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Rui Sun,1 Ruth A. Gault,2 Lisbeth A. Welniak,2 Zhigang G. Tian,1 Susan Richards,3 ... University of Nevada–Reno, Reno, Nevada; 3Genzyme Corporation, ...
Biology of Blood and Marrow Transplantation 9:426-434 (2003) 䊚 2003 American Society for Blood and Marrow Transplantation 1083-8791/03/0907-0002$30.00/0 doi:10.1016/S1083-8791(03)00107-1

Immunologic and Hematopoietic Effects of Recombinant Human Prolactin after Syngeneic Bone Marrow Transplantation in Mice Rui Sun,1 Ruth A. Gault,2 Lisbeth A. Welniak,2 Zhigang G. Tian,1 Susan Richards,3 William J. Murphy2 1

School of Life Sciences, University of Sciences and Technology of China, Anhui, China; 2School of Medicine, University of Nevada–Reno, Reno, Nevada; 3Genzyme Corporation, Framingham, Massachusetts

Correspondence and reprint requests: William J. Murphy, PhD, Department of Microbiology MS320, School of Medicine, University of Nevada–Reno, Reno, NV 89557 (e-mail: [email protected]). Received February 13, 2003; accepted March 24, 2003

ABSTRACT The period of immune deficiency following bone marrow transplantation (BMT) results in a susceptibility to opportunistic infections and remains a growing obstacle in improving the efficacy of BMT. Neuroendocrine hormones have been shown to affect numerous immunologic and hematologic responses after in vivo administration. We investigated whether neuroendocrine hormones, notably prolactin (PRL), could be administered after BMT and result in improved immunologic recovery. Mice were given lethal total body irradiation followed with a congeneic or a syngeneic BMT. Some groups then received recombinant human PRL (rhPRL) daily for 3 weeks. Effects on immune reconstitution and function were then monitored. The results show that PRL could increase thymic cellularity and donor T-cell reconstitution after congeneic BMT. Increases in B cells and myeloid progenitors were also observed. Mitogenic responses by both T and B cells were observed after PRL treatment. These results suggest that PRL may be of use to promote immune and myeloid reconstitution after BMT. © 2003 American Society for Blood and Marrow Transplantation

KEY WORDS Prolactin ● Hematopoiesis Neuroendocrine hormones

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Bone marrow transplantation

INTRODUCTION Autologous bone marrow transplantation (BMT) is currently used in the treatment of a variety of neoplastic diseases. However, there are several problems associated with autologous BMT (ABMT). The period of bone marrow aplasia leaves patients at risk for opportunistic infections, and the immunosuppression caused by the cytotoxic therapy results in a greater risk of tumor recurrence [1]. Cytokines such as granulocyte colony-stimulating factor (G-CSF) and granulocyte-monocyte colony-stimulating factor (GM-CSF) are currently used to accelerate neutrophil recovery post-BMT [2,3]. However, systematic administration of these cytokines are associated with significant toxicities including flu-like symptom, bone pain, fatigue, and microvascular leaking syndrome [4,5]. Importantly, these cytokines are not associated 426

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with the promotion of immunologic recovery. Therefore, agents need to be found that may be used alone or in combination with G-CSF or GM-CSF for accelerating the hematopoietic and immunologic reconstitution to decrease the dosage or to be used in place of G-CSF or GM-CSF for shortening the period of bone marrow aplasia and immunodeficiency after ABMT. It is known that differentiation and development of various blood cell lineages from hematopoietic progenitor cells and coordination of host immune response are regulated by a group of cytokines. The receptors for these factors have been grouped in a large family of structural-related molecules including IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, GM-CSF, G-CSF, M-CSF, erythropoietin (Epo), prolactin (PRL), and growth hormone (GH), which bind to distinct but

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related receptors [6]. Because of the structural similarities between adenopituitary hormones (GH and PRL) and conventional hematopoietic cytokines, GH and PRL had been shown to exert many immunohematopoietic-promoting effects [7-9]. Specific depression of PRL release induced by bromocriptin is associated with decreased T-cell function and the response of T cells in vitro is depressed in the presence of anti-PRL antibodies [10]. Prolactin increased the proliferating response of NK, T cells, and B cells to mitogen. We have found that PRL administration increased the antigen-specific proliferation of lymph node T cells of both normal and dwarf mice [8] as well as reversed the anemia and myelosuppression induced by azidothymidine [11]. Here, we wanted to further address the effects of PRL treatment on hematopoietic and immunologic recovery following syngeneic or congenic BMT (SBMT) in mice, affecting multiple hematopoietic lineages at various stages of differentiation. Therefore, recombinant human PRL (rhPRL) may be of potential clinical use after high-dose chemotherapy and ABMT to promote hematopoietic recovery. MATERIALS AND METHODS Mice

BALB/c, C57BL/6 (Ly5.1) mice, and C57BL/6 (Ly5.2) mice were obtained from the Animal Production Area (National Cancer Institute at Frederick, Frederick, MD). Mice were 8 to 12 weeks of age at the initiation of treatment. Animal care was provided in accordance with the procedures outlined in the “Guide for the Care and Use of Laboratory Animals” (National Institutes of Health Publication No. 86-23, 1985). Bone Marrow Transplantation

Recipient BALB/c mice received 850 cGy and C57BL/6-Ly5.1 mice received 950 cGy total body irradiation from a 137Cs irradiation source. BALB/c mice then received 1 ⫻ 106 syngeneic bone marrow cells and C57BL/6 mice received 1 ⫻ 106 C57BL/6Ly5.2 congenic bone marrow cells intravenously (IV). Experiments consisted of 4 to 5 mice per group at each time point and each experiment was performed 3 to 4 times. Hormone Treatment

BALB/c mice received 10 ␮g rhPRL/0.2 mL (Genzyme Corporation, Framingham, MA) in Dulbecco’s phosphate buffered saline (DPBS) by intraperitoneal injection 3 times per week for 3 weeks beginning at day 0 after BMT. C57BL/6 mice received 50 ␮g rhPRL/0.2 mL in DPBS 5 times per week for 3 weeks beginning at day 0 after BMT. Mice

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were weighed weekly. Immunologic and myeloid recovery was assessed at days 7, 14, 28, and 42 postBMT. The cells of the thymus, spleen, and femur, were analyzed for cellularity, hematopoietic progenitor assays, mitogen assays, and flow cytometric analysis for immune markers. Cellularity Analysis

Cell suspensions were prepared in RPMI-1640 media containing 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mmol/L L-glutamine. Bone marrow cells were flushed from the femurs and singlecell suspensions were obtained using a syringe and 22-gauge needle. Single-cell suspensions were prepared from the spleens and thymi by mechanical dissociation. The cellularities of bone marrow and spleens were assayed using a particle counter (Beckman Coulter, Hialeah, FL). Hematopoietic Progenitor Colony Assays

Spleen cells and bone marrow cells (BMCs) were washed and suspended in Iscove’s modified Dulbecco’s medium with 15% FBS, 2 mmol/L L-glutamine, 5 ⫻ 10-5 M 2-mercaptoethanol, 100 U penicillin/mL, and 100 ␮g streptomycin/mL. Colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-e) were determined by plating cells in 1.1% methylcellulose (Fisher Scientific) in triplicate 35-mm petri dishes at a concentration of 1 ⫻ 105 spleen cells or 5 ⫻ 104 BMC per plate. Colony formation was stimulated with predetermined doses of cytokines including 10 ng/mL each of recombinant murine GM-CSF (rmGM-CSF) and interleukin-3 (rmIL-3) (Peprotech, Rocky Hill, NJ), and 5 U/mL Epo (Stem Cell Technologies, British Columbia, Canada). Plates were incubated at 37°C for 7 days in 5% CO2with 100% humidity. Colonies were defined as aggregates of more than 50 cells. Colonies that contained only red cells in single or multiple bursts were scored as BFU-e. Colonies consisting of only white cells containing granulocytes and/or macrophages were scored as CFU-GM. Flow Cytometry Analysis

Thymic, splenic, and bone marrow single-cell suspensions were individually prepared and analyzed by double-color flow cytometry analysis. Reagents used included fluorescein isothiocyanate–labeled anti-CD4 (RM4-5), ⫺GR-1 (RB6-8C5), ⫺CD45.1 (Ly5.2; A20), phycoerythrin-labeled anti-CD8 (53-67), B220 (RA36B2), ⫺CD11b (M1/70), biotin-anti–CD4 (RM4-5), streptavidin-cychrome obtained from PharmingenBecton Dickinson (Mountain View, CA), and fluorescein isothiocyanate– goat anti-mouse IgM (Southern Technologies Associates, Birmingham, AL). 1 ⫻ 106cells were resuspended in staining buffer (DPBS 427

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containing 5% FBS) and incubated with antibody for 30 minutes at 4°C. After washing with staining buffer, cells were fixed in 1% paraformaldehyde and analyzed on a FacScan flow cytometer (Becton-Dickenson). Each fluorescence study included directly labeled isotype controls of the appropriate rat immunoglobulin isotype. Splenic Mitogen Proliferation Assays

Splenocytes (1 ⫻ 106/200 ␮L) per well were plated in 96-well flat-bottomed plates in triplicate and incubated at 37°C in 5% CO2 with 0.1 to 25 ␮g/mL of concanavalin A (ConA) or lipopolysaccharide (LPS) (Sigma, St. Louis, MO). After 3 days of culture at 37°C, the cultures were pulsed with 1 ␮Ci of 3Hthymidine (ICN, Aurora, OH) per well and incubated for an additional 12 hours. Cells were then harvested onto glass fiber filters and the incorporated radioactivity was quantified by a scintillation counter (Wallac LKB, Turku, Finland). Statistical Analysis

All experiments were performed 3 times with 4 to 5 mice per group. Data are presented as the mean ⫾ SEM. A Student’s t-test was performed to determine statistical significance of differences between experimental groups and control groups. RESULTS rhPRL Promotes Hematopoietic Progenitor Recovery in Mice after Syngenenic Bone Marrow Transplantation

To determine whether rhPRL could enhance hematopoietic engraftment and reconstitution following SBMT, BALB/c mice were injected with 10 ␮g of rhPRL 3 times per week for 3 weeks after myeloablative irradiation and BMC transfer. This dose and schedule of rhPRL administration has been shown to counteract the myelosuppressive activity of azidothymidine in mice [11]. Animals were assessed at weekly intervals for hematopoietic recovery. Toxicity was not observed at any level of rhPRL given in these studies. Administration of rhPRL did not significantly alter spleen and bone marrow cellularity in recovering mice with the exception of day 14 posttransplantation for both tissues and day 28 in the spleen (data not shown). rhPRL administration did result in significant increases in bone marrow hematopoietic progenitor cell, including myeloid (CFU-GM) and erythroid (BFU-e) progenitor content at various time-points after SBMT compared with the vehicle control treated group (Figure 1). The CFU-GM content of bone marrow significantly increased 1.9-fold at day 21 (P ⬍ .05) after SBMT (Figure 1A). Femoral BFU-e were significantly increased at days 21 (P ⬍ .001), 28 (P ⬍ .05), and 42 428

Figure 1. Effects of rhPRL on bone marrow hematopoietic progenitor content after syngeneic bone marrow transplantation. BALB/c mice received 850 cGy and 1 ⫻ 106 syngeneic BMC IV. The mice then received PBS or 10 ␮g rhPRL 3 times per week IP for 3 weeks. Hematopoietic progenitor content was analyzed at various time-points post-SBMT. Progenitor cell CFU-GM (A) and BFU-e (B) assays were performed. Colonies per femur were calculated from the colony frequency and BM cellularity. There were 4 mice for per group at each time point. Data shown are from 3 separate experiments. * ⫽ P ⬍ .05; ** ⫽ P ⬍ .01; *** ⫽ P ⬍ .001 compared with PBS-treated controls.

(P ⬍ .001) post-SBMT by 1.6- to 3.2-fold compared with PBS control-treated mice at the various time points (Figure 1B). Splenic hematopoietic progenitor cell content was significantly increased in mice receiving rhPRL at day 7 (P ⬎ .05) to day 42 (P⬍ .01) (CFU-GM), and day 7 (P ⬍ .05), 21 (P ⬍ .01), 28 (P ⬍ .001), and 42 (P ⬍ .01) (BFU-e) after SBMT (Figure 2). In both the rhPRL and PBS control-treated animals, peak CFU-GM (Figure 2A) and BFU-e (Figure 2B) recovery in spleen occurred on day 14 postBMT. Thus, the results indicated that treatment with rhPRL promoted BMC engraftment through improved development and expansion of hematopoietic progenitor cells. rhPRL Improves B-Cell Lineage Development and Function

A role for PRL on immune function has been suggested, but hormonal effects have been difficult to

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marked drop off in B-cell production as homeostasis is achieved. B-cell content in the spleen was significantly (P ⬍ .05) increased in PRL-treated mice compared with PBS treated mice (Figure 4A). Splenic IgM⫹ B cells were significantly increased at day 21 (P ⬍ .05) and day 28 (P ⬍ .01). The absolute number of IgM⫹ B cells in the spleen increased 1.4 fold at both time points in rhPRL treated mice, respectively, compared with PBS-treated animals (Figure 4B). IgM⫹ B cells peak at day 28 in both treatment groups. The results suggest that rhPRL accelerates B-cell engraftment and differentiation. We also tested B-cell function as measured by splenocyte response to the mitogen LPS. C57BL/6-Ly5.1 mice received 950

Figure 2. Effects of rhPRL on splenic hematopoietic progenitor after syngeneic bone marrow transplantation. BALB/c mice received 850 cGy followed by infusion of 1 million syngeneic BMC IV. The mice then received PBS or 10 ␮g rhPRL 3 times per week IP for 3 weeks. Mice were euthanized at various time-points post SBMT. Progenitor cell CFU-GM (A) and BFU-e (B) assays were performed. Colonies per spleen were calculated from the colony frequency and splenic cellularity. There were 4 mice for per group at each time point. Data shown are from 3 separate experiments. * ⫽ P ⬍ .05; ** ⫽ P ⬍ .01; *** ⫽ P ⬍ .001 compared with PBS-treated controls.

demonstrate with isolated cells and there are little data showing rhPRL effects on B-cell development and function in vivo. There has been a recent report that PRL could affect B-cell function [12]. We focused on the content of pre-B cells (B220⫹ IgM⫺) and B cells (B220⫹ IgM⫹) in the bone marrow and spleen after rhPRL treatment following SBMT. The results show that pre-B cells in the bone marrow are significantly increased in rhPRL treatment mice after SBMT at day 21 (Figure 3A). The absolute number of pre-B cells in the bone marrow increased 1.6 fold. It was also noted that B cells (B220⫹/sIgM⫹ cells) were significantly increased in the bone marrow at day 28 (P ⬍ .05) after SBMT compared with PBS control (Figure 3B). Following BMT, the spleen is an extramedullary site for both B lymphopoesis and myelopoiesis. Pre-B cells peaks at day 14 posttransplantation in animals that received either rhPRL or PBS followed by a

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Figure 3. Effects of rhPRL on bone marrow B-cell progenitor and mature B-cell content after syngeneic bone marrow transplantation. BALB/c mice received 850 cGy followed by infusion of 1 million syngeneic BMC IV. The mice then received PBS or 10 ␮g rhPRL 3 times per week IP for 3 weeks. Mice were euthanized at day 21 and day 28. Bone marrow cells were labeled and analyzed with 2-color flow cytometry to determine (A) pre–B cells and (B) B cells. There were 4 mice for per group at each time point. Data shown are from 3 separate experiments. * ⫽ P ⬍ .05 compared with PBS-treated controls. 429

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Figure 4. Effects of rhPRL on splenic B-cell progenitors and mature B-cell content after syngeneic bone marrow transplantation. BALB/c mice received 850 cGy followed by infusion of 1 million syngeneic BMC IV. The mice then received PBS or 10 ␮g rhPRL 3 times per week IP for 3 weeks. Mice were euthanized at various time-points and spleen cells were labeled and analyzed with 2-color flow cytometry to determine (A) pre–B cells and (B) B cells. There were 4 mice for per group at each time point. Data shown are from 3 separate experiments. * ⫽ P ⬍ .05; ** ⫽ P ⬍ .01; compared with PBS-treated controls.

cGy total body irradiation followed by infusion of 1 million C57BL/6-Ly5.2 congenic bone marrow cells. The relative frequency of B cells (donor and host derived) did not differ significantly between rhPRL and control treated groups (data not shown). As shown in Figure 5, a significant (P ⬍ .01 at maximal peak of 10 ␮g/mL) dose-dependent enhancement of 3H-thymidine incorporation was observed in the splenocytes stimulated by LPS in mice at day 21 after rhPRL administration in SBMT. The data presented here show that PRL can improve the development and function of B-cell lineage from hematopoietic progenitor cells after BMT. rhPRL Improves T-Cell Lineage Development and Function

T-cell recovery in particular is critical after BMT in adults [13]. To determine if rhPRL administration 430

could enhance donor T-cell engraftment, we used a congenic BMT (CBMT). C57BL/6 Ly5.2 BMC were transferred into congenic Ly 5.1 mice. After total body irradiation, our initial studies in SBMT BALB/c mice and 10 ␮g rhPRL given 3 times per week showed minimal effects in T cells (data not shown). Therefore, the dose was increased to 50 ␮g 5 times per week. Thymus cellularity was significantly increased at day 14 (P ⬍ .05) and day 60 (P ⬍ .01) after CBMT (Figure 6). The donor-derived T-cell content of the spleen (CD4⫹ T cells) was also significantly increased at day 21 (P ⬍ .05) and day 28 (P ⬍ .05) after CBMT (Figure 7) and donor derived CD8⫹ T cells were significantly (P ⬍ .05) elevated in PRL treatment on day 21, suggesting that T-cell development after SBMT was accelerated by rhPRL administration. In addition, we examined the T-cell function in mice receiving rhPRL after CBMT. We found that, in addition to increased numbers of donor-derived T cells, the splenocytes from mice receiving rhPRL showed significantly (P ⬍ .001 at maximal peak of 1 ␮g/mL) greater proliferating response to the T-cell mitogen, concanavalin A, at day 21 posttransplantation (Figure 8). This difference was observed despite the observation that the relative frequency of CD3⫹ cells (donor and host derived) did not differ significantly between rhPRL and control treated groups (data not shown). These data verify that rhPRL may improve the development and function of T-cell lineage after CBMT. DISCUSSION Prolactin, initially described as a peptide hormone secreted by the anterior pituitary, has been shown to exert a variety of biological effects in vivo and has been

Figure 5. Effects of rhPRL on splenic B-cell mitogen responsiveness after congenic bone marrow transplantation. C57BL/6-Ly5.1 mice received 950 cGy total body irradiation followed by infusion of 1 million C57BL/6-Ly5.2 congenic bone marrow cells. The mice then received PBS or 50 ␮g rhPRL 5 times per week IP for 3 weeks. Mice were euthanized at day 21 and spleen cells proliferative response to LPS was measured as described in “Materials and Methods.” Data shown are from 3 separate experiments. * ⫽ P ⬍ .05; ** ⫽ P ⬍ .01 compared with PBS-treated controls.

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Figure 6. Effects of rhPRL on donor engraftment in thymus after congenic bone marrow transplantation. C57BL/6-Ly5.1 mice received 950 cGy total body irradiation. Then received 1 ⫻ 106congenic bone marrow cells from C57BL/6-Ly5.2 donor IV. The mice then received PBS or 50␮g rhPRL 5 times per week IP for 3 weeks. Thymuses were harvested at various time-points post-CBMT. Donor cell engraftment was calculated from the CD45.1⫹ cell frequency and cellularity. Mean engraftment and standard error was determined with 4 to 5 mice per group. Data shown are from 3 separate experiments. * ⫽ P ⬍ .05; ** ⫽ P ⬍ .01 compared with PBS-treated controls.

suggested to also exert effects on hematopoietic and immune cell types [8]. We previously reported that ovine PRL could augment peripheral immune function in pituitary hormone-deficient dwarf mice [14] and PRL administration increased numbers of hematopoietic progenitors in mice stressed by azidothymidine, a myelotoxic drug [11]. The data presented support and extend these findings. The results indicate that administration of rhPRL to mice after BMT enhances hematopoietic reconstitution as determined by increased hematopoietic progenitor cell content of bone marrow and spleen, and numbers of granulocytes, T-cell progenitors, and B-cell progenitors by flow cytometric phenotypic analysis, accelerated recovery of red blood cells in the peripheral blood, and improved splenic T-cell and B-cell mitogen responses. We have reported that GH and PRL exerted differential effects on murine T-cell development and function in neuroendocrine hormone-deficient dwarf mice [15], which have been reported to have deficiencies in T-cell development and function [16]. In contrast to the thymopoietic effects of GH, ovine PRL administration resulted in accelerated thymic atrophy in these mice [15]. Interestingly, PRL administration had no effect on thymic size in normal mice and thymic recovery was more obvious at the 50 ␮g PRL dose than the 10 ␮g dose, suggesting that lack of other hormones in the dwarf mouse made it susceptible to the inhibitory effects of PRL. We speculate that there are important differences in immunohematopoiesis between neuroendocrine hormone-deficient dwarf

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mice and lethally irradiated and reconstituting mice. Dwarf mice have low circulating levels of pituitary hormones because of the lack of acidophilic anterior cells, but lethally irradiated mice do produce these hormones; therefore, we believe multiple pituitary hormones may influence hematopoiesis. While the mechanism by which rhPRL promotes thymopoiesis in either of these models is not known, PRL receptors are found on both thymocytes [17] and thymic epithlelium [18]. Prolactin administration post-BMT may induce sustained changes in the thymic stroma, which result in enhanced and prolonged thymopoiesis. In addition to effects on thymus, we also found that PRL exerted effects on B-cell development and function in vivo after BMT. It is also important to note that the hematopoietic growth-promoting effects of rhPRL

Figure 7. Effects of rhPRL on splenic T-cell subsets after congenic bone marrow transplantation. C57BL/6-Ly5.1 mice received 950 cGy total body irradiation followed by infusion of 1 million C57BL/ 6-Ly5.2 congenic bone marrow cells. The mice then received PBS or 50 ␮g rhPRL 5 times per week IP for 3 weeks. Mice were euthanized at day 21 and day 28. Splenocytes were labeled and analyzed with triple-color flow cytometry to determine donor origin and T-cell subsets. There were 4 mice per group at each time point. * ⫽ P ⬍ .05 compared with PBS-treated controls. Data shown are from 3 separate experiments. 431

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Figure 8. Effects of rhPRL on splenic T-cell mitogen responsiveness after congenic bone marrow transplantation. C57BL/6-Ly5.1 mice received 950 cGy total body irradiation followed by infusion of 1 million C57BL/6-Ly5.2 congenic mice then received PBS or 50 ␮g rhPRL 5 times per week IP for 3 weeks. Mice were euthanized at day 21 and spleen cells proliferative response to ConA was measured, as described in “Materials and Methods.” Data shown are from 3 separate experiments. *P ⬍ .05; ***P ⬍ .001 compared with PBS-treated controls.

administration after SBMT occurred at a dose and schedule that did not result in toxicity or significant weight gain. It was reported that systemic administration of IL-11 after SBMT [19], administration of IL-6 following chemotherapy [20,21], or treatment with IL-12 also increases hematopoietic recovery [22,23]. In a clinical setting, systemic administration of these cytokines may produce toxic side effects [19-23]. Therefore, the advantage of using rhPRL in combination with other cytokines after ABMT may be of benefit in the reduction of toxicity after systemic administration. The mechanism by which rhPRL promotes hematopoietic and immune reconstitution after SBMT is not yet clear. The stimulatory effects on hematopoietic progenitor cells may be because of its ability to exert either direct proliferative effects on the progenitor cells or indirect effects by improving the responsiveness of the marrow cells to growthpromoting reagents. There is mixed evidence from in vitro experiments to support the suggestion that PRL acts directly on hematopoietic progenitor via PRL receptors [24,25]. The PRL receptor is expressed on lymphoid and hematopoietic tissue [26,27]. Engagement of PRL with its receptor on a very early population of hematopoietic progenitors promotes the expression of an Epo receptor and thus improves the responsiveness to Epo [24]. The PRL receptor rescues Epo-R⫺/⫺ erythroid progenitors and replaces Epo-R in a synergistic interaction with c-kit [28]. Prolactin improves human CD34⫹ colony development in the semi-solid agar colony assay system [24] and enhances erythropoiesis and myelopoiesis in vitro [25]. Increasing of colony for432

mation was observed with 25 to 50 ng/mL of PRL in vitro [29]. Formation of BFU-e colonies from GM-CSF, IL-3, and Epo-stimulated CD34⫹ progenitors was significantly inhibited by an anti-PRL antibody [25]. We report here that mice receiving rhPRL exhibited significant increases in hematopoietic progenitor cell (CFU-GM, CFU-Mix, and BFU-e) content and resulted in significant increases in red blood cells and granulocytes after SBMT. Results suggest that PRL affects multiple hematopoietic lineages at various stages in differentiation. It was recently reported that human peripheral blood granulocytes express PRL mRNA in vitro [30]. Macrophages and monocytes could mediate the effects of PRL on hematopoietic progenitors through secretion of cytokines and chemokines. The activities of macrophages are enhanced by PRL [31]. We observed that PRL increased the number of granulocytic and monocytic progenitors (CFUGM) in mice after SBMT; indirect feedback through monocytes may explain the indirect effects of PRL on hematopoiesis. We also found that PRL exerted effects on B-cell development and function, further confirming the results that expansion/differentiation of normal BM pro-B cells was stimulated by PRL in vitro and in vivo [32]. Human GH has also been shown to be capable of binding the murine PRL receptor [33], and some of the in vivo effects of rhGH may be caused by this binding capability. Additionally, it is known that many of the growth-promoting effects of rhGH are mediated by IGF-1. IGF-1 stimulates rat PRL gene expression [34]. Studies have determined that IGF-1 promotes hematopoiesis after in vivo administration [35]. Growth hormone has also been shown to exert these effects [7] and may exert these effects in a manner similar to PRL. It is unclear that the effects of PRL on immunologic and hematopoietic development are direct or indirect. Prolactin has been postulated to have significant effects on the immune system [36], but more work needs to be performed to determine the effect of rhPRL on long-term immune reconstitution after SBMT. When ABMT is used in the treatment of cancer, recurrence of the underlying malignancy remains a major cause of treatment failure. Therefore, caution must be taken if using rhPRL to promote hematopoietic recovery in treating a cancer patient with ASBMT, when ABMT is used for the treatment of cancer because of potential effects of rhPRL on tumor growth [37]. However, the present report suggests that the pleiotropic actions of rhPRL on various stages and lineages of hematopoietic cell growth after systematic administration may be of considerable use to promote hematopoietic recovery after myelosuppressive therapy, and to

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stimulate the function of immune competent cell as adjuvant immunotherapy. 14.

ACKNOWLEDGMENTS This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-12400. We wish to acknowledge Steve Stull for his excellent technical assistance.

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