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The ability of an infusion of ex vivo expanded hematopoietic cells to ameliorate cytopenia following transplantation of hematopoietic stem cells (HSCs) is controversial. To address this issue, we measured the recovery of circulating leukocytes, erythrocytes, and platelets in lethally irradiated mice transplanted with 10(3) enriched HSCs, with or without their expanded equivalent (EE) generated after 7 days of culture in interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor and Steel Factor. Two HSC populations differing in their content of short-term repopulating progenitors were evaluated. Thy-1loLIN-Sca-1+ (TLS) bone marrow (BM) is enriched in colony-forming cells (CFCs), day 8 and day 12 spleen colony-forming units (CFU-S) (435 +/- 19, 170 +/- 30, and 740 +/- 70 per 10(3) cells, respectively), and stem cells with competitive long-term repopulating potential (> or = 1 per 43 cells). Thy-1loSca-1+H-2Khl cells (TSHFU) isolated from BM 1 day after treatment of donor mice with 5-fluorouracil (5-FU) are also highly enriched in competitive repopulating units (CRU, > or = 1 per 55 cells), but are depleted of CFCs, day 8 and day 12 CFU-S (171 +/- 8, 0 and 15 +/- 4 per 10(3) cells, respectively). Recipients of 10(3) TLS cells transiently recovered leukocytes to > or = 2,000/microL in 12 days, but sustained engraftment required 25 days. Platelets recovered to > or = 200,000/microL in 15 days, and erythrocytes never decreased below 50% of normal. Mice transplanted with 10(3) TSHFU cells recovered leukocytes in 15 days, and platelets and erythrocytes in 18 days. Recipients of unseparated normal or 5-FU-treated BM cells (containing 10(3) TLS or TSHFU cells) recovered safe levels of blood cells in 9 to 12 days, suggesting that unseparated marrow contains early engrafting cells that were depleted by sorting. Upon ex vivo expansion, total cells, CFCs and day 12 CFU-S were amplified 2,062-,83- and 13-fold, respectively, from TLS cells; and 1,279-, 259- and 708-fold, respectively, from TSHFU cells. Expanded cells could regenerate the majority of lymphocytes and granulocytes in primary (17 weeks) and secondary (26 weeks) hosts and were only moderately impaired compared to fresh HSCs. The EE of TSHFU cells was more potent than that of TLS cells, suggesting that more highly enriched HSCs are more desirable starting populations for this application. When mice were transplanted with 10(3) TSHFU cells and their EE, the duration of thrombocytopenia was shortened from 18 to 12 days, and anemia was abolished. Leukocytes were also elevated on days 9 to 12, although sustained recovery was not accelerated. Anemia was also abrogated in recipients of 10(3) TLS cells and their EE. Early platelet counts were slightly higher than with TLS cells alone, but leukocyte recovery was not improved. These data confirm that TLS cells contribute to early and sustained hematopoiesis, and demonstrate a benefit of ex vivo expanded cells in accelerating engraftment of more primitive TSHFU stem cells depleted of progenitors.
Sepsis is a constellation of clinical signs and symptoms resulting from excessive systemic host inflammatory response to infection. This inflammatory response is largely mediated by cytokines, which are released into the systemic circulation. Plasma concentrations of specific cytokines, TNF alpha, IL-1 beta, IL-6 and IL-8 are frequently elevated in human sepsis and cytokine concentrations correlate with severity and outcome of sepsis. In addition to pro-inflammatory cytokines, soluble cytokine receptors, cytokine receptor antagonists and counter-inflammatory cytokines are also produced in large quantities in patients with sepsis; however, the specific role of these molecules in sepsis remains undefined. A complex interaction of cytokines and cytokine-neutralizing molecules probably determines the clinical presentation and course of sepsis. Intervening in this sequence of events to modify the host inflammatory responses may prove to be a beneficial treatment strategy for sepsis, but currently tested anticytokine therapies have been largely unsuccessful.
Animal studies suggest that acute phase reactant cytokines and polymorphonuclear leukocytes (PMN) may play a critical role in ischemia-reperfusion injury. To evaluate whether similar mechanisms are operative in human liver, six cirrhotic and nine noncirrhotic patients undergoing right hepatectomy were randomized for utilization of hepatic vascular exclusion (HVE) as a model of ischemia-reperfusion injury. Portal and systemic levels of acute reactant cytokines (interleukin 6 [IL-6], interleukin 1 [IL-1], tumor necrosis factor alpha [TNF-alpha]) and neutrophil adhesion in serial liver biopsy specimens were studied. Correlations among mediators, leukocyte adhesion, and markers of liver injury were also evaluated. Hepatic vascular exclusion resulted in substantial and reproducible changes in portal and arterial IL-6 levels in both cirrhotic and noncirrhotic patients. Portal and systemic cytokine levels were comparable in most instances, whereas levels were usually higher in cirrhotic patients than in noncirrhotic patients. Negative correlations were found between IL-6 levels at the time of reperfusion and later TNF-alpha levels. IL-6 levels correlated negatively with numerous markers of hepatocellular injury and the number of postoperative complications. Hepatic vascular exclusion increased neutrophils adhesion after reperfusion in cirrhotic patients but not in noncirrhotic patients. In cirrhotic patients, the degree of leukocyte adhesion after reperfusion correlated with several postoperative markers of liver injury. This study in humans shows that acute reactant cytokines are released during liver ischemia and, interestingly, that IL-6 levels strongly correlate with clinical and laboratory measures of injury. Further studies to evaluate possible causal relationship with hepatic injury are warranted, with emphasis on the role of IL-6 and PMN adhesion.
Attempts to maintain or expand primitive hematopoietic stem cells in vitro without the concomitant loss of their differentiative and proliferative potential in vivo have largely been unsuccessful. To investigate this problem, we compared the ability of three cloned bone marrow (BM) stromal cell lines to support the growth of primitive Thy-1lo Sca-1+H-2Khi cells isolated by fluorescence-activated cell sorting from the BM of Ly-5.2 mice treated 1 day previously with 5-fluo- rouracil. Sorted cells were highly enriched in cobblestone area-forming cells (CAFC), but their frequency was dependent on the stromal cell lines used in this assay (1 per 45 cells on SyS-1; 1 per 97 cells on PA6). In the presence of recombinant leukemia inhibitory factor (LIF), CAFC cloning efficiency was increased to 1 per 8 cells on SyS-1 and 1 per 11 cells on PA6, thus showing the high clonogenicity of this primitive stem cell population. More primitive stem cells with competitive repopulating potential were measured by injecting the sorted cells into lethally irradiated Ly-5.1 mice together with 10(5) radioprotective Ly-5.1 BM cells whose long-term repopulating ability has been "compromised" by two previous cycles of marrow transplantation and regeneration. Donor-derived lymphocytes and granulocytes were detected in 66% of animals injected with 50 sorted cells. To quantitate the maintenance of competitive repopulating units (CRU) by stromal cells, sorted cells were transplanted at limiting dilution before and after being cultured for 2 weeks on adherent layers of SyS-1, PA6, or S17 cells. CRU represented 1 per 55 freshly sorted cells. CRU could be recovered from cocultures supported by all three stromal cell lines, but their numbers were approximately-sevenfold less than on day 0. In contrast, the addition of LIF to stromal cultures improved CRU survival by 2.5-fold on S17 and PA6 cells (approximately two-fold to threefold decline), and enabled their maintenance on SyS-1. LIF appeared to act indirectly, because alone it did not support the proliferation of Thy-1lo Sca-1+H-2Khi cells in stroma-free cultures. Polymerase chain reaction (RT-PCR) analysis revealed that Interleukin-1beta (IL-1 beta) IL-2, IL-6, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, transforming growth factors, LIF, and Steel Factor (SLF) mRNAs were upregulated in SyS-1 within 1 to 6 hours of LIF-stimulation. To determine if increased expression of SLF by LIF-stimulated SyS-1 cells could account for their capacity to support stem cells, sorted calls were cocultured on simian CV-E cells that were transfected with an expression vector encoding membrane-bound SLF, or supplemented with soluble SLF. In both cases, SLF synergized with IL-6 produced endogenously by CV-E cells enabling CAFC growth equivalent to that on LIF-stimulated SyS-1. CAFC development on LIF-stimulated SyS-1 could also be completely abrogated by an anti-SLF antibody. These data provide evidence for a role of LIF in the support of long-term repopulating stem cells by indirectly promoting cytokine expression by BM stroma. Furthermore, we have used quantitative assays to show a maintenance of CRU numbers, with retention of in vivo function following ex vivo culture.
IL-4 inhibits production of certain proinflammatory cytokines, including IL-1 beta, TNF-alpha, and IL-6, by activated monocytes. Although monocytes are a major source of IL-6, other cell types such as fibroblasts and endothelial cells can also express this cytokine. To determine whether IL-4 inhibits IL-6 expression in non-hemopoietic cells, we investigated the effects of IL-4 on IL-6 production in both primary human fibroblasts and fibroblast lines. Rheumatoid synovial fibroblasts were evaluated in these studies because, like monocytes, they produce high levels of IL-6 when stimulated with IL-1. Although peripheral blood monocytes did not constitutively express IL-6 mRNA or protein, stimulation with IL-1 or LPS induced de novo IL-6 expression in these cells. In contrast, synovial fibroblasts displayed a significant basal level of IL-6 production, which was markedly increased after stimulation with IL-1. IL-4 suppressed IL-6 expression in monocytes, but did not inhibit IL-6 production in synovial fibroblasts. The inability of IL-4 to suppress IL-6 synthesis in rheumatoid synovial fibroblasts was not caused by a lack of IL-4R and was not unique to these cells because IL-4 also failed to inhibit IL-6 production in normal fibroblast lines derived from other tissues. Inhibition of IL-6 production by IL-4 in monocytes was associated with decreased nuclear NF-kappa B levels. However, IL-4 does not globally suppress the activity of all DNA-binding proteins because IL-4 treatment did not reduce the levels of NF-IL-6 or NF-IL-1 beta B in the same cells. Because NF-kappa B activation is required for transcription of many cytokine genes, including IL-6, the ability of IL-4 to suppress NF-kappa B activity in monocytes suggests a potential mechanism by which this molecule may inhibit the expression of multiple cytokines.
Interleukin 6 (IL-6) is an important regulator of the acute phase response, T cell function, and terminal B cell differentiation. Excessive or inappropriate production of this cytokine may be involved in a variety of autoimmune and neoplastic disorders. To investigate the consequences of dysregulated synthesis of IL-6 in vivo, a high-titer recombinant retroviral vector produced in psi-2 packaging cells was used to introduce the coding sequences of murine IL-6 into mouse hematopoietic cells. Congenitally anemic W/Wv mice reconstituted with bone marrow cells transduced with the retroviral vector developed a syndrome characterized by anemia, transient granulocytosis, hypoalbuminemia, and polyclonal hypergammaglobulinemia, with marked splenomegaly and peripheral lymphadenopathy. Extensive plasma cell infiltration of lymph nodes, spleen, liver, and lung was noted. The similarity of these findings to those of multicentric Castleman's disease, taken together with the observation that lymph nodes from these patients elaborate large amounts of this cytokine, suggest that the inappropriate synthesis of IL-6 has a primary role in the pathogenesis of this systemic lymphoproliferative disorder.