Abnormal function of the bone marrow microenvironment in chronic myelogenous leukemia: role of malignant stromal macrophages

The bone marrow microenvironment supports and regulates the proliferation and differentiation of hematopoietic cells. Dysregulated hematopoiesis in chronic myelogenous leukemia (CML) is caused, at least in part, by abnormalities in CML hematopoietic progenitors leading to altered interactions with the marrow microenvironment. The role of the microenvironment itself in CML has not been well characterized. We examined the capacity of CML stroma to support the growth of long-term culture-initiating cells (LTC-IC) obtained from normal and CML marrow. The growth of normal LTC-IC on CML stroma was significantly reduced compared with normal stroma. This did not appear to be related to abnormal production of soluble factors by CML stroma because normal LTC- IC grew equally well in Transwells above CML stroma as in Transwells above normal stroma. In addition, CML and normal stromal supernatants contained similar quantities of both growth-stimulatory (granulocyte colony-stimulating factor (CSF), interleukin-6, stem cell factor, granulocyte-macrophage CSF, and interleukin-1 beta) and growth- inhibitory cytokines (transforming growth factor-beta, macrophage inflammatory protein-1 alpha, and tumor necrosis factor-alpha). The relative proportion of different cell types in CML and normal stroma was similar. However, polymerase chain reaction and fluorescence in situ hybridization studies showed the presence of bcr-abl-positivo cells in CML stroma, which were CD14+ stromal macrophages. To assess the effect of these malignant macrophages on stromal function, CML and normal stromal cells were separated by fluorescence-activated cell sorting into stromal mesenchymal cell (CD14-) and macrophage (CD14+) populations. CML and normal CD14- cells supported the growth of normal LTC-IC equally well. However, the addition of CML macrophages to normal or CML CD14- mesenchymal cells resulted in impaired progenitor support. This finding indicates that the abnormal function of CML bone marrow stroma is related to the presence of malignant macrophages. In contrast to normal LTC-IC, the growth of CML LTC-IC on allogeneic CML stromal layers was not impaired and was significantly better than that of normal LTC-IC cocultured with the same CML stromal layers. These studies demonstrate that, in addition to abnormalities in CML progenitors themselves, abnormalities in the CML marrow microenvironment related to the presence of malignant stromal macrophages may contribute to the selective expansion of leukemic progenitors and suppression of normal hematopoiesis in CML.     

Therapy for ongoing graft-versus-host disease induced across the major or minor histocompatibility barrier in mice with anti-CD3F(ab')2-ricin toxin A chain immunotoxin

A new pharmacologic agent, anti-CD3F(ab')2-ricin toxin A chain (RTA), was synthesized for the purpose of targeting T cells and as a means of treating established graft-versus-host disease (GVHD). The Fc region of anti-CD3 monoclonal antibody (MoAb) was removed to prevent its ability to activate T cells. The resulting F(ab')2 fragments were conjugated to deglycosylated RTA (dgRTA), a catalytic and potent phytotoxin. The resulting immunotoxin (IT) was potent (greater than 95% inhibition) and selective in inhibiting T-cell mitogenesis in vitro. In vivo, the IT depleted 80% of T cells in mice receiving bone marrow (BM) transplants. Transplantation in an aggressive acute GVHD model using C57BL/6 donor cells and H-2 disparate B10.BR recipients resulted in an infiltration of CD3-expressing cells and a median survival time (MST) of 20 to 30 days. A 5-day course of anti-CD3F(ab')2-RTA (30 micrograms/d intraperitoneally) beginning 7 days after GVHD induction was beneficial in treating established GVHD in these mice, as evidenced by significantly prolonged survival (MST, greater than 80 days), superior mean weight values, and improved clinical appearance. Neither intact anti-CD3, unconjugated anti-CD3 F(ab')2 fragments, nor a mixture of anti-CD4 and anti-CD8 MoAbs (which are highly effective in prophylactic models) were as effective. F(ab')2 fragments made from anti-Lyt-1 (the murine homologue of human anti-CD5) linked to RTA were also not effective, despite the fact that both anti-CD3F(ab')2-RTA and anti-Lyt- 1F(ab')2-RTA had similar half-lives of about 9 hours. The IT also increased MST in two aggressive models of GVHD across non-H-2 minor histocompatibility barriers, indicating that the usefulness of anti- CD3F(ab')2-dgRTA is not limited to a single-strain combination. This agent should be further investigated as an alternative to current strategies for treating steroid refractory GVHD.     

Assessment of clinical significance of anti-Ge in an untransfused man

A 19-year-old, untransfused Melanesian man from Papua New Guinea was admitted to the hospital for repair of an atrial septal defect. His serum contained an alloantibody that reacted strongly on the indirect antiglobulin test and was identified as anti-Ge. Gerbich-negative blood was transfused following urgent surgery. A 51Cr red cell survival study performed 2 weeks after surgery yielded zero survival of Gerbich-positive cells after 24 hours. A monocyte-driven, antibody-dependent, cell-mediated cytotoxicity assay performed on both pretransfusion and posttransfusion serum samples and on concentrated serum showed less than 1 percent specific lysis of Gerbich-positive cells. This did not correlate with the indication of clinical significance predicted by the 51Cr study. Red cell adherence and phagocytosis, not evident in a monocyte monolayer assay using native serum, were demonstrable in 16 percent of monocytes by the use of concentrated serum.     

Human fibroblasts downregulate plasminogen activator inhibitor type-1 in cultured human macrovascular and microvascular endothelial cells

We have previously reported that plasminogen activator inhibitor type-1 (PAI-1) expression in endothelial cells (ECs) can be modulated differently by smooth muscle cells depending on their origin. Human pulmonary artery smooth muscle cells (HPASMCs) strongly downregulated PAI-1 expression in ECs. Fibroblasts (FBs) are another cell type that could come in close contact with ECs. Therefore, it was the aim of this study to investigate whether FBs could also influence the fibrinolytic potential of ECs. As in the case of HPASMCs, PAI-1 antigen produced by human umbilical vein ECs (HUVECs) cocultured with human skin FBs (HSFBs) was significantly lower as compared with the sum of PAI-1 secreted by the respective cell types cultured separately. Not only HUVECs but also human skin microvascular ECs (HSMECs) responded in a dose-dependent way to serum-free conditioned media (CM) from HSFBs from one individual donor. Similar results were obtained when CM from HSFBs from four other individual donors were used. PAI-1 mRNA decreased in HUVECs incubated for 6 hours with HSFB-CM to 24% to 55% of control, depending on the preparation of HSFBs used. A significant PAI-1 downregulatory effect was only observed when CM from low-passage HSFBs (up to passage no. 5) was used, whereas no reduction in EC PAI-1 production was observed with CM obtained from HSFBs in passage no. 8. This PAI-1 downregulatory activity present in HSFB-CM was heat-labile and had a molecular mass of approximately 5 kD. When CM from HPASMCs was analyzed in the same way, an almost identical elution profile was found. In conclusion, our data showed that FBs can decrease the expression of PAI-1 in ECs. Such an effect could be operative during wound-healing and at other capillary sites where FBs could render ECs profibrinolytic, thereby facilitating processes requiring an increase in proteolytic activity such as EC migration and proliferation.     

A monoclonal antibody-defined membrane antigen complex is required for neutrophil-neutrophil aggregation

We examined the aggregation responses of normal neutrophils treated with the murine monoclonal antibody (MoAb) 60.3. Addition of MoAb 60.3 to normal neutrophils produced dose-dependent inhibition of neutrophil aggregation in response to phorbol myristate acetate, zymosan-activated plasma, and N-formyl-methionylleucylphenylalanine. We conclude that the membrane glycoprotein complex recognized by MoAb 60.3--designated CDw18- -is required for neutrophil-neutrophil aggregation in vitro.