Postthymectomy wasting associated with autoimmune phenomena. I. Antiglobulin-positive anemia in A and C57BL-6 Ks mice.

1. Mice of A and C(57)BL/6 Ks strains, thymectomized at birth acquire wasting disease in 84.1% (A) and 77.1% (C(57)BL/6 Ks) of the cases. There is no sex predelection. 2. Anemia in these animals is characterized by shortened red cell survival and increased fragility to hypotonic salt solutions. Among thymectomized A mice reticulocytosis is absent and extramedullary hematopoiesis is found in the spleen in the presence of bone marrow hypoplasia for the erythroid and lymphocyte series. 3. Positive antiglobulin tests of the red cells were observed in all the thymectomized C(57)BL/6 Ks (7/7) and 71.2% of the A strains (62/87). Normal mice do not show positive Coombs' tests. 4. The globulin coat on the A strain consists of IgM, whereas beta(1C) and IgG are not detectable. By contrast, red cell coats of NZB mice developing spontaneous autoimmune hemolytic anemia show IgM and beta(1C), but these erythrocytes do not react with anti-gamma chain antibodies. Another difference in the globulin coats of the two types of erythrocytes is that the IgM on NZB red cells has available light chain determinants but these are apparently hidden or absent in the case of sensitized erythrocytes. The difference in antibody coating, association with a component of complement in one but not the other, suggests a different mechanism for the immune surface phenomenon in each instance. 5. Anemia in NZB mice is associated with reticulocytosis while that in thymectomized A mice is not. 6. Thymectomy appears to initiate a chain of events leading to a series of autoimmune phenomena which may be due to alteration in host response consequent to loss of thymic tissue and thymic dependent functions or alternatively to infection to which increased susceptibility exists as a result of thymic extirpation.

Human megakaryocytes. I. Characterization of the membrane and cytoplasmic components of isolated marrow megakaryocytes.

Human marrow megakaryocytes have been isolated with high purity and yield by processing marrow cells sequentially through density centrifugation and velocity sedimentation. Analysis of the isolated cells for various platelet-associated components by immunofluorescence demonstrated that fibrinogen, plasma factor VIII antigen (factor VIII:AGN) platelet myosin, platelet glycoproteins I and III are present on the membrane and in the cytoplasm of over 90% of marrow megakaryocytes. Parallel studies of human and mouse megakaryocytes and platelets for IgG receptor (FcR), complement receptor type one (CR1) (C3b receptor), complement receptor type two (CR2) (C3d receptor), and Ia antigen by fluorescence and (or) rosette formation methods were performed. FcR were present on most human megakaryocytes and platelets. The Ia antigen was detected on a proportion (10-15%) of human megakaryocytes but it was undetectable on human platelets. CR1 was found on 20-40% of mouse megakaryocytes and also on a proportion of mouse platelets. These differentiation markers may be of use in monitoring megakaryocyte maturation.

Expression of transforming growth factor-beta 1 in specific cells and tissues of adult and neonatal mice.

We have used immunohistochemical techniques to detect transforming growth factor-beta 1 (TGF-beta 1) in many tissues of adult and neonatal mice. Each of two antibodies raised to the amino-terminal 30 amino acids of TGF-beta 1 selectively stained this molecule in either intracellular or extracellular locations. Strong intracellular staining was found in adrenal cortex, megakaryocytes and other cells of the bone marrow, cardiac myocytes, chondrocytes, renal distal tubules, ovarian glandular cells, and chorionic cells of the placenta. Marked staining of extracellular matrix was found in cartilage, heart, pancreas, placenta, skin, and uterus. Staining was often particularly intense in specialized cells of a given tissue, suggesting unique roles for TGF-beta within that tissue. Levels of expression of mRNA for TGF-beta 1 and its histochemical staining did not necessarily correlate in a given tissue, as in the spleen. The present data lend further support to the concept that TGF-beta has an important role in controlling interactions between epithelia and surrounding mesenchyme.

Regulation of megakaryocyte phenotype in human erythroleukemia cells.

Induction of human erythroleukemia (HEL) cells with nanomolar tumor-promoting phorbol myristate acetate (PMA) diesters results in the synchronous acquisition of multiple markers of the megakaryocyte phenotype. Induced cells markedly increase their content of cytoplasm and show features of morphological maturation. At the ultrastructural level, PMA-treated cells show increases in cytoplasm, nuclear lobulation and nucleolar content, and free ribosomes. Limited numbers of cells also express alpha-granules and nascent demarcation membrane systems. Functionally, PMA-stimulated HEL cells express increased amounts of the megakaryocyte/platelet proteins: glycoprotein IIb/IIIa, platelet factor 4, von Willebrand factor, glycoprotein Ib, and thrombospondin. No changes are observed in antigenic markers of the erythroid (glycophorin A) or macrophage lineages (MO-1 or MO-2). The increases in antigenic expression are rapid, reaching maximum levels within 3-4 d under serum-free conditions. Treatment with PMA also abruptly (within 1-2 d) inhibits cellular division in these cells. Washout studies indicate that phorbols exert their effect within 18-24 h, the approximate cell cycle time for these cells. Consistent with proliferative arrest, c-myc proto-oncogene transcripts begin to decline within 8 h of PMA treatment, although transcripts of c-myb are unaffected. Importantly, megakaryocyte differentiation is associated with endomitotic DNA synthesis (i.e., continued DNA synthesis in the absence of mitosis and cytokinesis), with HEL cells reaching a DNA content of 3-12 times that of unstimulated cells. Endomitosis is coordinately regulated with changes in antigenic expression and cell size such that those cells having the highest DNA content are the largest and also express the greatest levels of antigen.

Elevated level of p60c-src in virus-transformed murine megakaryocytic cell lines.

Megakaryocytic cell lines derived from mouse bone marrow cells transformed by the Myeloproliferative Leukemia Virus (MPLV) contain elevated levels of p60c-src. Northern blot analysis revealed the presence of a 4 kb normal sized c-src transcript only in MPLV-transformed megakaryocytic cell lines containing a high percentage of acetylcholinesterase positive cells (AChE+ greater than 10%), but not in MPLV-transformed erythroblastic or myeloblastic cell lines. The p60c-src protein was identified in lysates from in vivo labelled cells and in in vitro labelled membrane extracts by immunoblotting analysis and by immunoprecipitations with specific anti-src antibodies. In dimethylsulfoxide (DMSO) treated cells, the number of AChE+ cells increased together with p60c-src kinase activity indicating a possible correlation between p60c-src expression/activity and megakaryocytic differentiation.

Lipid composition of guinea pig platelets and megakaryocytes. The megakaryocyte as a probable source of platelet lipids.

Platelets are formed by fragmentation of the cytoplasm and plasma membrane of the megakaryocyte in the bone marrow. This study has compared the lipid composition of guinea pig platelets and megakaryocytes. Phospholipids were quantitated by TLC and measurement of lipid phosphorus. Cholesterol and fatty acids were quantitated by GLC. The cholesterol/phospholipid molar distribution in megakaryocytes was: 9.8% phosphatidylserine, 6.7% phosphatidylinositol, 14.2% sphingomyelin, 40.0% phosphatidylcholine and 29.3% phosphatidylethanolamine. Platelets continued 11.2% phosphatidylserine, 5.1% phosphatidylinositol, 16.1% sphingomyelin, 38.6% phosphatidylcholine and 29.0% phosphatidylethanolamine. The major megakaryocyte fatty acids were 20.0% palmitic, 16.4% stearic, 20.6% oleic, 13.2% linoleic and 8.2% arachidonic. The major platelet fatty acids were 17.4% palmitic, 17.5% stearic, 11.6% oleic, 12.4% linoleic and 14.6% arachidonic. The major and minor fatty acid compositions of the individual platelet phospholipids reflected those of the megakaryocyte counterparts. The increased arachidonic acid and decreased oleic acid in platelets relative to megakaryocytes were found in all four glycerophospholipids. The similarity of the phospholipid and fatty acid composition of megakaryocytes and platelets suggests that the lipid composition of the platelet is determined by the megakaryocyte.

A quantitative and dynamic study of endothelial cells and megakaryocytes in human long-term bone marrow cultures.

The quantitative evolution of endothelial cells (ECs) in Dexter-type human long-term bone marrow cultures (HLTBMCs) was investigated. Using monoclonal antibodies directed against von Willebrand factor (vWF) and against membrane antigens (EN-4 and PAL-E), a low percentage--usually less than 1% of stromal cells--of ECs was detected in all confluent cultures established from 11 different bone marrow samples. Generally these cells are not associated directly with the areas of myelopoiesis ("cobblestone areas"). ECs cannot be demonstrated in the adherent layer of most young, non-confluent, and of some old, HLTBMCs. In some instances, morphological features suggestive of dynamic behavior were seen (sprouting, canal formation). In addition, a very low proportion of vWF-positive megakaryocytic cells was found in 4 of 11 cultures, always in direct contact with the stromal fibroblastic cells.

Acquired hypomegakaryocytic thrombocytopenic purpura. Occurrence in a patient with absent thrombopoietic stimulating factor.

A 49-year-old woman had purpura and thrombocytopenia not associated with drugs or identifiable underlying disease. The platelet survival was normal and the marrow showed a sharp reduction in megakaryocytes with preservation of other cell lines. There was no response to steroids or infusion of fresh frozen plasma. Lithium carbonate therapy similarly had no effect. Thrombopoietic activity was absent in serum and urine samples. Erythropoietin activity was normal. In vitro formation of granulocyte-macrophage colonies in soft agar was normal. The case represents a unique incidence of selective megakaryocytic hypoplasia, though to result from a failure in stem cell differentiation.

Human megakaryocytes. VII. Analysis of megakaryocytes for nuclear DNA content distribution in whole marrow cell suspensions by flow cytometry.

These studies were designed to quantitate and determine the DNA content distribution of human marrow megakaryocytes using whole bone marrow. Cellular DNA content within megakaryocytic cells was established by measuring propidium iodide staining in marrow cells expressing platelet glycoprotein IIb/IIIa (Gp IIb/IIIa). These studies were based on the development of a method for rapid analysis of whole marrow cell preparations by a dual fluorescent system. DNA values ranging from 2 to 64 C were observed in all samples tested, with 16 C corresponding to the modal ploidy class representing almost one-half of the cells containing Gp IIb/IIIa. The second most frequent ploidy class corresponded to 32 C, followed by 8 C, with 20% and 15%, respectively. Virtually all high-ploidy megakaryocytes (greater than or equal to 8 C) were of low density (less than 1.050 g/cm3), whereas 2 and 4 C megakaryocytes were evenly distributed between less than or equal to 1.050 and greater than 1.050 g/cm3 marrow cells. These studies conclusively establish DNA content distribution of normal human marrow megakaryocytes and provide a basis for the study of states of altered megakaryocytopoiesis.

Three stages of differentiation in mouse megakaryocyte progenitor cells (CFU-Meg).

Mouse megakaryocyte colonies developed in a fibrin clot culture system were morphologically classified into three types: immature homogeneous (IH), heterogeneous (H), and mature homogeneous (MH) colonies. The colony size (number of megakaryocytes per colony), the ploidy distribution of megakaryocytes in colonies, and the ratios of their progenitors (megakaryocyte colony-forming units, CFU-Meg) in the S-phase of the cell cycle were compared among the three types. Also, morphological changes in colonies were examined in time sequence. The mode of the number of megakaryocytes per colony on day 6 of culture was greater than 64, between 17 and 32, and between 4 and 8, in IH, H, and MH colonies, respectively. The mean DNA content of megakaryocytes was 3.1N, 4.2N, and 7.2N on day 6 of incubation, in IH, H, and MH colonies, respectively, these values being significantly different from each other (p less than 0.001). The mean proportion of CFU-Meg synthesizing DNA was 21.9%, 26.0%, and 48.6% for CFU-Meg forming IH, H, and MH colonies, respectively (p less than 0.01; IH- versus MH-CFU-Meg and H- versus MH-CFU-Meg). Successive observation of the colony morphology from days 5 to 11 of culture suggested a transformation of the colony types from IH colony to MH colony through H colony. These observations indicate that the three types of megakaryocyte colonies classified on the basis of their morphological features reflect various stages of differentiation of mouse CFU-Meg.

Ultrastructural localization of platelet factor 4 in rat megakaryocytes and platelets by gold-labeled antibody detection.

The pattern of distribution of platelet factor 4 (PF-4) was studied in rat platelets and megakaryocytes following immunohistochemical labeling with a monoclonal antibody (2E7) specific for PF-4 and visualization by a protein A-gold complex. We observed a heterogeneity in PF-4 expression among alpha granules with a minority of them being unlabeled by immunoelectron microscopy, a pattern similar in both mature, circulating platelets and developing megakaryocytes. Furthermore, the majority of the labeled alpha granules in both cell types displayed a unique, eccentric localization of the PF-4 that was mainly over the nucleoid region within each granule. This localization is similar to the microtubular localization in alpha granules reported previously for von Willebrand factor and yet distinct from the reported random distribution of fibrinogen. We also observed significant labeling of small vesicular structures in developing megakaryocytes that may be involved in the transport of PF-4 and its packaging in platelet alpha granules. This new information is important in relating patterns of PF-4 biogenesis in megakaryocytes to conditions of alpha granular dysfunction in platelets.

Immunogold probing of platelet factor 4 in different ploidy classes of rat megakaryocytes sorted by flow cytometry.

Different ploidy classes of rat megakaryocytes were sorted by flow cytometry from highly purified perfusion-fixed megakaryocyte cell suspensions prepared by sequential centrifugal elutriation and Percoll gradient centrifugation. Sorted cell populations were studied for the localization of platelet factor 4 (PF-4) probed with the monoclonal antibody 2E7 in order to clarify the relevance of PF-4 localization to the cytoplasmic and nuclear development of megakaryocytes. The relative numbers of labeled alpha granules and labeled alpha granule-related small vesicular structures (AGR-SVS) were quantitated using the gold-labeled antibody detection method and correlated with DNA content and cytoplasmic maturation in individual megakaryocytes. We determined that the stage of cytoplasmic maturation exerted a significant effect on the proportion of labeled alpha granules and labeled AGR-SVS. A significant interaction effect of stage and ploidy class resulted in the stage effect on proportion of labeled alpha granules being significant only in two of the three ploidy classes. The least mature cells present within each ploidy group exhibited PF-4 labeling mostly in SVS that were not related to alpha granules. During subsequent cytoplasmic maturation, more of the labeled SVS were seen related to alpha granules, with more of the mature alpha granules themselves becoming labeled. Polyploidization also affected the proportion of labeled AGR-SVS. Our data suggest that SVS play a role in the intramegakaryocytic transport of PF-4 into alpha granules. These data provide evidence of the complexity of megakaryocytic differentiation involving both cytoplasmic maturation and nuclear endoreduplication as reflected in PF-4 expression.

Occurrence of platelet basic protein, a precursor of low affinity platelet factor 4 and beta-thromboglobulin, in human platelets and megakaryocytes.

beta-thromboglobulin antigen, a platelet-specific secreted protein, occurs in three forms: platelet basic protein, low affinity platelet factor 4, and beta-thromboglobulin. The combined level of beta-thromboglobulin antigen in megakaryocytes, measured by radioimmunoassay, was 13 +/- 7 micrograms/10(6) cells (SD, n = 6). The relative proportions of the three forms of beta-thromboglobulin antigen present within platelets and megakaryocytes were determined in cells lysed with trichloroacetic acid to minimize artifactual proteolysis. Samples were analyzed by isoelectric focusing in polyacrylamide gel with quantitative immunological detection on a nitrocellulose transfer of the gel. In platelets, the major species found was low affinity platelet factor 4 with precursor platelet basic protein as only 25% +/- 11% (SD, n = 16) of total beta-thromboglobulin antigen. In megakaryocytes partially purified both from normal bone marrow aspirates and from whole marrow specimens obtained after surgery, platelet basic protein was a higher proportion of beta-thromboglobulin antigen (49% +/- 13% SD, n = 11) than was the case in platelets. beta-thromboglobulin itself was never detected under the conditions of cell lysis used. Our results suggest that platelet basic protein is synthesized in megakaryocytes and that its cleavage is associated with an earlier stage of cell development than simply maturation to platelets. Further support for the precursor status of platelet basic protein was found in the expression of predominantly this antigenic form in a human erythroleukemia cell line.

A monoclonal antibody to a megakaryoblast-like cell line detects a protein found in blood cells and in the epithelial cell lining of various rat tissues.

Megakaryoblasts of bone marrow differentiate into megakaryocytes that in turn are the source of blood platelets. We have raised monoclonal antibodies to a megakaryoblast-like cell line derived from rat bone marrow (RPM cells). One antibody (Mab 213) and the corresponding antigen has been characterized by Western blotting and immunohistochemistry. Biosynthetic labeling with [35S]methionine showed that this antigen is synthesized by the RPM cells. In Western blots the antibody recognized proteins of about 90 kDa and 160 kDa in Triton extracts of RPM cells, whereas it recognized proteins of about 160 kDa and 200 kDa in Triton extracts of rat platelets and one of about 200 kDa in Triton extracts of various rat tissues (kidney, lung, intestine, and heart). By immunohistochemistry, the antigen was localized to the apical part of the epithelium lining certain parts of kidney tubuli, bronchi and large intestine.

Familial essential thrombocythemia.

Primary or essential thrombocythemia is rarely observed in childhood, and familial occurrence has been reported only once. In this study, essential thrombocythemia is documented in five members of both sexes from two to 62 years of age in three successive generations. The propositus had a persistent elevation of the platelet count, splenomegaly, a normal hemoglobin level, a normal white blood cell count, and abnormal platelet aggregation. Platelet arachidonic acid metabolites assayed by high-performance liquid chromatography and serum thrombopoietin levels were normal. Megakaryocytes were increased in number and size. Both mature and early immature megakaryocytes, but no atypical megakaryocytes, were identified by surface immunofluorescence. Bone marrow cultures showed normal myeloid and erythroid colony formation, and chromosome studies revealed a normal female karyotype. These findings support the concept that familial essential thrombocythemia is a myeloproliferative disorder that is transmitted by an autosomal dominant mode of inheritance, and that untreated young women and children with essential thrombocythemia have long survival.

Immunohistochemistry in bone marrow diagnosis. Value of a panel of monoclonal antibodies on routinely processed bone marrow biopsies.

To see if immunohistochemistry can be used on routinely processed bone marrow biopsies for diagnostic purposes, 73 biopsy specimens, fixed in sublimate-formaldehyde, decalcified in an acetic acid-formaldehyde mixture, and embedded in paraffin, were studied with a panel of antibodies. The specimens included "normal," lymphomatous, and myeloproliferative disorders as well as some biopsies with metastatic carcinoma. The results show that the different cell lines and their localization in the bone marrow can be easily identified and quantitative and qualitative changes can be assessed. Megakaryopoiesis is identified with Factor VIII-related antigen and Ulex europaeus agglutinin (UEA); myelopoiesis stains with MT-1, elastase, Leu M-1, LN-2, LN-3, HECA 452, and 115D8; and in myeloproliferative conditions, myeloblasts and promyelocytes stained with leukocyte common antigen (LCA). Erythroid cells stained with UEA, glycophorin A, and LN-1. Lymphocytes were marked with LCA, MB-2, and LN-2. Plasma cells were stained best with immunoglobulin light chain antisera; only occasional reactivity with LCA and 115D8 was observed. Carcinomas all reacted with MB-2; occasional reactivity with 115D8, HECA 452, LN-1, LN-2, MT-1, and Ber H-2 was seen. A small panel of selected antibodies, such as UEA, Leu M-1, and LCA, and the immunoglobulin light chain antisera can be very helpful in bone marrow diagnosis and would cover most indications.

The bleeding time is inversely related to megakaryocyte nuclear DNA content and size in man.

The relation between the bleeding time and the megakaryocyte nuclear DNA content and size was evaluated in eleven consecutive patients with normal steady state thrombopoiesis undergoing thoracotomy. A statistically significant inverse correlation was found between the bleeding time and both megakaryocyte DNA content (r = -0.71, p less than 0.05), megakaryocyte total size (r = -0.58, p less than 0.05), megakaryocyte cytoplasmic size (r = -0.64, p less than 0.05) and megakaryocyte nuclear size (r = -0.58, p less than 0.05). The megakaryocyte total size and the megakaryocyte cytoplasmic size were statistically significantly larger in men than women (p less than 0.02 and p less than 0.03 respectively). Changes in the megakaryocytes in the bone marrow are associated with changes in primary haemostasis in normal individuals.

Megakaryocytic leukemia; identification utilizing anti-factor VIII immunoperoxidase.

Coagulation Factor VIII is produced by endothelial cells and megakaryocytes. Antibody directed against Factor VIII or its associated antigens has been shown to be a sensitive marker for both normal and neoplastic endothelial cells. Its use to identify neoplastic megakaryocytes has not been described. We used immunoperoxidase localization of Factor VIII to identify megakaryocytes and immature megakaryocytic precursors in a case of megakaryocytic leukemia.

Presence of cytoadhesins (IIb-IIIa-like glycoproteins) on human metastatic melanomas but not on benign melanocytes.

Glycoproteins IIb and IIIa, a heterodimer complex, play a vital role in blood platelet aggregation and are members of a wide family of membrane receptors known as integrins or cytoadhesins. Cellular interaction to extracellular matrix (ECM) adhesive proteins is mediated by integrins. Certain tumor cells are known to interact with ECM and blood platelets in the process of metastasis. However, it is not known if tumor cells, compared with their normal counterparts, acquire IIb-IIIa-like receptors to help them in their metastatic spread. In this study, monoclonal antibodies directed against the IIb-IIIa platelet glycoprotein complex were used on frozen biopsies of normal and various tumor tissues to detect the presence of these integrins. These studies demonstrate the presence of IIb-IIIa-like glycoproteins on the cells of metastatic malignant melanoma but not on benign melanocytes and rarely on other tumors. The presence of integrins on melanomas may help explain their propensity for frequent metastasis.

Immunohistochemical localisation of thrombospondin in human megakaryocytes and platelets.

Thrombospondin (TSP) is a glycoprotein released from platelets after thrombin-induced aggregation. Endothelial culture studies suggest that it or a similar protein may also be produced by endothelial cells. The origin of the glycoprotein is therefore of some importance. This report concerns the immunohistochemical localisation of TSP within megakaryocytes and platelets. TSP would appear to be a primary platelet protein of megakaryocyte origin, rather than a substance taken up by the platelet in the circulation. The relation of this molecule to the endothelial glycoprotein remains to be established, but our preliminary immunohistochemical studies suggest that the molecule is also located in vascular endothelium.