Effects of prolactin on hematopoiesis.

The presence of extra-pituitary prolactin and its cognitive receptors in the hematopoietic micro-environment raises the question of whether prolactin plays a role in lympho-hematopoiesis and under what conditions. Current studies suggest that endogenous prolactin does not play a significant role under normal steady-state conditions. Rather, prolactin has been implicated as a 'stress hormone', functioning to restore hematopoietic homeostasis under conditions of dysregulation. The stress response of prolactin as well as its complex relationship with other hormones and factors has resulted in conflicting reports in the literature regarding prolactin's role in lympho-hematopoiesis. A review of this literature is provided as well as discussion of conditions under which lymphohematopoietic activity of prolactin may be evident.

The diverse role of chemokines in tumor progression: prospects for intervention (Review).

Chemokines, proteins chemotactic for leukocytes and non-leukocytes, have been intensively studied for their role in tumor growth and metastasis. Recent work has shown that particular chemokines may have multiple effects on tumors including promoting growth, angiogenesis, metastasis, and suppression of the immune response to cancer, while other chemokines inhibit tumor mediated angiogenesis and promote anti-tumor immune responses. Increasing biological evidence supports the hypothesis that tumor-generated chemokines provide more than simply angiogenic signals. Tumor-derived chemokines may potentially act as inhibitors of anti-tumor immune responses as well as autocrine growth factors for the tumor. The complexity and redundancy of tumor chemokine expression suggests that a single chemokine target for tumor therapy may not be appropriate. Indeed, multiple target therapy including blockade of tumor enhancing chemokines while delivering or inducing the secretion of anti-tumor chemokines is the approach that currently holds the most promise. The role of chemokines in tumor biology as well as various means of blocking chemokines in cancer models in order to develop successful therapeutic strategies will be discussed.

Augmentation of antitumor effects by NK cell inhibitory receptor blockade in vitro and in vivo.

Subsets of natural killer (NK) cells are characterized by the expression of inhibitory and/or stimulatory receptors specific for major histocompatibility complex (MHC) class I determinants. In mice, these include the Ly49 family of molecules. One mechanism by which tumor cells may evade NK cell killing is by expressing the appropriate MHC class I and binding inhibitory Ly49 receptors. Therefore, the question of whether blocking the interaction between the Ly49 inhibitory receptors on NK and MHC class I cells on tumor cells augments antitumor activity was investigated. Blockade of Ly49C and I inhibitory receptors using F(ab')(2) fragments of the 5E6 monoclonal antibody (mAb) resulted in increased cytotoxicity against syngeneic tumors and decreased tumor cell growth in vitro. The effect of 5E6 F(ab')(2) was specific for the MHC of the tumor, as the use of F(ab')(2) of the mAb against Ly49G2 failed to increase NK activity. Treatment of leukemia-bearing mice with 5E6 F(ab')(2) fragments or adoptive transfer of NK cells treated ex vivo with the F(ab')(2) resulted in significant increases in survival. These results demonstrate that blockade of NK inhibitory receptors enhances antitumor activity both in vitro and in vivo, suggesting that NK inhibitory receptors can be responsible for diminishing antitumor responses. Therefore, strategies to block inhibitory receptors may be of potential use in increasing the efficacy of immunotherapy. (Blood. 2001;97:3132-3137)

Thymic emigrants isolated by a new method possess unique phenotypic and functional properties.

T cells that emigrate from the thymus have primarily been studied in vivo using fluorescent dye injection of the thymus. This study examined the properties of thymocytes that emigrate from cultured thymic lobes in organ culture. Under these conditions, thymic emigrants displayed the expected phenotype, that of mature thymocytes expressing high levels of T-cell receptor (TCR-alphabeta) and either CD4 or CD8, and were observed to emigrate within 24 hours of positive selection. Emigration was inhibited by cytochalasin D, pertussis toxin, or Clostridium difficile toxin B, implicating an active motility process. Most of the surface markers on alphabeta-thymic emigrants (Thy1, CD44, CD69, CD25, leukocyte functional antigen-1, intercellular adhesion molecule-1, alpha(4)-integrin, alpha(5)-integrin, CD45, and CD28) were expressed at a surface density similar to that on mature intrathymic cells and peripheral splenic T cells. Heterogeneous expression of L-selectin and heat-stable antigen (HSA) suggested that subsets emerge from the thymus with a commitment to different migration patterns. The only marker on emigrants not found on either intrathymic cells or mature spleen T cells was CTLA-4, which could dampen the response of emigrants to peripheral antigens. Antigen responsivenes measured in vitro against allogeneic dendritic cells showed a proliferative response comparable to that of splenic T cells. In vivo, however, thymic emigrants failed to induce an acute graft-versus-host reaction in allogeneic severe combined immunodeficiency recipients. This suggests that a mechanism operating in vivo, perhaps tolerance or migration pattern, attenuates the response of emigrants against antigens that did not induce their deletion in the thymus.

Gastrointestinal cells of IL-7 receptor null mice exhibit increased sensitivity to irradiation.

IL-7 is a critical cytokine in the development of T and B cells but little is known about its activity on nonhematopoietic cells. An unexpected finding was noted in allogeneic bone marrow transplant studies using IL-7 receptor null (IL-7R alpha(-/-)) mice as recipients. These mice exhibited a significantly greater weight loss after total body irradiation compared with wild type, IL-7R alpha(+/+), mice. Pathological assessment indicated greater intestinal crypt damage in IL-7R alpha(-/-) recipients, suggesting these mice may be predisposed to gut destruction. Therefore, we determined the effect of the conditioning itself on the intestinal tract of these mice. IL-7R alpha(-/-) mice and IL-7R alpha(+/+) mice were irradiated and examined for lesions and apoptosis within the small intestine. In moribund animals, IL-7R alpha(-/-) mice had extensive damage in the small intestine, including marked ablation of the crypts and extreme shortening of villi following 1500 cGy total body irradiation. In contrast, by 8 days after irradiation, the small intestines of IL-7R alpha(+/+) mice had regenerated as distinguished by normal villus length and hyperplastic crypts. Following 750 cGy irradiation, IL-7R alpha(-/-) mice had a higher proportion of apoptotic cells in the crypts and an accompanying increase in the pro-apoptotic protein Bak was expressed in intestinal epithelial cells. These results demonstrate the increased radiosensitivity of intestinal stem cells within the crypts in IL-7R alpha(-/-) mice and a role for IL-7 in the protection of radiation-induced apoptosis in these same cells. This study describes a novel role of IL-7 in nonhematopoietic tissues.

Keratinocyte growth factor facilitates alloengraftment and ameliorates graft-versus-host disease in mice by a mechanism independent of repair of conditioning-induced tissue injury.

We have previously shown that pretreatment of mice with keratinocyte growth factor (KGF), an epithelial tissue repair factor, can ameliorate graft-versus-host disease (GVHD) after intensive chemoradiotherapeutic conditioning and allogeneic bone marrow transplantation (BMT). To determine whether this effect was dependent on a KGF-mediated mechanism affecting repair of conditioning-induced epithelial cell injury, we studied GVHD in the absence of conditioning using BALB/c severe combined immune-deficient (SCID) recipients given C57BL/6 T cells. KGF (5 mg/kg per day, subcutaneously) given either before or after T-cell transfer enhanced body weights and extended survival. KGF-treated recipients had elevated serum levels of the Th2 cytokine interleukin 13 (IL-13) on day 6 after T-cell transfer concomitant with reduced levels of the inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon gamma (IFN-gamma). A 3-day KGF pretreatment also depressed the secondary in vitro mixed lymphocyte response (MLR) of C57BL/6 splenocytes taken 7 days after in vivo alloimmunization with irradiated BALB/c spleen cells. To determine whether KGF would inhibit host-antidonor-mediated BM rejection, pan-T-cell-depleted BALB/c BM cells were infused into sublethally irradiated C57BL/6 mice and administered KGF either before or before and after BMT. Surprisingly, all KGF schedules tested actually resulted in enhanced alloengraftment. The presence of KGF receptor on donor antihost alloreactive T cells could not be detected by binding studies with radiolabeled KGF, reverse transcriptase-polymerase chain reaction, and Western blotting. Therefore, the mechanism of action of KGF on inhibiting T-cell-mediated immune effects may not be due to a direct effect of KGF on T cells. These studies demonstrate that KGF, by mechanisms independent of repair of conditioning-induced injury, has great potential as an anti-GVHD therapeutic agent with the added benefit of inhibiting the rejection of pan-T-cell-depleted donor BM allografts. (Blood. 2000;96:4350-4356)

Opposing roles of interferon-gamma on CD4+ T cell-mediated graft-versus-host disease: effects of conditioning.

Although alloreactive T cells are required for the induction of graft-versus-host disease (GVHD), other factors can influence outcome in murine models of the disease. Lethal total body irradiation (TBI) conditioning regimens followed by reconstitution with allogeneic lymphohematopoietic cells results in the generation of donor anti-host cytotoxic T lymphocyte (CTL)-mediated solid organ (gut, liver, skin) destruction. In contrast, donor anti-host CTL-mediated hematopoietic failure is the primary cause of morbidity following sublethal TBI. To determine the role of interferon (IFN)-gamma in graft-versus-host reactions against hematopoietic and solid organ targets, we used IFN-gamma knockout mice as donors in both lethal TBI and bone marrow transplantation (BMT) rescue and sublethal TBI models. In this report, we show that CD4+ T cells from IFN-gamma knockout (KO) mice resulted in accelerated GVHD after lethal TBI/BMT using a single major histocompatibility class II mismatch model. In marked contrast, the use of these same IFN-gamma KO CD4+ donor cells in combination with sublethal TBI significantly ameliorated GVHD-associated mortality. In these recipients, severe anemia, bone marrow aplasia, and intestinal lesions were observed in the presence but not the absence of donor-derived IFN-gamma. Administration of anti-IFN-gamma antibodies to sublethally irradiated recipients of wild-type donor cells confirmed the role of IFN-gamma depletion in CD4+ T cell-mediated GVHD. In conclusion, the extent of conditioning markedly affects the role of IFN-gamma in GVHD lesions mediated by CD4+ T cells. In models using sublethal TBI, the absence of IFN-gamma is protective from GVHD, whereas in lethal TBI situations, the loss is deleterious.

Adoptive cellular immunotherapy: NK cells and bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) has been increasingly used for the treatment of both neoplastic and non-neoplastic disorders. However, serious obstacles currently limit the efficacy and thus more extensive use of BMT. These obstacles include: graft-versus-host disease (GVHD), relapse from the original tumor, and susceptibility of patients to opportunistic infections due to the immunosuppressive effects of the conditioning regimen. Overcoming these obstacles is complicated by dual outcome of existing regimens; attempts to reduce GVHD by depleting T cells from the graft, result in increased rates of tumor relapse and failure of engraftment. On the other hand, efforts to increase graft-versus-tumor (GVT) effects of the transplant also promote GVHD. In this review, the use of natural killer (NK) cells to overcome some of these obstacles of allogeneic BMT is evaluated. Adoptive immunotherapy using NK cells after allogeneic BMT has several potential advantages. First, NK cells can promote hematopoiesis and therefore engraftment by production of hematopoietic growth factors. Second, NK cells have been shown to prevent the incidence and severity of GVHD. This has been shown to be at least partially due to TGF-beta, an immunosuppressive cytokine. Third, NK cells have been shown to augment numerous anti-tumor effects in animals after BMT suggesting a vital role of NK cells in mediating GVT effects. Finally, NK cells have been demonstrated to affect B cell recovery and function in mice. Therefore, understanding the mechanisms of beneficial effects of NK cells after BMT may lead to significant increases in the efficacy of this procedure.

Effects of growth hormone and prolactin on hematopoiesis.

The use of the neuroendocrine hormones growth hormone (GH) and prolactin (PRL) in preclinical models, demonstrating promotion of hematopoietic recovery and immune function, offers promise for several clinical situations. These hormones do not appear to produce the same extent of immune/hematopoietic effects when compared to conventional hematopoietic and immune stimulating cytokines (i.e. G-CSF or interleukin-2). However, their pleiotropic effects and limited toxicity after systemic administration makes them attractive to test in myeloablative situations. More work needs to be performed to understand the mechanism(s) of GH and PRL action, particularly with regard to hematopoietic progenitor cell expansion and differentiation both in normal and pathologic situations.

Prolactin exerts hematopoietic growth-promoting effects in vivo and partially counteracts myelosuppression by azidothymidine.

Prolactin (PRL) is a neuroendocrine hormone that influences immune and hematopoietic development. The mechanism of action of this hormone in vivo remains unclear; therefore, we assessed the effects of PRL on hematopoiesis in vivo and in vitro. Normal resting mice were treated with 0, 1, 10, or 100 microg of recombinant human prolactin (rhPRL) for 4 consecutive days and euthanized on the fifth day for analysis of myeloid and erythroid progenitors in the bone marrow and spleen. Both frequencies and absolute numbers of splenic colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-e) were significantly increased in mice receiving rhPRL compared to the controls that had received saline only. Bone marrow cellularities were not significantly affected by any dose of rhPRL, but the absolute numbers and frequencies of bone marrow CFU-GM and BFU-e were augmented by rhPRL. These results suggest that rhPRL can promote hematopoiesis in vivo. Because rhPRL augments myeloid development in vivo, we examined the potential of the hormone to reverse the anemia and myelosuppression induced by azidothymidine (AZT). Mice were given rhPRL injections concurrent with 2.5 mg/mL AZT in drinking water. rhPRL partially restored hematocrits in the animals after 2 weeks of treatment and increased CFU-GM and BFU-e in both spleens and bone marrow. The experiments with AZT and rhPRL support the conclusion that the hormone increases myeloid and erythroid progenitor numbers in vivo, and they suggest that the hormone is clinically useful in reversing myelosuppression induced by AZT or other myeloablative therapies.

Use of neuroendocrine hormones to promote reconstitution after bone marrow transplantation.

A survey of the previous literature and the data shown here indicate that neuroendocrine hormones such as growth hormone and prolactin may be of potential clinical use after bone marrow transplantation (BMT) to promote hematopoietic and immune recovery. The amounts of hormones used in our model do not promote weight gain suggesting that their lymphohematopoietic actions were independent of their anabolic effects. While the hormones may not produce the same extent of immune/hematopoietic effects when compared to conventional hematopoietic and immune stimulating cytokines (i.e. IL-2 or G-CSF), their pleiotropic effects and limited toxicity after systemic administration makes them attractive to test in the post-BMT setting. However, more work needs to be performed to understand the mechanism(s) of their action, particularly with regard to T-cell function and development.

Differential effects of the absence of interferon-gamma and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice.

Graft-versus-host disease (GVHD), in which immunocompetent donor cells attack the host, remains a major cause of morbidity after allogeneic bone marrow transplantation (BMT). To understand the role of cytokines in the pathobiology of GVHD, we used cytokine knockout (KO) mice as a source of donor T cells. Two different MHC-disparate strain combinations were examined: BALB/c (H2(d)) donors into lethally irradiated C57BL/6 (H2(b)) recipients or C57BL/6 (H2(b)) donors into B10.BR (H2(k)) recipients. Donor cells were from mice in which either the interferon-gamma (IFN-gamma) or the IL-4 gene was selectively disrupted to understand the role of these cytokines in acute GVHD. In both strain combinations the same pattern was noted with regard to GVHD onset and morbidity. All mice exhibited the classic signs of acute GVHD: weight loss with skin, gut, and liver pathology resulting in morbidity and mortality. Surprisingly, donor cells obtained from mice lacking IFN-gamma gave rise to accelerated morbidity from GVHD when compared with cells from wild-type control donors. Similar results were obtained using normal donors when neutralizing antibodies to IFN-gamma were administered immediately after the BMT. These results suggest that IFN-gamma plays a role in protection from acute GVHD. In marked contrast, cells obtained from IL-4 KO mice resulted in protection from GVHD compared with control donors. Splenocytes from IFN KO mice stimulated with a mitogen proliferated to a significantly greater extent and produced more IL-2 compared with splenocytes obtained from IL-4 KO or control mice. Additionally, there was increased IL-2 production in the spleens of mice undergoing GVHD using IFN-gamma KO donors. These results therefore indicate, with regard to the TH1/ TH2 cytokine paradigm, the absence of a TH1-type cytokine can be deleterious in acute GVHD, whereas absence of a TH2 cytokine can be protective.

Recombinant human growth hormone promotes hematopoietic reconstitution after syngeneic bone marrow transplantation in mice.

Recombinant human growth hormone (rhGH) was administered to mice after syngeneic bone marrow transplantation (BMT) to determine its effect on hematopoietic reconstitution. BALB/c mice were given 10 microg intraperitoneal injections of rhGH every other day for a total of 10 injections following syngeneic BMT. Mice that received rhGH exhibited significant increases in total hematopoietic progenitor cell content (colony-forming unit-culture) in both bone marrow and spleen. Erythroid cell progenitor content (burst-forming unit-erythroid) was also significantly increased after rhGH treatment. Analysis of peripheral blood indicated that administration of rhGH resulted in significant increases in the rate of white blood cell and platelet recovery. Granulocyte marker 8C5+ cells were also increased in the bone marrow and spleens of treated mice. Red blood cell, hematocrit, and hemoglobin levels were increased at all time points after rhGH treatment. No significant pathologic effects or weight gain were observed in mice receiving repeated injections of 10 microg rhGH. Thus, rhGH administration after syngeneic BMT promoted multilineage hematopoietic reconstitution and may be of clinical use for accelerating hematopoiesis after autologous BMT.

Expression of receptors for C5a anaphylatoxin (CD88) on human bronchial epithelial cells: enhancement of C5a-mediated release of IL-8 upon exposure to cigarette smoke.

Results are presented that demonstrate a heightened responsiveness of human bronchial epithelial cells (HBECs) toward the complement-derived anaphylatoxin C5a when these cells are exposed to cigarette smoke. This C5a response is possible because we show at both the protein and mRNA levels that HBECs constitutively express receptors for C5a (C5aR, CD88). Control (untreated) HBECs responded to C5a (50 nM) by releasing the proinflammatory cytokine IL-8 at low but significant levels. However, exposure of HBECs to 5% cigarette smoke extract (CSE) for at least 15 min resulted in an increase in the ability of an anti-human C5aR Ab to bind to the cell surface. CSE-treated HBECs responded in a dose-dependent fashion to human recombinant C5a and to a conformationally biased decapeptide agonist of C5a (YSFKPMPLaR) by releasing IL-8. The levels of IL-8 released in response to C5a were significantly greater in CSE-treated HBECs than in control HBECs. Moreover, this C5a-mediated release of IL-8 from CSE-treated HBECs was significantly reduced in the presence of the anti-human C5aR Ab. These results indicate that HBECs constitutively express C5aRs and that exposure to environmental irritants such as cigarette smoke modulates the expression and responsiveness of these C5aRs toward the C5a-mediated release of IL-8.