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Murine acquired immunodeficiency syndrome (MAIDS) induced by defective LP-BM5 murine leukemia virus (MuLV) is a disease with many similarities to human AIDS. Our previous studies demonstrated that the depressed hematopoiesis observed in LP-BM5-infected marrow cultures could be attributed to a defective hematopoietic stroma. We report now the generation of permanent stroma cell lines from noninfected and LP-BM5-infected marrow cultures. Retrovirus infection was confirmed by the polymerase chain reaction for detecting viral genome expression of the p12 envelope glycoprotein. The ability of these cell lines to support in vitro hematopoiesis was evaluated. The results demonstrated that when cocultured with normal or infected nonadherent mononuclear cells, noninfected cell lines efficiently supported the production of hematopoietic progenitors, whereas in virus-infected progenitors was suppressed. Expression of cytokine genes in stromal cell lines was also examined. All cell lines expressed equivalent levels of transcripts for interleukin (IL)-1 beta, IL-2, IL-3, IL-6, IL-7, IL-10, interferon, tumor necrosis factor-alpha and stem cell factor. However, infection was associated with higher expression of IL-4 and transforming growth factor-beta 1. These findings demonstrate that infected stomal cell lines generate a defective hematopoietic microenvironment to produce altered cytokine expression and faulty hematopoiesis. Further characterization of these defective cell lines should assist elucidation of the mechanism(s) whereby retroviruses alter hematopoiesis ultimately leading to the generation of immunodeficiency.

We have previously demonstrated that continuous administration of dose-escalation zidovudine (AZT) in either normal or LP-BM5 MuLV immunodeficient virus-infected mice (MAIDS) was associated with the development of anemia, neutropenia, and thrombocytopenia. Hematopoietic growth factors/cytokines are being evaluated to determine their efficacy in ameliorating the hematopoietic toxicity associated with AZT. In normal mice receiving AZT, an increase in only plasma erythropoietin and not GM-CSF, Meg-CSF or TNF-alpha has been reported. This article describes studies that investigated the effect of combination interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) administered in normal non-viral, viral-infected, and viral-infected C57BL6 mice receiving dose-escalation AZT, i.e. 0.1 mg/ml, 1.0 mg/ml, and 2.5 mg/ml placed in the drinking water. Non-viral control mice responded to IL-3/GM-CSF by increasing erythropoiesis, myelopoiesis and platelet production measured by increased bone marrow and spleen derived erythroid, myeloid and platelet precursor stem cells cultured in semi-solid media. Virus-infected control mice not receiving IL-3/GM-CSF developed pancytopenia. Administration of IL-3/GM-CSF to virus-infected mice receiving dose-escalation AZT did not ameliorate the peripheral pancytopenia associated with immunodeficiency disease and AZT treatment, even though erythroid, myeloid and platelet precursor progenitor cells were increased at certain times when compared to either normal or viral-infected mice receiving IL-3/GM-CSF. These results indicate that the combination use of IL-3 and GM-CSF in vivo is only a partially effective growth factor/cytokine treatment to ameliorate the hematopoietic toxicity associated with the use of the anti-viral drug zidovudine.

Lithium salts have been demonstrated to induce the production of hematopoietic cells following administration in vivo and to minimize the reduction of these cells following treatment with either radiation, chemotherapeutic or antiviral drugs. We have previously demonstrated that lithium, when administered in vivo to immunodeficient mice infected with LP-BM5 MuLV (MAIDS) significantly reduced the development of lymphadenopathy, splenomegaly, and the lymphoma associated with late-stage immunodeficiency disease in this model, and increased the survival of these animals compared to virus-infected controls not receiving lithium. We report here the results of in vivo studies in the MAIDS model that determined the effect of lithium on peripheral blood indices and the number of myeloid (CFU-GM), erythroid (BFU-E) and megakaryocyte (CFU-Meg) hematopoietic progenitors from bone marrow and spleen harvested from immunodeficient mice receiving lithium carbonate (1 mM) placed in their drinking water compared to virus-infected controls not receiving lithium. Time-points evaluated were at weeks 1, 5, 9, 13, 17, and 21 postviral infection. Virus-control mice not receiving lithium demonstrated all the signs that are characteristic of MAIDS, i.e., splenomegaly, lymphadenopathy, hypergammaglobulinemia, reduced hematopoiesis, and death. Infected mice receiving lithium demonstrated diminished presence of splenomegaly, lymphadenopathy, hypergammaglobulinemia, no suppression of hematopoiesis nor mortality. Enhanced hematopoiesis was demonstrated by neutrophilia, lymphocytosis, thrombocytosis, and erythrocytosis that was evident by increased myeloid, erythroid, and megakaryocyte progenitor cells cultured from bone marrow and spleen. These studies further demonstrate that lithium influences the disease process in the MAIDS model and restricts the development of hematopoietic suppression that develops in this retroviral animal model of immunodeficiency.

Murine acquired immunodeficiency syndrome (MAIDS) induced by defective LP-BM5 murine leukemia virus is a disease with many similarities to human AIDS. Previous studies indicated that the depressed hematopoiesis observed in LP-BM5-infected marrow cultures may be attributable to a defect of hematopoietic stroma. We report here the generation of permanent stromal cell lines from noninfected and LP-BM5-infected marrow cultures. Retrovirus infection was confirmed by polymerase chain reaction for viral genome. The ability of these cell lines to support in vitro hematopoiesis was studied. Results indicated that, when cocultured with normal or infected nonadherent mononuclear cells, noninfected cell lines efficiently supported the production of hematopoietic precursors, whereas viral-infected cell lines induced suppression of both normal and viral-infected progenitors. Expression of cytokine genes in stromal cell lines was also examined. All cell lines expressed equivalent levels of transcripts for stem cell factor and tumor necrosis factor alpha. However, infection was associated with higher levels of interleukin-4 and transforming growth factor beta 1 transcript expression. These findings suggest that infected stromal cell lines exhibit a defective hematopoietic microenvironment that produced altered cytokine expression resulting in faulty hematopoiesis. Further characterization of the defective cell lines should prove valuable for studies of the pathogenesis of murine AIDS.

The drug zidovudine (AZT), a synthetic thymidine analog, has been used in the treatment of acquired immunodeficiency syndrome (AIDS). Clinical use of zidovudine has been associated with the development of hematopoietic toxicity manifested by anemia, neutropenia, and on occasion thrombocytopenia. This toxicity has resulted in the development of alternative dideoxynucleoside drugs capable of exerting anti-viral potency while minimizing the risk for inducing organ toxicities. One such dideoxynucleoside drug is 2',3'-dideoxyinosine (ddI). Clinical trials are currently evaluating the effect of combination anti-viral drug treatment such as zidovudine plus ddI. We report here the results of studies designed to evaluate the effect of interleukin-3 (IL-3) on its ability to influence the hematopoietic toxicity associated with zidovudine and ddI following combination with retroviral-infected murine bone marrow cells. Toxicity was evaluated by quantitating several classes of hematopoietic progenitor stem cells such as granulocyte-macrophage (CFU-GM), erythroid (CFU-E and BFU-E) and megakaryocyte (CFU-Meg). Dose-escalation IL-3 provided protection of anti-viral drug induced suppression of progenitor cells when combined in the presence of the ID50 concentration of either zidovudine or ddI; however, when zidovudine and ddI were combined, IL-3 was less effective in providing protection against drug-induced toxicity at any concentration examined. These results indicate that IL-3 is effective in reducing anti-viral drug-induced hematopoietic toxicity associated with single-agent use; however, IL-3 is less effective when such drugs are used in combination.

Use of the anti-viral drug zidovudine in the treatment of acquired immunodeficiency syndrome (AIDS) has been associated with the development of hematopoietic toxicity. Several hematopoietic growth factors have been investigated in their ability to modulate such toxicity; however, no single factor has been demonstrated to produce restoration of hematopoiesis following use with zidovudine. We report results describing the effect of combination interleukin-1 (IL-1) and erythropoietin (Epo) in their ability to modulate the hematopoietic toxicity associated with dose-escalation zidovudine administered in normal mice. When administered over a six-week period, IL-1 and Epo raised the packed red cell volume, white blood cell and platelet counts in control mice and mice receiving dose-escalation zidovudine. These effects were attributed in part to the ability of combination IL-1 and Epo to increase erythroid, myeloid and megakaryocyte progenitor stem cells from bone marrow and spleen. These results indicate that use of combined IL-1 and Epo may be efficacious in ameliorating the hematopoietic toxicity associated with the use of zidovudine.

Zidovudine (AZT) has been the drug of choice in the treatment of human AIDS; however, associated with the use of zidovudine has been the development of hematopoietic toxicity, the mechanism of which is not clearly defined. We report here studies designed to evaluate dose-escalation of zidovudine, i.e. 0.1 and 1.0 mg/ml placed in the drinking water on hematopoiesis in C57BL/6 normal and LP-BM5 immunodeficiency virus-infected mice. Over a 6-week evaluation period, compared to normal, non-virus-infected controls, murine immunodeficiency (MAIDS) infection was associated with reduced hematopoietic progenitors, i.e. CFU-E, BFU-E, CFU-GM, and CFU-Meg from bone marrow and spleen. Following zidovudine treatment, further suppression of marrow-derived progenitors was observed, while increased numbers of progenitors were obtained from the spleen. Spleen-derived erythroid progenitors, i.e. CFU-E, were increased by 950% (P < 0.001) from MAIDS-infected animals receiving 1.0 mg/ml of drug following 4-weeks exposure compared to non-drug-treated MAIDS control animals. Splenic BFU-E were increased 654% following 6-weeks exposure compared to non-drug-treated MAIDS-infected mice. This study suggests that the bone marrow is particularly sensitive to zidovudine toxicity which, at least early in exposure, appears to be compensated by splenic-derived hematopoiesis, in particular, erythropoiesis. Overt toxicity develops when, at least in this immunodeficiency model, the spleen is unable to provide progenitors in response to continued zidovudine exposure in vivo.

Lithium is an agent capable of influencing many aspects of blood cell production, in particular, the formation of granulocytes. Because of this property, lithium has been demonstrated to be an effective agent whenever granulocyte production is either faulty or inadequate. The anti-viral drug zidovudine (AZT) has used been extensively in the treatment of acquired immune deficiency syndrome (AIDS). However, its effectiveness is limited because of the myelosuppression and bone marrow toxicity associated with its use. We have previously demonstrated that lithium, when combined with AZT in vitro with normal bone marrow cells or when administered in vivo to mice receiving dose-escalation AZT, reduced the myelosuppression and marrow toxicity of AZT significantly. We report here further studies designed to evaluate the extent of lithium's capacity to modulate AZT toxicity by investigating the ability of lithium to influence blood cell production when administered to normal mice during an initial exposure to AZT. C57BL6 were administered dose-escalation AZT (1.0 mg/ml and 2.5 mg/ml) for a period of 4-weeks in the presence or absence of lithium carbonate (1 mM). This was followed by an additional 4-week period during which mice received only AZT. Animals were analyzed on a weekly basis for their peripheral blood indices. Animals receiving dose-escalation AZT demonstrated anemia, thrombocytopenia, and neutropenia which was dose-related. During the period when animals received combination lithium/AZT, there was significantly less anemia, thrombocytopenia, and neutropenia as compared to the AZT controls.(ABSTRACT TRUNCATED AT 250 WORDS)

We report here the results of studies examining the ability of zidovudine (AZT) to influence the establishment and maintenance of long-term marrow cultures (LTMC) using marrow from murine immunodeficient mice (MAIDS). Normal C57BL6 mice were infected with LP-BM5 (MuLV) immunodeficiency virus (10 micrograms total protein) intraperitoneally. Five weeks after viral infection, mice were sacrificed and marrow was harvested from normal non-virus-infected and virus-infected animals. LTMC were established in the presence or absence of dose escalation of AZT, that is, 10(-6), 5 x 10(-7), and 10(-7) M in vitro. Compared with controls prepared from normal bone marrow, LTMC using MAIDS-infected marrow failed to establish and subsequently release supernatant-derived mononuclear cells. The addition of AZT was ineffective in either establishing LTMC or consistently producing mononuclear cells. Measurements of erythroid (BFU-E), myeloid (CFU-GM), and megakaryocyte (CFU-Meg) precursors were all depressed and none were observed after 5 weeks of culture. Treatment with AZT failed to reverse this depression of stem cell progenitors. Microscopic examination of cultures at 10 weeks demonstrated a failure of MAIDS-LTMC to establish an adequate stromal layer compared to LTMC prepared form non-virus-infected controls. This data indicate that LP-BM5 MuLV infection alters the establishment of a normal functioning hematopoietic microenvironment or stroma. Acknowledging that important differences between MAIDS and human AIDS exist, the implications of these findings concerning the establishment of the immunodeficiency disease state in human immunodeficiency virus infection is discussed.

We studied the effect of interleukin-1 (IL-1 alpha) either alone or administered with GM-CSF on the induction of hematopoietic toxicity associated with zidovudine (AZT) in vivo, as determined by peripheral blood indices, and assays of hematopoietic progenitors, i.e., erythroid (BFU-E), myeloid (CFU-GM), and megakaryocyte (CFU-Meg) cultured from bone marrow and spleen. Previous results reported from this laboratory have established dose-escalation of zidovudine to normal mice induced a dose-dependent decrease in hematocrit, WBC, and platelets with altered populations of marrow and splenic erythroid, myeloid and megakaryocyte progenitors. Daily administration of IL-1 alpha (recombinant murine, 5 u/animal) with or without GM-CSF (recombinant murine (10 micrograms/kg/bw) was associated with reduced AZT-toxicity as measured by increases in peripheral blood indices and progenitor stem cells, i.e., CFU-GM, CFU-Meg and BFU-E cultured from either bone marrow and spleen. The presence of GM-CSF amplified the effect observed with IL-1 especially with respect to myelopoiesis. These results demonstrate IL-1 with or without GM-CSF reverses AZT-hematopoietic toxicity when used in vivo.

We studied the long-term effect of continued zidovudine exposure in mice on hematopoiesis, as determined by peripheral blood indices, assays of erythroid (colony-forming unit-erythroid [CFU-E] and burst-forming unit-erythroid [BFU-E]), myeloid (CFU-granulocyte-macrophage [GM]), megakaryocyte (CFU-Meg), and plasma titers of erythropoietin, granulocyte-macrophage colony-stimulating factor, megakaryocyte colony-stimulating factor, and tumor necrosis factor-alpha. Dose-escalation of zidovudine (0.1, 1.0, and 2.5 mg/ml) induced a dose-dependent decrease in hematocrit, white blood cells, and platelets. High-dose drug, i.e., greater than 1.0 mg/ml, reduced marrow CFU-E; splenic CFU-E was increased after 1 week, then declined. BFU-E was increased at Weeks 1 and 2, then declined to control levels. Splenic BFU-E rose during the examination period that was dose-dependent. Femoral CFU-GM was cyclic, i.e., low-dose drug, 0.1 mg/ml, was increased gradually, the declined; higher doses of 1.0 and 2.5 mg/ml were lower until Week 5, then were above controls. Splenic CFU-GM was increased initially at Week 2 (1.0 mg/ml), then declined; the higher dose (2.5 mg/ml) increased initially, then declined below controls (Week 6). Femoral CFU-Meg was increased after low-dose drug and inhibited after high dose (2.5 mg/ml). Splenic CFU-Meg was reduced initially, followed by an increase at Week 4. Plasma titer of erythropoietin was elevated, proportional to dose escalation of drug, and inversely proportional to the hematocrit. No difference was observed in plasma levels of granulocyte-macrophage colony-stimulating factor, megakaryocyte colony-stimulating factor, or tumor necrosis factor-alpha. This study demonstrates that zidovudine-induced anemia results from: (i) inadequate numbers of bone marrow-derived, erythropoietin-dependent hematopoietic progenitors, i.e., CFU-E; and (ii) a shift in erythropoietin-responsive progenitors from bone marrow to spleen capable of responding to obligatory growth factors.

Bone marrow transplantation (BMT) has become a widely used procedure in the treatment of numerous hematological and non-hematological clinical disorders. The preparative regimen used for marrow recipients is not without risk as the immunodeficient recipient is susceptible to life-threatening infections due to the inability of the marrow to engraft properly. The monovalent cation lithium has been demonstrated to influence regenerating hematopoiesis following the use of several agents known to suppress hematopoiesis. The following report summarizes our studies which have been designed to determine the rate of hematopoietic reconstitution in lethally irradiated mice that were transplanted with bone marrow cells, harvested from either syngeneic or allogeneic donor animals, treated with lithium or phosphate buffered saline (PBS). Transplanted recipients receiving marrow cells from lithium treated donors were evaluated for their survival, peripheral blood indices and several classes of hematopoietic progenitors (granulocyte, erythroid, and megakaryocyte). Transplanted animals that received marrow cells from either syngeneic or allogeneic donors treated with lithium demonstrated greater survival, increased recovery of peripheral indices and hematopoietic progenitors compared to PBS-treated controls. These results indicate that the use of lithium to treat the donor may be an effective procedure to enhance hematopoietic recovery and engraftment in the transplanted recipient. Because of its wide-ranging effects, the use of lithium as a single agent, may be more efficacious than administering several hematopoietic growth factors in order to achieve a similar response.

The drug 3'-azido-3'-deoxythymidine (AZT), a synthetic thymidine analogue, has been used clinically in the management of acquired immune deficiency syndrome (AIDS). The drug is an effective antiviral agent due to its ability to block reverse transcriptase activity. This action of AZT was demonstrated in the Rauscher leukemia virus (RLV)-induced murine erythroleukemia model system. Unfortunately, associated with AZT has been the development of hematopoietic toxicity manifested by anemia, neutropenia, and overall bone marrow suppression. Hematopoietic growth factors (GM-CSF, erythropoietin), cytokines (interleukin-1), and agents known to potentiate hematopoiesis (lithium) have been demonstrated to modulate drug and/or radiation-induced hematopoietic toxicity. We report the results of further studies designed to investigate the ability of GM-CSF, erythropoietin, interleukin-1, and lithium to modulate AZT toxicity on murine hematopoietic granulocyte-macrophage (CFU-GM), megakaryocytic (CFU-Meg), and erythroid (BFU-E) progenitors cultured from bone marrow and spleen cells from mice infected with RLV. Hematopoietic progenitors from either normal or RLV-infected animals when exposed to AZT demonstrated concentration-dependent toxicity and differed for each progenitor with BFU-E being the most sensitive (ID50 concentration, 5 x 10(-9) M) and CFU-GM the least sensitive (ID50 concentration, 5 x 10(-5) M). As has been previously demonstrated using normal murine hematopoietic progenitors, when cultured with RLV-infected marrow or spleen cells, addition of GM-CSF, Meg-CSF or erythropoietin failed to inhibit AZT toxicity in vitro on CFU-GM, CFU-Meg, and BFU-E, respectively. However, in the presence of interleukin-1 (recombinant human IL-1 alpha, 30 ngm) or lithium chloride (ultra-pure, 1.0 mM), AZT toxicity CFU-GM, CFU-Meg, and BFU-E cultured from RLV-infected marrow or spleen cells was reduced. These results further demonstrate interleukin-1 and lithium are effective in modulating the toxic action of AZT on hematopoietic progenitors and that RLV-infected animals serve as a useful viral model system to study the effect of agents capable of modulating hematopoiesis in the presence of the anti-viral drug AZT.