF-2, whereas the signal for production of the cytokine was provided by HA binding to CD44H, the standard hematopoietic form of the molecule.

The role of phospholipases A2 in the stimulation of neutrophil motility by cobra venoms.

Neutrophil (PMN) accumulation frequently occurs at the site of snakebite as part of the inflammatory response to envenoming. We demonstrate here that the venoms of the cobras, Naja naja and N. mossambica, and two purified venom phospholipase A(2)s (PLA(2)s) isolated from the latter venom, stimulate CD11b translocation from the PMN granule store to the plasma membrane and enhance neutrophil motility on collagen-coated surfaces. These effects were partially attenuated by the PLA(2) inhibitor, aristolochic acid, and almost completely abolished by the specific cytosolic PLA(2) inhibitor, methylarachidonylfluorophosphonate (MAFP). Annexin V and inhibitors of collagenase, cyclo-oxygenase and lipo-oxygenase, all inhibited PMN motility to a variable extent. FACS analysis and confocal microscopy showed that Annexin V interfered with binding and rapid endocytosis of the venom PLA(2). These results indicate that venom and venom PLA(2) preparations first caused a non-enzymatic stimulation of PMN leading to the activation of cytosolic PMN PLA(2) and production of arachidonate metabolites involved in stimulation of PMN degranulation and motility. The evidence suggests that venom PLA(2) then interacts with anionic phospholipids exposed on stimulated PMN, becomes endocytosed, and then contributes itself to the production of chemoattractants responsible for PMN accumulation at the site of the snakebite.