Immunohistochemical localization of membrane and alpha-granule proteins in plastic-embedded mouse bone marrow megakaryocytes and murine megakaryocyte colonies

Using an immunoperoxidase technique that permits optimal antigen localization at the light microscope level, we have detected two platelet alpha-granule constituents and three platelet membrane glycoproteins in mouse bone marrow megakaryocytes and in murine megakaryocyte colonies grown in soft agar culture for three to seven days. Using polyclonal antibodies prepared against human platelet proteins, we have demonstrated labeling for von Willebrand factor, fibrinogen, and the membrane glycoproteins IIIa and GMP-140 in both bone marrow megakaryocytes and megakaryocyte colonies after seven days of culture. Using monoclonal antibodies to membrane glycoproteins IIb and GMP-140, we have demonstrated label in mouse bone marrow megakaryocytes. Granulocyte and macrophage colonies were negative for each of these markers. Murine bone marrow megakaryocytes and megakaryocyte colonies demonstrated a similar enzyme histochemical pattern: weakly positive for alpha-naphthyl acetate esterase and negative for chloroacetate esterase. These data indicate that megakaryocytes grown in soft agar culture express many of the same glycoproteins as bone marrow megakaryocytes. Furthermore, the ability of antibodies directed against human platelet membrane glycoproteins to identify murine megakaryocyte glycoproteins indicates that these constituents have been highly conserved during evolution.     

Measurement of ploidy distribution in megakaryocyte colonies obtained from culture: with studies of the effects of thrombocytopenia

Microdensitometric measurement of the DNA content of individual megakaryocytes was performed using megakaryocyte colonies obtained following culture, in soft agar, of hematopoietic cells from C57BL/6J mice. Two types of colonies were detected. After 7 days of culture, the big cell type contained 16 /+- 2.3 acetylcholinesterase (AChE) positive cells/colony, with a mean ploidy level of 16.8 /+- 0.8/cell and the ploidy distribution characteristic of recognizable megakaryocytes in bone marrow. The heterogeneous type contained 44 /+- 9.6 cells/colony (some of which were AChE negative), with a mean ploidy level of 6.8 /+- 0.7/cell. The ploidy distribution of heterogeneous colonies differed markedly from big cell colonies, with preponderance of 2N and 4N cells. Colony-forming cells, obtained 4-5 days after induction of acute thrombocytopenia, gave big cell colonies with a marked increase in DNA content. Mean ploidy level increased to 21.5 /%- 1.8/cell; the frequency of 32N cells increased from 17% to 30% and 64N cells from 0% to 6%. This is the pattern of change observed in bone marrow, in vivo, 24 to 48 hr after induction of acute thrombocytopenia. The number of cells/colony did not increase. In contrast, acute thrombocytopenia did not alter the ploidy of heterogeneous colonies. The different responses to the stimulus of acute thrombocytopenia suggest that there are at least two types of Meg-CFC. The delayed appearance of altered Meg-CFC that produced big cell colonies indicates that the pool of stem cells, from which committed megakaryocyte precursors are derived, may respond indirectly to the stimulus of platelet depletion.     

Characteristics of rat megakaryocyte colonies and their progenitors in agar culture

The characteristics of megakaryocyte colonies that develop from megakaryocyte progenitors of rat bone marrow stimulated by rat spleen-conditioned medium (SCM) in agar culture were investigated. Colony frequency was optimal on day 7 and increased relative to both the number of cells plated and the concentration of SCM used. Plating efficiencies averaged 72 +/- 16 megakaryocyte colonies/10(5) cells with 0.1 ml SCM/culture. Colonies were categorized as small cell and big cell. Small-cell colonies had a greater proliferative potential, with a mean of 25 cells/colony. Big-cell colonies averaged 15 cells/colony. The ratio of big-cell to small-cell colonies was 0.69 +/- 0.29. Granulocyte-macrophage colonies, which were also stimulated by SCM, accounted for 70% +/- 15% of the total colonies in the cultures. Cytocidal experiments with tritiated thymidine reduced megakaryocyte colony formation by 45% and granulocyte-macrophage colony formation by 21%. The properties of rat, mouse, and human megakaryocyte progenitors as assayed in vitro are compared.     

Evidence for secretion of human eosinophil granule major basic protein and Charcot-Leyden crystal protein during eosinophil maturation

Prior electron microscopic studies have suggested that immature eosinophils degranulate during normal maturation in human bone marrow. This hypothesis was tested by measuring levels of eosinophil granule major basic protein (MBP) and Charcot-Leyden crystal (CLC) protein in bone marrow sinusoidal blood. MBP and CLC protein levels were elevated initially in bone marrow sinusoidal blood from normal volunteers when compared with peripheral blood, and levels of both proteins decreased during serial sampling; CLC protein levels remained significantly elevated, while MBP levels declined to equal those of peripheral blood. To investigate whether MBP and CLC protein were secreted during eosinophil maturation, bone marrow cells were cultured in soft agar; MBP and CLC protein levels were measured in culture supernatants by RIA. There was a significant positive correlation between eosinophil colony growth and levels of each protein. Electron micrographs of cells in soft agar colonies provided ultrastructural evidence for secretion of granule products by immature eosinophils. These results support prior observations that eosinophil promyelocytes degranulate during maturation.     

Effects of culture matrix on differentiation of murine mast cells

The effects of culture matrix (agar, collagen, and methylcellulose) on differentiation of mast cells were investigated. Because berberine sulfate-positive colonies in agar were composed of macrophages but not of mast cells, the naphthol AS-D chloroacetate esterase reaction was used to identify mast-cell colonies. When bone marrow cells of WBB6F1 mice were cultured with conditioned media containing interleukin 3 (IL-3), numbers of mast-cell colonies were greater in collagen cultures than in agar and methylcellulose cultures. Electron microscopic examination revealed that mast cells developing in collagen and agar cultures were more mature than those developing in methylcellulose cultures. However, when bone marrow-derived cultured mast cells or purified peritoneal mast cells were plated, the efficiency of colony formation and size of colonies were comparable among agar, collagen, and methylcellulose cultures. Therefore, all three matrices tested had similar effects on the proliferation of mast cells. Collagen appeared to be suitable for differentiation of bone-marrow precursors and their maturation. Agar appeared to be suitable only for maturation.     

Characteristics of megakaryocyte colony formation in normal individuals and in primary thrombocythemia: studies using an optimal cloning system

Colony formation by megakaryocyte (MK) progenitors was studied in 36 normal individuals and in 26 patients with primary thrombocythemia (PT) using an improved plasma clot cloning system. MK colonies were identified by immunoperoxidase staining using a monoclonal antibody against human platelet glycoprotein IIb/IIIa complex. In normal individuals, a frequency of 194 +/- 23 MK colony-forming units (CFU-MK) per 5 X 10(5) bone marrow nonadherent mononuclear cells and 11 +/- 4 CFU-MK per 5 X 10(5) blood mononuclear cells was found. These CFU-MK grew as large (greater than 20 cells), mean (11-20 cells), small (3-10 cells), or mix-MK colonies (at least two MK) that comprised 15%, 23%, 62%, or 5% of all MK colonies in bone marrow and 0%, 8%, 92%, or 1% in peripheral blood, respectively. In PT, several abnormalities of MK colony formation were observed: 1) increased circulating CFU-MK numbers, 2) increased mix-MK colony formation, 3) spontaneous MK colony formation without phytohemagglutinin-stimulated leukocyte-conditioned medium and normal serum, 4) decreased proportion of larger MK colonies (greater than 11 cells) in PT bone marrow, and 5) failure of PT plasma or serum to stimulate MK colony formation by normal marrow cells in a normal fashion. These results indicate some of the characteristics of quantitative and qualitative abnormalities of in vitro megakaryocytopoiesis in PT.     

In vivo administration of antibody to interleukin-5 inhibits increased generation of eosinophils and their progenitors in bone marrow of parasitized mice

Bone marrow of mice parasitized with Nippostrongylus brasiliensis showed increased numbers of eosinophils as early as 4 days after infection. By day 7, their bone marrow also contained elevated numbers of progenitors that form small eosinophil colonies (20 to 50 cells) in soft agar cultures supplemented with interleukin-5 (IL-5). However, when mice were infused with anti-IL-5 antibodies at the time of infection, the number of recognizable eosinophils present in bone marrow remained low and eventually dropped below normal levels. The antibody treatment also prevented increased generation of IL-5- responsive precursors capable of differentiating into mature eosinophils in liquid culture and inhibited the generation of progenitor cells capable of forming small eosinophil colonies or clusters in soft agar cultures. The results of these in vivo experiments directly show that IL-5 is an essential regulatory molecule required for the bone marrow-dependent phase of a parasite-induced eosinophilia.     

Uncoordinated expression of fibrinogen compared with thrombospondin and von Willebrand factor in maturing human megakaryocytes

The localization of three known alpha-granule proteins, thrombospondin (TSP), von Willebrand factor (vWF), and fibrinogen (Fg) has been studied in human megakaryocytes (MK) by immunofluorescence and immunoelectron microscopy. For this study, highly purified populations of MK were prepared from human bone marrow either by counterflow centrifugal elutriation or by cell culture from normal subjects and from two patients with megakaryoblastic leukemia. In normal bone marrow immature MK, TSP, and vWF were observed in the Golgi-associated vesicles and in small immature alpha-granules; in mature MK, they were found in the matrix of the mature large alpha-granules. Surprisingly, Fg was detected neither in the Golgi area, nor in the small precursors of alpha-granules; it was only found in the mature alpha-granules but this labeling was generally weaker than in blood platelets. In order to confirm these differences between the expression of Fg and vWF or TSP additional studies were performed on cultured maturing MK: immunofluorescent and ultrastructural immunogold labeling confirmed that vWF appeared early in the maturation while the same immature MK were negative for Fg. In the late maturation stage, the three proteins were detected in the alpha-granules. In order to know whether Fg was lately synthesized or endocytosed from the outside medium, normal MK were grown in the presence of either normal or afibrinogenemic plasma, and normal serum. Fg was detected only in the alpha-granules of MK grown in normal plasma. Similar results were observed with malignant MK, whose maturation was independent of the culture conditions. In conclusion, this study brings immunocytochemical evidence that vWF and TSP are synthesized by immature MK, whereas Fg appears later in the MK alpha-granules and its expression is dependent of the presence of an exogenous Fg source.     

Biology of human megakaryocyte factor V

To learn more about human megakaryocyte coagulation cofactor V (FV), we studied the expression of this protein in normal bone marrow megakaryocytes and in megakaryocytes cloned from their colony-forming unit in FV-depleted plasma clot cultures. Mouse monoclonal antibodies directed against either the light chain or an activation peptide of human FV and a rabbit polyclonal, monospecific FV antiserum were used as probes for these experiments in conjunction with a variety of immunochemical detection techniques. All morphologically recognizable megakaryocytes were shown to contain FV. The origin of this protein appeared to be both from FV bound to the cell as well as from endogenous FV in the majority of cells examined. The existence of a population of small bone marrow mononuclear cells that simultaneously expressed platelet glycoproteins and FV was also noted. Such cells represented approximately 70% of all small cells positive for platelet glycoproteins. In contrast, only about 40% of megakaryocyte colonies cloned in FV-deficient medium contained cells with immunochemically detectable FV. FV expression was most clearly demonstrated in large cells in the colonies, whereas smaller, presumably less mature cells labeled weakly or not at all. Synthesis of FV by human megakaryocytes was documented using elutriation-enriched cells incubated in 35S-methionine-containing medium. Megakaryocyte lysates and medium conditioned by these cells were subjected to immunoaffinity column purification. Column eluates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed radioactive bands comigrating with the heavy and light chains of thrombin-activated FV. These studies suggest that human megakaryocytes both bind and synthesize FV. Expression of these traits appears to be related to cell maturation, with binding ability appearing earlier than the ability to synthesize this protein. Finally, although the ability to bind FV appears to be universal among megakaryocytes, our culture data suggest that synthesis may be a restricted, or constitutively expressed property of these cells.     

Developmental changes in human megakaryocyte ploidy

Megakaryocytes (MK) obtained from the differentiation of MK colony-forming units (CFU-MK) were grown from fetal liver, cord blood, and adult marrow in liquid culture containing aplastic plasma. Ploidy distribution was studied by a double-staining technique and flow cytometry and MK maturation by ultrastructural techniques. Cultured MK from fetuses and neonates were small sized (about 10 microns) in comparison to adult MK. They were mature cells that contained large membrane complexes as previously found in vivo. Only 2N and 4N MK were usually present in 8- to 10-week-old fetus cultures; 8N MK were detected at 20 weeks of gestation and in neonates. Higher ploidy classes were present in culture from adults but with a much lower frequency than in marrow. Therefore, a progressive shift to higher ploidy and an increase in MK size were observed simultaneously during development. Interleukin 3 (IL-3) increased MK proliferation as in adults but abrogated MK ploidization of 20-week-old fetus culture. The present results suggest that the changes occurring during ontogenesis are related to intrinsic MK modifications because no inhibitor of MK ploidization could be detected in fetal cultures.     

Proliferation and maturation of murine megakaryocyte progenitor cells in a liquid culture system

Murine megakaryocytes generated in a liquid culture system containing pokeweed-mitogen-stimulated spleen-cell-conditioned medium were classified into four groups: heterogeneous colonies, homogeneous colonies, two-cell colonies, and solitary megakaryocytes. The ploidy distribution and ultrastructural cytoplasmic maturation were compared among the four groups. The mean ploidy of heterogeneous colonies was low and remained unchanged during the culture. In contrast, an intensive increase in DNA content of homogeneous colonies, two-cell colonies, and solitary megakaryocytes was observed. In ultrastructural analyses, type-I megakaryocytes (early immature megakaryocytes) were predominant in heterogeneous colonies. Type-II megakaryocytes (immature megakaryocytes) increased in homogeneous colonies and two-cell colonies. Type-III megakaryocytes (mature megakaryocytes) increased in two-cell colonies and solitary megakaryocytes. In the liquid culture system, megakaryocytes from two-cell colonies and solitary megakaryocytes could mature into platelet-producing megakaryocytes, whereas megakaryocytes from heterogeneous colonies and homogeneous colonies remained immature.     

Inhibitory effect of highly purified human platelet beta-thromboglobulin on in vitro human megakaryocyte colony formation.

The effect of highly purified human beta-thromboglobulin (beta TG), a glycoprotein present in platelet alpha granules, was tested on human bone marrow in vitro megakaryocyte (MK) colony formation. A concentration of 5 micrograms/ml of beta TG induced a 50% reduction in the number of MK colonies, and concentrations of 10 and 20 micrograms/ml completely inhibited MK growth. This inhibition was of importance for MKs because a higher concentration of beta TG (10 micrograms/ml) was needed to obtain a nonsignificant decrease in erythroblastic progenitors (erythroid burst-forming units, BFU-E), and no inhibition of granulocyte-macrophage colony-forming units (CFU-GM) was observed. beta TG acts mainly on maturation of MKs. These results indicate that beta TG could play a role in the physiological regulation of platelet production by MKs.     

Effect of infusing rat monoclonal antibodies to the murine GPIb-IX-V complex on platelet and megakaryocyte morphology in mice

In the Bernard-Soulier syndrome, the absence of GPIb-IX-V leads to thrombocytopenia and giant platelets. In autoimmune thrombocytopenia in man, anti-platelet antibodies are associated with changes in megakaryocyte (MK) morphology and platelet size heterogeneity. We have compared the ultrastructural changes in mature MK following the infusion of rat monoclonal antibodies (MoAbs) to different epitopes of the murine GPIb-IX-V complex in mice. Blood and marrow samples were examined during both the acute thrombocytopenic phase and during the recovery phase. A MoAb to GPV induced neither thrombocytopenia nor changes in platelet morphology. During the acute thrombocytopenic phase with anti-GPIbalpha MoAbs, the size of residual platelets was heterogeneous and included large forms and platelets with few granules. During recovery, platelet size heterogeneity continued, and some platelets showed signs of activation. But only rare platelets were giant forms with ultrastructural defects resembling BSS. Megakaryocytopoiesis during acute thrombocytopenia was already abnormal, with some mature cells often showing vacuoles and an irregular development of the demarcation membrane system which varied in extent. These changes continued into the recovery phase. The anti-GPV MoAb had no effect on MK. Thus, anti-platelet antibodies can induce a different medullary response even when binding to the same receptor.     

Serum from patients with various thrombopoietic disorders alters terminal cytoplasmic maturation of human megakaryocytes in vitro

Human bone marrow was depleted of progenitors (CFU-MK), but enriched for recognizable megakaryocytes (MK), and placed in cultures with serum from either normal donors (NABS) or patients with primary (PTS) or secondary (STS) thrombocytosis, autoimmune thrombocytopenia (ATS) or aplastic anemia (AAS). Mean MK diameters shifted during the 3-4 days of incubation. Endomitotic figure were visible and mean ploidy increased slightly during cytoplasmic maturation, where decreases in immature cells (stages 1 and 2) were accompanied by increases in the mature MK (stages 3 and 4). Cytoplasmic maturation was faster in AAS, ATS and STS than PTS or NABS; mean size and ploidy were similar in all cultures. Recognizable MK were not forced to undergo additional endoreduplication in response to stimulation. Only AAS augmented MK colony formation, which indicated that at least two humoral factors can regulate megakaryocytopoiesis at separate levels, the progenitors and morphologically recognizable MK.     

Effect of infusing rat monoclonal antibodies to the murine GPIb-IX-V complex on platelet and megakaryocyte morphology in mice

In the Bernard-Soulier syndrome, the absence of GPIb-IX-V leads to thrombocytopenia and giant platelets. In autoimmune thrombocytopenia in man, anti-platelet antibodies are associated with changes in megakaryocyte (MK) morphology and platelet size heterogeneity. We have compared the ultrastructural changes in mature MK following the infusion of rat monoclonal antibodies (MoAbs) to different epitopes of the murine GPIb-IX-V complex in mice. Blood and marrow samples were examined during both the acute thrombocytopenic phase and during the recovery phase. A MoAb to GPV induced neither thrombocytopenia nor changes in platelet morphology. During the acute thrombocytopenic phase with anti-GPIb f MoAbs, the size of residual platelets was heterogeneous and included large forms and platelets with few granules. During recovery, platelet size heterogeneity continued, and some platelets showed signs of activation. But only rare platelets were giant forms with ultrastructural defects resembling BSS. Megakaryocytopoiesis during acute thrombocytopenia was already abnormal, with some mature cells often showing vacuoles and an irregular development of the demarcation membrane system which varied in extent. These changes continued into the recovery phase. The anti-GPV MoAb had no effect on MK. Thus, anti-platelet antibodies can induce a different medullary response even when binding to the same receptor.     

Complementary and antagonistic effects of IL-3 in the early development of human megakaryocytes in culture

The effect of IL-3 on the early steps in the growth and development of megakaryocytes (MK) in culture has been studied. Although thrombopoietin (TPO) by itself could support the development of mature CD41⁺ MK from pre-MK, the number of cells produced was greatly augmented by the addition of IL-3 and SCF. IL-3 was also able to support the growth of MK colonies in semi-solid media (CFU-MK). The CD41⁺ cells that developed in suspension cultures containing IL-3 differed phenotypically from those that developed without this agent. Cells grown in the presence of IL-3 lost CD34 expression more rapidly, expressed lower levels of the platelet glycoproteins gpIIb-IIIa and Ib and achieved lower degrees of polyploidy than in the absence of IL-3. The inhibitory effects of IL-3 were not a consequence of the dilution of the mature cells by increased numbers of immature cells since it was observed under conditions in which IL-3 did not stimulate MK growth. The results obtained in these cultures suggest that IL-3 plays an important role in early MK development, but inhibits further maturation after endoreduplication begins. Thus, prolonged contact with IL-3 results in the appearance of cells that do not mature normally.     

Immunophenotypic analyses of cultured hemopoietic mast cells

Immunophenotypic analyses of immature stage (day 19-23), intermediate stage (day 28-32), mature stage (day 34-37), and older stage (day 42-44) human hemopoietic mast cells from colonies grown in semi-solid agar cultures were performed to study the ontogeny and identity of this cell type and its relationship to other leukocytes. Intermediate to mature stage mast cells were positive with the YB5.B8 mouse monoclonal antibody, (McAb) specific for human mast cells, whereas the reactivity of immature mast cells with this McAb was inconsistent and older cells were generally negative. Mast cells at all stages of maturation were strongly positive for IgE receptor sites and negative with the Bsp-1 McAb, specific for human basophils. Mast cells at all stages of maturation were also strongly positive with the monocyte McAbs RPA-M1 (CD11), positive with the monocyte McAb OKM5 and the monocyte/granulocyte McAbs BMA-210 and MY7 (CD13), strongly positive with the B-cell markers J5 (CD10) and anti-IgM, and positive with the plasma cell marker PCA-1 and to a lesser extent with the activated B-cell marker CD23. The mast cells were also strongly positive with anti-CD45 to the common leukocyte antigen and positive with an antibody to HLA-DR and an antibody to FVIIIC. They were negative for specific T-cell markers. The diversity of this phenotype supports the current concept that mast cells originate from the pluripotential progenitor cells in the bone marrow.     

Correlation of in vitro and in vivo biological activities during the partial purification of thrombopoietin.

We have evaluated three in vitro assays for megakaryocyte maturation as monitors of biological activities of thrombopoietin (TPO). The in vitro measurements, that is, potentiation of murine megakaryocyte colonies in soft agar cultures, growth of single murine megakaryocytes in soft agar, and acetylcholinesterase production in liquid cultures of murine bone marrow cells, were correlated with measurements of thrombopoiesis stimulatory activity in vivo, based on labeling of newly formed platelets with [75Se]selenomethionine. Protein fractions produced during the purification of TPO from the plasma of thrombocytopenic rabbits were used to evaluate the in vitro assays. Potentiation of murine megakaryocyte colony growth in soft agar was least valuable as a TPO assay, due to variability. Growth of single megakaryocytes in vitro, in a serum-free, agar culture system, correlated well with measurement of thrombopoiesis-stimulating activity in vivo, in regard to increases in specific activity during the purification, and was the most sensitive of the three assays. The serum-free, liquid culture assay also correlated well with the in vivo assay, and it had the additional advantage of being feasible for evaluation of the large numbers of protein fractions produced by high-resolution chromatographic procedures. Using the liquid culture system to assay fractions from gel permeation high performance liquid chromatography, a biologically active fraction with a molecular weight range of 40-47 kd was identified.     

Spontaneous formation of megakaryocyte progenitors (CFU-MK) in primary thrombocythaemia

An improved plasma clot culture method for CFU megakaryocytes (MK) has been developed with a higher plating efficiency and easier identification and enumeration of MK colonies by an indirect immunoperoxidase staining using a monoclonal antibody specific to the platelet glycoprotein IIb/IIIa complex. This technique has been used to study megakaryocytopoiesis in 20 normal individuals, 4 recently diagnosed patients with untreated primary thrombocythaemia (PT) and 2 patients with secondary thrombocytosis (ST). An increased number of MK colonies was evident in peripheral blood (mean 115 +/- 31 CFU-MK/5 X 10(5) seeded cells or 206 +/- 91/ml) and to a lesser extent in bone marrow (mean 188 +/- 26 CFU-MK/5 X 10(5) seeded cells or 696 +/- 103/ml) of PT patients as compared to controls (mean 11 +/- 4/5 X 10(5) seeded cells or 13 +/- 3/ml of peripheral blood and 153 +/- 15/5 X 10(5) seeded cells or 319 +/- 43/ml of bone marrow). There was a very obvious difference between PT patients and the others (controls plus ST patients) because CFU-MK growth with no added stimulus (PHA-LCM or PHA-LCM and normal serum) could be seen in PT patients only.     

Long-term culture of B lymphocytes and their precursors from murine bone marrow.

Growth of mature B cells and their precursors from mouse bone marrow was maintained in culture for greater than 1 year. Feeder layers of adherent bone marrow cells, established in medium containing fetal calf serum and no exogenous steroids, were used to provide an in vitro environment that supported continuous growth and development of these cells. In such bone marrow cultures, the number of cells bearing membrane immunoglobulin increased gradually for 4 wk and then decreased. Between 10 and 14 wk, some of the cultures gave rise to continuously dividing B-cell populations that contained pre-B cells (producing mu heavy chains only and sensitive to transformation by Abelson murine leukemia virus) as well as mature B cells (synthesizing both light and heavy chains of IgM). Immunoglobulin molecules synthesized by cells in these populations were heterogeneous in two-dimensional gel analysis. This suggests that mature B cells arose via maturation of pre-B cells in the cultures that involved rearrangement and expression of different variable region gene segments.