Activation-induced cellular accumulation of histamine in immature but not mature murine mast cells.

Mast cell activation involves the rapid release of inflammatory mediators, including histamine, from intracellular granules. The cells are capable of regranulation and multiple rounds of activation. The goal of this study was to determine if there are changes in the content of pre-formed mast cell mediators after a round of activation. After 24 h, the histamine content of bone marrow-derived mast cells (BMMC), but not that of peritoneal mast cells, exceeded the amount in resting cells. Accumulation of histamine in BMMC peaked at 72 h of activation, and returned toward preactivation levels by 96 h. The increase in histamine content was accompanied by an increase in the gene expression of histidine decarboxylase. No increases in beta hexosaminidase or murine mast cell protease-6 were observed. These findings indicate that BMMC respond to activation by increasing total cell-associated histamine content. This increase may be important to the response of these cells upon subsequent exposure to antigens.

Autocrine regulation of IL-12 receptor expression is independent of secondary IFN-gamma secretion and not restricted to T and NK cells.

The biological response to IL-12 is mediated through specific binding to a high affinity receptor complex composed of at least two subunits (designated IL-12Rbeta1 and IL-12Rbeta2) that are expressed on NK cells and activated T cells. The selective loss of IL-12Rbeta2 expression during Th2 T cell differentiation suggests that regulation of this receptor component may govern IL-12 responsiveness. In murine assays, down-regulation of IL-12Rbeta2 expression can be prevented by treatment with IFN-gamma, indicating that receptor expression and hence IL-12 responsiveness may be regulated, at least in part, by the local cytokine milieu. In this study, we report that cellular expression of both IL-12Rbeta1 and beta2 mRNA is increased in the lymph nodes of naive mice following systemic administration of murine rIL-12 (rmIL-12). Changes in IL-12R mRNA were associated with increased IFN-gamma secretion following ex vivo activation of lymph node cells with rmIL-12, indicating the presence of a functional receptor complex. Expression of IL-12R mRNA was not restricted to lymph node T cells, and its autocrine regulation was independent of secondary IFN-gamma secretion. Data from fractionated lymph node cells as well as rmIL-12-treated B cell-deficient mice suggest that IL-12-responsive B cells may represent an alternative cellular source for IFN-gamma production. However, the strength of the biological response to rmIL-12 is not governed solely by receptor expression, as rmIL-12-induced IFN-gamma secretion from cultured lymph node cells is accessory cell dependent and can be partially blocked by inhibition of B7 costimulation.

Dose and timing of interleukin (IL)-12 and timing and type of total-body irradiation: effects on graft-vs.-host disease inhibition and toxicity of exogenous IL-12 in murine bone marrow transplant recipients.

Paradoxically, a single injection of recombinant murine interleukin (IL)-12 on the day of bone marrow transplantation (BMT) inhibits graft-vs.-host disease (GVHD) while preserving graft-vs.-leukemia (GVL) effects in lethally irradiated mice receiving fully MHC-mismatched bone marrow and spleen cells. These protective effects are mediated by interferon (IFN)-gamma, whose early secretion is induced by IL-12 treatment. We investigated the relationship of IL-12 dose and timing of administration, as well as timing and type of total-body irradiation (TBI), with the ability of IL-12 to inhibit GVHD or mediate toxicity. The results show that a relatively low dose of IL-12 (as little as 50 U in a single injection) can mediate significant GVHD protection. The timing of IL-12 administration, however, is a critical factor. IL-12 administered 1 hour before BMT was most protective, but protection was still observed when it was administered 1-12 hours after BMT. Delaying IL-12 administration to 36 hours post-BMT completely obviated its protective effect. Administration of a second IL-12 injection 6 days after BMT negated the protective effect of an initial injection at the time of BMT. While IL-12 protection was evident when TBI was administered by 137Cs-irradiator in one or two fractions on day -1 or day 0, the use of an X-irradiator to deliver TBI on day -1 was associated with marked IL-12 toxicity. Whereas the protective effect of IL-12 against GVHD depended on donor-derived IFN-gamma, toxicity depended on the ability of host cells to produce IFN-gamma. Careful studies are warranted to test the effects of IL-12 in the context of BMT with various conditioning regimens in large animal preclinical models before this novel approach to GVHD protection can be applied clinically.

Additive effects of human recombinant interleukin-11 and granulocyte colony-stimulating factor in experimental gram-negative sepsis.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is widely used to promote granulocyte recovery from a variety of pathologic states. Recombinant human interleukin-11 (rhIL-11) has recently become available clinically as a platelet restorative agent after myelosuppressive chemotherapy. Preclinical data has shown that rhIL-11 limits mucosal injury after chemotherapy and attenuates the proinflammatory cytokine response. The potential efficacy of combination therapy with recombinant human forms of rhIL-11 and rhG-CSF was studied in a neutropenic rat model of Pseudomonas aeruginosa sepsis. At the onset of neutropenia, animals were randomly assigned to receive either rhG-CSF at a dose of 200 micrograms/kg subcutaneously every 24 hours for 7 days; rhIL-11 at 200 micrograms/kg subcutaneously every 24 hours for 7 days; the combination of both rhG-CSF and rhIL-11; or saline control. Animals were orally colonized with Pseudomonas aeruginosa 12.4.4 and then given a myelosuppressive dose of cyclophosphamide. rhG-CSF resulted in a slight increase in absolute neutrophil counts (ANC), but did not provide a survival advantage (0 of 12, 0% survival) compared with the placebo group (1 of 12, 8% survival). rhIL-11 was partially protective (4 of 10, 40% survival); the combination of rhG-CSF and rhIL-11 resulted in a survival rate of 80% (16 of 20; P <.001). rhIL-11 alone or in combination with rhG-CSF resulted in preservation of gastrointestinal mucosal integrity (P <.001), lower circulating endotoxin levels (P <.01), and reduced quantitative levels of P. aeruginosa in quantitative organ cultures. These results indicate that the combination of rhIL-11 and rhG-CSF is additive as a treatment strategy in the prevention and treatment of experimental Gram-negative sepsis in immunocompromised animals. This combination may prove to be efficacious in the prevention of severe sepsis in neutropenic patients.

Regulation of the inflammatory response in animal models of multiple sclerosis by interleukin-12.

Interleukin 12 (IL-12), a novel heterodimeric protein produced primarily by antigen-presenting cells, serves as a key regulator of innate and adaptive immune responses. In addition to being a potent inducer of IFN-gamma, IL-12 is widely considered to be the principal cytokine that regulates the generation of Th1 type effector cells. As the successful induction of experimental autoimmune encephalomyelitis (EAE) is associated with a strong Th1 type cellular response, we have evaluated the role of IL-12 in regulating the pathogenesis of EAE in SJL/J mice and Lewis rats. In both settings, treatment with IL-12 was found to accelerate the onset and increase the severity and duration of clinical disease. More importantly, administration of IL-12 to Lewis rats that had recovered from primary disease was found to trigger clinical relapse. In all instances, IL-12-induced exacerbation was associated with a profound increase in iNOS positive macrophages within the perivascular lesions. Although IL-12-induced IFN-gamma does not appear to be required for exacerbation of disease, neutralizing antibodies against murine IL-12 delay the onset and reduce the severity of adoptively transferred EAE, indicating a role for endogenous IL-12 as regulator of disease. Based on the above findings, effective inhibition of IL-12 in vivo may have great therapeutic value in the treatment of MS and other Th1-associated inflammatory disorders.

Immunoregulation by interleukin-12 in MB49.1 tumor-bearing mice: cellular and cytokine-mediated effector mechanisms.

Administration of recombinant murine interleukin (rmIL)-12 to MB49.1 tumor-bearing mice results in dose-dependent regression of the primary tumor and the generation of protective antitumor immunity in the majority of animals. rmIL-12 administration is associated with a marked increase in lymph node cellularity that is predominantly due to the expansion of B220+ B cells as well as CD8+ T cells. Stimulation of lymph node cells from rmIL-12-treated, but not control tumor-bearing mice, with MB49.1 tumor cells in vitro was shown to enhance the secretion of interferon (IFN)-gamma. The magnitude of this in vitro response was dependent on the dose of rmIL-12 administered in vivo and mirrored the change in circulating serum IFN-gamma. Furthermore, at the height of the in vitro response to tumor stimulation, the addition of a neutralizing antibody to murine IL-12 suppressed IFN-gamma production, indicating a role for endogenous IL-12 in this antigen-specific cytokine response. Although studies in SCID mice confirmed that an appropriate T cell response was required for rmIL-12-mediated antitumor activity, in immunocompetent animals early tumor regression was not accompanied by cellular infiltration of the tumor. In contrast, a profound increase in tumor-associated inducible nitric oxide synthase (iNOS) was observed in mice receiving rmIL-12 which preceded T cell infiltration of the tumor which could be detected during the second week of IL-12 treatment. Direct tumor killing through the cytotoxic actions of NO via the iNOS pathway may serve as a way of generating tumor antigen which enables the host to mount a subsequent T cell response against the tumor.

Interleukin-11.

Interleukin-11 (IL-11) is a cytokine which interacts with a variety of haemopoietic and non-haemopoietic cell types. Recombinant human IL-11 (rhIL-11; oprelvekin) is produced in Escherichia coli and differs from the naturally occurring protein only in the absence of the amino-terminal proline residue. In synergy with other factors, rhIL-11 stimulates the growth of myeloid, erythroid, and megakaryocyte progenitor cells in vitro. In vivo, rhIL-11 is active in mice, rats, dogs, guinea pigs, hamsters and non-human primates, where the principal activity measured was stimulation of megakaryocytopoiesis and thrombopoiesis. rhIL-11 has shown benefit in 2 clinical trials by significantly reducing severe chemotherapy-induced thrombocytopenia. In addition to its thrombopoietic activity, rhIL-11 has also shown activity in models of acute gastrointestinal mucosal damage. rhIL-11 enhanced survival in mice following cytoablative therapy and in a hamster model of chemotherapy-induced oral mucositis, where treatment with rhIL-11 was associated with decreased mucosal damage, accelerated healing and reduced numbers of deaths. rhIL-11 is currently in clinical trials for the treatment of chemotherapy-induced mucositis. In rat models of acute colonic injury and inflammatory bowel disease, rhIL-11 treatment reduced intestinal mucosal damage and alleviated clinical signs. rhIL-11 has direct effects on activated macrophages to reduce the production of pro-inflammatory mediators. In animal models of endotoxaemia, rhIL-11 treatment reduced serum levels of pro-inflammatory cytokines and blocked hypotension. rhIL-11 increased survival in models of Gram-negative sepsis and toxic shock. Based on these studies, rhIL-11 is currently in clinical trials for treatment of Crohn's disease. Other inflammatory conditions are being further evaluated. Mechanistically, rhIL-11 functions at many levels to control inflammation, ameliorate tissue damage and maintain haemostasis in the face of trauma or infection. rhIL-11 has direct effects on hepatocytes, inducing the production of acute phase reactant proteins, haem oxygenase and tissue inhibitor of metalloproteinase-1 (TIMP-1). TIMP-1 expression can also be induced in synoviocytes and chondrocytes by treatment with rhIL-11. rhIL-11 administration has been associated with increased plasma levels of von Willebrand factor and fibrinogen. rhIL-11 treatment potentially offers multiple benefits for cancer chemotherapy patients, such as prevention of thrombocytopenia, gastrointestinal epithelial protection and subsequent reduction of mucositis, and amelioration of inflammatory complications. In addition, rhIL-11 is being evaluated further in the treatment of inflammatory disorders such as inflammatory bowel disease, rheumatoid arthritis and sepsis.

Regulation of experimental autoimmune encephalomyelitis by interleukin-12.

We have evaluated the effects of rmIL-12 on the course of adoptively transferred EAE. When mice were injected with LNC that had been stimulated in vitro with PLP in the presence of rmIL-12, the subsequent course of disease was more severe and prolonged than controls. In vitro stimulation with PLP in the presence of IL-12 was associated with an increase in IFN-gamma and decrease in IL-4-producing cells, indicating a preferential expansion of Th1 effector cells. At peak disease, no notable differences in either the cellular composition or cytokine expression within CNS lesions was seen between groups. However, the frequency of macrophages that stained positively for inducible nitric oxide synthase (iNOS) was increased in animals challenged with rmIL-12 treated LNC. These data suggest that in addition to promoting the preferential expansion of IFN-gamma-producing cells by rmIL-12 treatment in vitro, in vivo effects leading to macrophage activation and iNOS expression may contribute to the severe, protracted course of CNS inflammation in this model. In contrast, treatment of mice with an antibody to murine IL-12 following cell transfer completely prevented paralysis with only 40% of the mice developing mild disease. These data suggest that endogenous IL-12 plays a pivotal role in the pathogenesis of this model of autoimmune disease.

Thrombopoietic potential and serial repopulating ability of murine hematopoietic stem cells constitutively expressing interleukin 11.

Based on transplantation studies with bone marrow cultured under various conditions, a role of interleukin 11 (IL-11) in the self-renewal and/or the differentiation commitment of hematopoietic stem cells has been indicated. To better evaluate the in vivo effects of IL-11 on stem/progenitor cell biology, lethally irradiated mice were serially transplanted with bone marrow cells transduced with a defective retrovirus, termed MSCV-mIL-11, carrying the murine IL-11 (mIL-11) cDNA and the bacterial neomycin phosphotransferase (neo) gene. High serum levels (i.e., > 1 ng/ml) of mIL-11 in all (20/20) primary and 86% (12/14) of secondary long-term reconstituted mice, as well as 86% (12/14) of tertiary recipients examined at 6 weeks posttransplant, demonstrated persistence of vector expression subsequent to transduction of bone marrow precursors functionally definable as totipotent hematopoietic stem cells. In agreement with results obtained with human IL-11 in other myeloablation models, ectopic mIL-11 expression accelerated recovery of platelets, neutrophils, and, to some extent, total leukocytes while preferentially increasing peripheral platelet counts in fully reconstituted mice. When analyzed 5 months posttransplant, tertiary MSCV-mIL-11 recipients had a significantly greater percentage of G418-resistant colony-forming cells in their bone marrow compared with control MSCV animals. Collectively, these data show that persistent stimulation of platelet production by IL-11 is not detrimental to stem cell repopulating ability; rather, they suggest that IL-11 expression in vivo may have resulted in enhanced maintenance of the most primitive hematopoietic stem cell compartment. The prolonged expression achieved by the MSCV retroviral vector, despite the presence of a selectable marker, contrasts with the frequent transcriptional extinction observed with other retroviral vectors carrying two genes. These findings have potentially important implications for clinical bone marrow transplantation and gene therapy of the hematopoietic system.

Thrombopoietic activity of recombinant human interleukin 11 (rHuIL-11) in normal and myelosuppressed nonhuman primates.

We have extensively characterized the hematological response of normal and myelosuppressed nonhuman primates to treatment with recombinant human interleukin 11 (rHuIL-11) in vivo. In normal cynomolgus monkeys, rHuIL-11 significantly increased peripheral platelet counts when administered at doses of 10 micrograms/kg/day to 100 micrograms/kg/day either by constant i.v. infusion or s.c. injection. As few as four days of rHuIL-11 treatment were sufficient to increase peripheral platelet counts significantly. In addition, extending the treatment period enhanced both the magnitude and the duration of the response. Bone marrow megakaryocytes from animals treated with 100 micrograms/kg/day of rHuIL-11 were increased in size compared to controls and were ultrastructurally normal. A nonhuman primate myelosuppression model using carboplatin, which causes severe thrombocytopenia with platelet counts of < or = 20 x 10(3) platelets/microliters, was developed. This novel model was used to evaluate the effectiveness of rHuIL-11 in platelet restoration. rHuIL-11, administered s.c. at a dose of 125 micrograms/kg/day either concurrently or following chemotherapy, prevented severe thrombocytopenia in addition to accelerating platelet recovery compared to control animals receiving no rHuIL-11. These data demonstrate that rHuIL-11 has potent in vivo thrombopoietic effects when administered to normal and myelosuppressed nonhuman primates, and that rHuIL-11 can be an important therapy to reduce the severity and duration of thrombocytopenia following chemotherapy.

Recombinant human macrophage colony-stimulating factor in nonhuman primates: selective expansion of a CD16+ monocyte subset with phenotypic similarity to primate natural killer cells.

The CD16 receptor (Fc gamma R-III) is found on many tissue macrophages (M phi s), but its expression on circulating monocytes is restricted to a small, phenotypically distinct subset. The number of these CD16+ monocytes may be markedly increased in response to sepsis, human immunodeficiency virus infection, or metastatic malignancy. We have recently shown that the CD16+ monocyte population is selectively expanded by administration of recombinant human macrophage colony-stimulating factor (rhM-CSF). In the current study, we used the highly rhM-CSF-responsive cynomolgus primate model to further characterize this novel monocyte population. Animals treated with rhM-CSF underwent a progressive and essentially complete conversion to the CD16+ monocyte phenotype, with up to a 50-fold increase in the number of CD16+ cells. This increase was paralleled by the emergence of a population of circulating cells that morphologically resembled large granular lymphocytes (LGLs). However, quantitatively, this population corresponded closely to the number of CD16+ monocytes, and fluorescence-activated cell sorting (FACS) confirmed that they were the same. In addition to their LGL-like morphology, many rhM-CSF-induced CD16+ monocytes showed a pattern of size, granularity, and quantitative cell surface marker expression that closely resembled the pretreatment LGL/natural killer (NK) cell population but that did not resemble the pretreatment monocyte population. However, rhM-CSF-induced CD16+ monocytes could be distinguished from LGL/ NK cells by fact that they all expressed cell surface receptors for rhM-CSF, and many of them showed reduced but detectable phagocytic and respiratory burst activity. Studies of human subjects treated with rhM-CSF also showed an analogous population of "LGL-appearing" CD16+ mononuclear cells. Thus, our studies reveal a previously unsuspected ability of cells in the monocyte lineage to adopt a phenotype similar to that of LGL/NK cells. The extent of this phenotypic convergence suggests that the two lineages retain access to elements of a similar developmental pathway.

Adoptive transfer of experimental allergic encephalomyelitis after in vitro treatment with recombinant murine interleukin-12. Preferential expansion of interferon-gamma-producing cells and increased expression of macrophage-associated inducible nitric oxide synthase as immunomodulatory mechanisms.

In an adoptive transfer model of experimental allergic encephalomyelitis, stimulation of lymph node cells with proteolipid protein and recombinant murine interleukin (rmIL)-12 before cell transfer accelerated the onset and exacerbates clinical disease. In vitro stimulation with proteolipid protein in the presence of rmIL-12 was associated with an increase in interferon-gamma-producing cells and a decrease in IL-4-producing cells, indicating a preferential expansion of Th1 effector cells. This was supported by the finding that severe disease with rapid onset could be transferred with as few as 10 x 10(6) rmIL-12-stimulated lymph node cells. Immunohistochemical analysis confirmed that the accelerated onset of disease after in vitro stimulation with rmIL-12 coincided with an acute inflammatory response in the central nervous system. At peak disease, both control and rmIL-12 treatment groups exhibited extensive cellular infiltration with characteristic perivascular cuffing. No notable differences in either the cellular composition or cytokine expression within the lesions were seen between groups. However, the frequency of macrophages that stained positively for inducible nitric oxide synthase was increased in animals challenged with rmIL-12-treated lymph node cells. The results suggest that, in addition to promoting the preferential expansion of interferon-gamma-producing cells by rmIL-12 in vitro, secondary in vivo effects leading to macrophage activation and inducible nitric oxide synthase expression may contribute to the severe and protracted course of central nervous system inflammation in this model.

Constant subcutaneous infusion of rhIL-11 in mice: efficient delivery enhances biological activity.

Previous studies have shown that subcutaneous (SC) bolus administration of recombinant human interleukin-11 (rhIL-11) stimulates megakaryocytopoiesis and increases peripheral platelet counts in naive mice. This study was designed to determine whether administration of rhIL-11 by constant SC infusion altered either the magnitude of the nature of the hematologic response. Female C57BL/6 mice were implanted subcutaneously with 7-day Alzet mini-osmotic pumps containing either rhIL-11 with 0.5% homologous mouse serum (delivery rate of 250 microg/kg/d) or vehicle alone. Mice were sacrificed on days 3, 7, 10, and 13 after pump implantation, and the hematopoietic response was compared to mice receiving an equivalent dose of rhIL-11 administered by SC injection (250 microg/kg/d, 7 days) or vehicle controls. Subcutaneous injection of rhIL-11 resulted in a significant increase in peripheral platelet counts with a maximum platelet increase of 44% over controls observed on day 7 of the study. Platelet counts subsequently declined (24% by day 10) returning to control values by day 13. The increase in peripheral platelet counts was accompanied by an increase in reticulated platelets on day 7 and a shift to higher ploidy bone marrow megakaryocytes on days 3 and 7. Compared to SC injection, both the magnitude and duration of the platelet increase were significantly enhanced following continuous SC infusion of rhIL-11. Maximum platelet counts were detected on day 10 (115% above vehicle controls), and platelets remained significantly elevated on day 13 (84%), 6 days after rhIL-11 administration had stopped. Consistent with the platelet response, the modal ploidy of bone marrow megakaryocytes was shifted from 16N to 32N on days 3 and 7, with increases in 32N megakaryocytes still apparent on days 10 and 13. There was also a significant increase in reticulated platelets detected in the peripheral blood on days 3, 7, and 10 compared to mice administered rhIL-11 by SC injection, The changes in reticulated platelets and bone marrow megakaryocyte ploidy are consistent with the increased and prolonged platelet response following SC infusion of rhIL-11. In addition to the effects observed on peripheral platelet counts, constant SC infusion of rhIL-11 dramatically enhanced splenic hematopoietic activity, increasing spleen weight and cellularity as well as splenic megakaryocyte, erythroid, granulocyte, and macrophage progenitors compared to mice receiving rhIL-11 by SC injection.

The role of recombinant interleukin 11 in megakaryocytopoiesis.

Recombinant human interleukin 11 (rHuIL-11) is a multifunctional cytokine with activities on a broad range of hematopoietic cells including primitive stem cells and mature progenitor cells. Analysis of rHuIL-11 in vitro has revealed that its hematopoietic activities are predominantly a result of synergistic interactions with other early-acting factors such as IL-3 and Steel factor. Studies indicate that rHuIL-11 acts directly on purified stem and progenitor cell populations and can support the growth of colony forming units-megakaryocyte in these cultures. In normal animals, rHuIL-11 has a potent effect on cells of the megakaryocyte (MK) lineage. Administration of rHuIL-11 results in a two- to threefold increase in circulating platelets, stimulation of bone marrow (BM) and spleen progenitor numbers, and enhanced MK maturation as measured by a shift to higher ploidy values. rHuIL-11 administration in preclinical models of myelosuppression induced by chemotherapy and/or irradiation has shown a reproducible acceleration of platelet recovery and, in some models, enhanced neutrophil and red blood cell recovery. rHuIL-11 has been tested in a non-human primate myelosuppression model using carboplatin. Administration of rHuIL-11 following carboplatin treatment was found to eliminate the period of severe thrombocytopenia (<20,000 platelets/ml) and enhance the recovery of platelets to normal levels (>100,000/ml). Recently, human clinical trials conducted with rHuIL-11 in patients treated with chemotherapy have demonstrated its potent thrombopoietic activity, including improved platelet nadirs, enhanced platelet recovery and a significant decrease in the number of patients who require platelet transfusions. Combined with the preclinical results, these studies confirm that this cytokine will be an effective agent in the treatment of myelosuppression and thrombocytopenia associated with cancer chemotherapy and BM transplantation.

Hematopoietic stem cells in the blood after stem cell factor and interleukin-11 administration: evidence for different mechanisms of mobilization.

Peripheral blood stem cells and progenitor cells, collected during recovery from exposure to cytotoxic agents or after cytokine administration, are being increasingly used in clinical bone marrow transplantation. To determine factors important for mobilization of both primitive stem cells and progenitor cells to the blood, we studied the blood and splenic and marrow compartments of intact and splenectomized mice after administration of recombinant human interleukin-11 (rhlL-11), recombinant rat stem cell factor (rrSCF), and IL-11 + SCF. IL-11 administration increased the number of spleen colony-forming units (CFU-S) in both the spleen and blood, but did not increase blood long-term marrow-repopulating ability (LTRA) in intact or splenectomized mice. SCF administration increased the number of CFU-S in both the spleen and blood and did not increase the blood or splenic LTRA of intact mice, but did increase blood LTRA to normal marrow levels in splenectomized mice. The combination of lL-11 + SCF syngeristically enhanced mobilization of long-term marrow-repopulating cells from the marrow to the spleen of intact mice and from the marrow to the blood of splenectomized mice. These data, combined with those of prior studies showing granulocyte colony-stimulating factor mobilization of long-term marrow repopulating cells from the marrow to the blood of mice with intact spleens, suggest different cytokine-induced pathways for mobilization of primitive stem cells.

Preclinical biology of interleukin 11: a multifunctional hematopoietic cytokine with potent thrombopoietic activity.

Interleukin 11 (IL-11) is a multifunctional hematopoietic cytokine which was originally identified as a factor produced by an IL-1-stimulated primate stromal cell line. The in vitro biological activities of recombinant human (rHu)IL-11 result predominantly from synergistic interactions with other growth factors. In combination with other cytokines, rHuIL-11 has been shown to support the formation of primitive hematopoietic and lymphohematopoietic progenitor colonies from bone marrow, to promote erythroid burst formation and to stimulate both early and late stages of megakaryocyte proliferation and differentiation. rHuIL-11 is biologically active in mice, rats, dogs and primates when administered as a single agent in vivo. The predominant effect of rHuIL-11 in naive mice was on cells of the megakaryocytic lineage, increasing the number of bone marrow megakaryocyte progenitors, stimulating megakaryocyte endoreplication and increasing peripheral platelet counts in a dose-dependent fashion. Similar megakaryocytic stimulatory activity was seen in nonhuman primates treated with rHuIL-11 where platelet counts were increased by as much as 300%. In several models of severe myelosuppression induced by chemotherapy and/or irradiation and in bone marrow transplant models, there were multilineage hematopoietic stimulation following rHuIL-11 treatment. In these models, accelerated recovery of platelets was a consistent observation, while some models show enhanced neutrophil and red blood cell recovery as well. These results from preclinical studies confirm the broad spectrum of biological activities exhibited by rHuIL-11 in vitro, and suggest that this cytokine may be an effective agent in the treatment of myelosuppression and thrombocytopenia associated with cancer chemotherapy and bone marrow transplantation.

CD16+ monocytes in patients with cancer: spontaneous elevation and pharmacologic induction by recombinant human macrophage colony-stimulating factor.

The small subset of circulating monocytes that express the maturation-associated CD16 antigen has recently been reported to be elevated in patients with bacterial sepsis. We now show that this novel CD16+ monocyte population is also spontaneously expanded in patients with cancer. We studied 14 patients with metastatic gastrointestinal carcinoma enrolled ina clinical trial of recombinant human macrophage colony-stimulating factor (rhMCSF) plus monoclonal antibody D612. We found that before any cytokine treatment, 12 of 14 patients constitutively displayed significant elevations in both the percentage and the absolute number of CD16+ monocytes, as compared with both normal subjects and ill patients with elevated monocyte counts but without malignancy. CD16+ monocytes accounted for 46% +/- 22% of total monocytes in the patients with cancer versus 5% +/- 3% for controls (P < .01). The increase was not attributable to infection or intercurrent illness and appeared to be associated with the underlying malignancy itself. A similar spontaneous elevation of CD16+ monocytes was observed in 35 of 44 additional patients diagnosed with a variety of other solid tumors. When patients with gastrointestinal carcinoma were treated with rhMCSF, there was a marked further increase in the percentage of CD16+ monocytes (to 83% +/- 11%), as well as in the absolute number of CD16+ cells and the level of CD16 antigen expression. In every case, the patients with cancer showed a greater CD16+ monocyte response than the maximal response obtained in normal volunteer subjects treated witha similar regimen of rhMCSF (n = 5, P < .001), suggesting that the presence of malignancy primed patients for enhanced responsiveness to rhMCSF. We hypothesize that spontaneous expansion of the CD16+ monocyte population may represent a novel biologic marker for a widespread and previously unsuspected host immune response to malignancy.