Tumor necrosis factor-dependent segmental control of MIG expression by high endothelial venules in inflamed lymph nodes regulates monocyte recruitment.

Monocytes recruited from the blood are key contributors to the nature of an immune response. While monocyte recruitment in a subset of immunopathologies has been well studied and largely attributed to the chemokine monocyte chemoattractant protein (MCP)-1, mechanisms mediating such recruitment to other sites of inflammation remain elusive. Here, we showed that localized inflammation resulted in an increased binding of monocytes to perifollicular high endothelial venules (HEVs) of lymph nodes draining a local inflammatory site. Quantitative PCR analyses revealed the upregulation of many chemokines in the inflamed lymph node, including MCP-1 and MIG. HEVs did not express detectable levels of MCP-1; however, a subset of HEVs in inflamed lymph nodes in wild-type (but not tumor necrosis factor [TNF] null mice) expressed MIG and this subset of HEVs preferentially supported monocyte binding. Expression of CXCR3, the receptor for MIG, was detected on a small subset of peripheral blood monocytes and on a significant percentage of recruited monocytes. Most importantly, in both ex vivo and in vivo assays, neutralizing anti-MIG antibodies blocked monocyte binding to inflamed lymph node HEVs. Together, these results suggest that the lymph node microenvironment can dictate the nature of molecules expressed on HEV subsets in a TNF-dependent fashion and that inflammation-induced MIG expression by HEVs can mediate monocyte recruitment.

CTACK, a skin-associated chemokine that preferentially attracts skin-homing memory T cells.

In contrast to naive lymphocytes, memory/effector lymphocytes can access nonlymphoid effector sites and display restricted, often tissue-selective, migration behavior. The cutaneous lymphocyte-associated antigen (CLA) defines a subset of circulating memory T cells that selectively localize in cutaneous sites mediated in part by the interaction of CLA with its vascular ligand E-selectin. Here, we report the identification and characterization of a CC chemokine, cutaneous T cell-attracting chemokine (CTACK). Both human and mouse CTACK are detected only in skin by Southern and Northern blot analyses. Specifically, CTACK message is found in the mouse epidermis and in human keratinocytes, and anti-CTACK mAbs predominantly stain the epithelium. Finally, CTACK selectively attracts CLA(+) memory T cells. Taken together, these results suggest an important role for CTACK in recruitment of CLA(+) T cells to cutaneous sites. CTACK is predominantly expressed in the skin and selectively attracts a tissue-specific subpopulation of memory lymphocytes.

The CC chemokine receptor-7 ligands 6Ckine and macrophage inflammatory protein-3 beta are potent chemoattractants for in vitro- and in vivo-derived dendritic cells.

Dendritic cell migration to secondary lymphoid tissues is critical for Ag presentation to T cells necessary to elicit an immune response. Despite the importance of dendritic cell trafficking in immunity, at present little is understood about the mechanisms that underlie this phenomenon. Using a novel transwell chemotaxis assay system, we demonstrate that the CC chemokine receptor-7 (CCR7) ligands 6Ckine and macrophage inflammatory protein (MIP)-3 beta are selective chemoattractants for MHC class IIhigh B7-2high bone marrow-derived dendritic cells at a potency 1000-fold higher than their known activity on naive T cells. Furthermore, these chemokines stimulate the chemotaxis of freshly isolated lymph node dendritic cells, as well as the egress of skin dendritic cells ex vivo. Because these chemokines are expressed in lymphoid organs and 6Ckine has been localized to high endothelial venules and lymphatic endothelium, we propose that they may play an important role in the homing of dendritic cells to lymphoid tissues.

IL-12, but not IFN-gamma, plays a major role in sustaining the chronic phase of colitis in IL-10-deficient mice.

IL-10-deficient (IL-10(-/-)) mice develop chronic enterocolitis mediated by CD4+ Th1 cells producing IFN-gamma. Because IL-12 can promote Th1 development and IFN-gamma production, the ability of neutralizing anti-IL-12 mAb to modulate colitis in IL-10(-/-) mice was investigated. Anti-IL-12 mAb treatment completely prevented disease development in young IL-10(-/-) mice. Treatment of adult mice resulted in significant amelioration of established disease accompanied by reduced numbers of mesenteric lymph node and colonic CD4+ T cells and of mesenteric lymph node T cells spontaneously producing IFN-gamma. In contrast, anti-IFN-gamma mAb had minimal effect on disease reversal, despite a significant preventative effect in young mice. These findings suggested that IL-12 sustains colitis by supporting the expansion of differentiated Th1 cells that mediate disease independently of their IFN-gamma production. This conclusion was supported by the finding that anti-IL-12 mAb greatly diminished the ability of a limited number of CD4+ T cells expressing high levels of CD45RB from diseased IL-10(-/-) mice to expand and cause colitis in recombination-activating gene-2(-/-) recipients, while anti-IFN-gamma mAb had no effect. Furthermore, IL-12 could support pathogenic IL-10(-/-) T cells stimulated in vitro in the absence of IL-2. While these studies show that IL-12 plays an important role in sustaining activated Th1 cells during the chronic phase of disease, the inability of anti-IL-12 mAb to abolish established colitis or completely prevent disease transfer by Thl cells suggests that additional factors contribute to disease maintenance.

Radioprotective effects of flk2/flt3 ligand.

The radioprotective properties of flk2/flt3 ligand (FL) were evaluated in lethally irradiated mice. Optimum survival rates (70-80%) were observed when 5 to 20 microg of FL was administered at both 20 and 2 hours before LD100/30 radiation. Administration of FL well in advance of irradiation was essential for conferring most of the radioprotection, since a single dose given at -20 hours still resulted in a significant survival rate (65%), whereas a single dose given at -2 hours was relatively nonprotective. Histopathologic examination at 7 and 9 days postirradiation revealed significant myelopoietic activity in the bone marrow (BM) of FL-treated mice, suggesting that their survival might be due to sparing of radiosensitive hematopoietic cells. By comparison, the BM of mice treated with phosphate-buffered saline was extremely hypocellular and remained that way until they died of bacterial infection. Hematopoietic assays confirmed a marked stimulation of early white blood cell (WBC) recovery in the BM and blood of FL-protected mice relative to PBS-treated controls. By day 21, FL-protected mice showed circulating WBC numbers that were higher than preirradiation values; however, their BM colony-forming units in culture were still depressed. Moreover, these mice experienced a prolonged anemia and thrombocytopenia. These findings are discussed in light of the restricted subset of hematopoietic progenitors shown to be responsive to FL in vitro.

flk2/flt3 ligand is a potent cofactor for the growth of primitive B cell progenitors.

We have investigated the role of flk2/flt3 ligand (FL) in B cell lymphopoiesis. The ability of FL to stimulate the growth of immature B cells was assessed using distinct populations: CD43lowB220+ pre-B cells, CD43+B220+ pro-B cells, and CD43+B220low progenitors. FL failed to affect the growth of the pro-B or pre-B cells whether used alone or in combination with stem cell factor (SCF) or IL-7. In striking contrast, FL was a potent cofactor for the CD43+B220low progenitor cells, interacting with either IL-7 and/or SCF to stimulate their growth. The combination of FL with IL-7 plus SCF stimulated maximum expansion of these cells, albeit, less than that stimulated in stromal cell cultures. When the CD43+B220low progenitors were divided based on expression of heat stable Ag (CD24) into a CD24- and a CD24+ subset, the FL-responsive cells were contained only within the CD24- subset. FL interacted with SCF or with IL-7 to stimulate their growth resulting in a 20- and 50-fold increase in cellularity, respectively. Since the CD24- subset was the most immature of the B cell populations studied, our data suggest that FL costimulates the expansion of very primitive B cell progenitors.

FLT3/FLK2 ligand promotes the growth of murine stem cells and the expansion of colony-forming cells and spleen colony-forming units.

The effect of FLT3/FLK2 ligand (FL) on the growth of primitive hematopoietic cells was investigated using ThyloSca1+ stem cells. FL was observed to interact with a variety of factors to initiate colony formation by stem cells. When stem cells were stimulated in liquid culture with FL plus interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor (G-CSF), or stem cell factor (SCF), cells capable of forming colonies in secondary methylcellulose cultures (CFU-c) were produced in high numbers. However, only FL plus IL-6 supported an increase in the number of cells capable of forming colonies in the spleens of irradiated mice (CFU-s). Experiments with accessory cell-depleted bone marrow (Lin- BM) showed that FL alone lacks significant colony-stimulating activity for progenitor cells. Nevertheless, FL enhanced the growth of granulocyte-macrophage progenitors (CFU-GM) in cultures containing SCF, G-CSF, IL-6, or IL-11. In these assays, FL increased the number of CFU-GM initiating colony formation (recruitment), as well as the number of cells per colony (synergy). Many of the colonies were macroscopic and contained greater than 2 x 10(4) granulocytes and macrophages. Therefore, FL appears to function as a potent costimulus for primitive cells of high proliferative potential (HPP). FL was also observed to costimulate the expansion of CFU-GM in liquid cultures of Lin- BM. In contrast, FL had no growth-promoting affects on progenitors committed to the erythrocyte, megakaryocyte, eosinophil, or mast cell lineages.

Ligand for FLT3/FLK2 receptor tyrosine kinase regulates growth of haematopoietic stem cells and is encoded by variant RNAs.

The FLT3/FLK2 receptor tyrosine kinase is closely related to two receptors, c-Kit and c-Fms, which function with their respective ligands, Kit ligand and macrophage colony-stimulating factor to control differentiation of haematopoietic and non-haematopoietic cells. FLT3/FLK2 is thought to be present on haematopoietic stem cells and found in brain, placenta and testis. We have purified to homogeneity and partially sequenced a soluble form of the FLT3/FLK2 ligand produced by mouse thymic stromal cells. We isolated several mouse and human complementary DNAs that encode polypeptides with identical N termini and different C termini. Some variants contain hydrophobic transmembrane segments, suggesting that processing may be required to release soluble ligand. The purified ligand enhances the response of mouse stem cells and a primitive human progenitor cell population to other growth factors such as interleukins IL-3 and IL-6 and to granulocyte-macrophage colony-stimulating factor, and also stimulates fetal thymocytes.

Interleukin-10 promotes the growth of megakaryocyte, mast cell, and multilineage colonies: analysis with committed progenitors and Thy1loSca1+ stem cells.

The growth-promoting activities of interleukin-10 (IL-10) were assessed in hematopoietic colony-forming assays. We found that IL-10 failed to support the clonal growth of normal and lineage-depleted (Lin-) bone marrow (BM) cells. Furthermore, IL-10 neither enhanced nor suppressed colony formation by eosinophil, neutrophil, or macrophage progenitors when combined with a variety of factors. IL-10 stimulated a modest increase in erythropoietin (Epo)-dependent erythroid colonies but had no effect on the burst-promoting activities of IL-3. However, the combination of IL-10 plus IL-3 resulted in the enhanced growth of mast cell progenitors. In addition to its mast cell stimulating activity, IL-10 promoted the growth of megakaryocyte (Mk) and Mk-mixed colonies when combined with Epo or with Epo plus IL-3, IL-6, or IL-11. Comparative studies showed that the megakaryocyte potentiating activity of IL-10 is roughly equivalent to that of IL-6 and IL-11. In experiments using Thy1loSca1+ stem cells, IL-10 was shown to enhance the number of cells initiating IL-3-dependent colony formation. IL-10 also costimulated increased colony formation when used with IL-3 and another factor such as IL-1, IL-6, and granulocyte colony-stimulating factor (G-CSF). Cellular analysis of the resulting colonies indicated that IL-10 increases the formation of multilineage colonies containing erythrocytes, megakaryocytes, and/or mast cells. The ability of IL-10 to cooperatively regulate various stages of hematopoietic development is discussed.

Anti-IL-6 antibodies suppress myeloid cell production and the generation of CFU-c in long-term bone marrow cultures.

Interleukin 6 (IL-6) is one of several hemopoietic growth factors produced by stromal cell lines derived from the adherent layer of long-term bone marrow cultures (LTBMCs). To evaluate the potential role of IL-6 in stromal cell-dependent myelopoiesis, we established LTBMCs and verified that IL-6 mRNA is transcribed by heterogeneous adherent cell layers and that IL-6 protein is present in culture supernatants. Established LTBMCs were then depleted of IL-6 by using a specific neutralizing monoclonal antibody (mAb). Cultures treated for 2-3 weeks with anti-IL-6 mAb showed decreased production of maturing myeloid cells and colony-forming progenitor cells (colony-forming units in culture, CFU-c) but not stem cells (spleen colony-forming units, CFU-s). In parallel experiments, it was also found that the addition of IL-6 to LTBMCs stimulated a marked increase in total cell production, CFU-c, and day-8 CFU-s. In sum, it appears that endogenous production of IL-6, although limiting, is essential for the normal level of myelopoiesis associated with stromal cell function in LTBMCs.

The in vitro response of phenotypically defined mouse stem cells and myeloerythroid progenitors to single or multiple growth factors.

Pluripotential stem cells (Thylo Lin- Sca+; referred to as Sca+) and primitive myeloerythroid progenitor cells (Thylo Lin- Sca-; referred to as Sca-), defined by their in vivo repopulating properties, have been purified from mouse bone marrow. In this study, the growth factor requirements of these two subsets were compared in colony-forming assays. Sca- progenitor cells grew well in interleukin (IL) 3 alone and showed maximum growth when two factors, IL-3 plus IL-1 or IL-3 plus IL-6, were combined. In contrast, Sca+ stem cells were generally not responsive to any single factor tested. Some colony formation was found when IL-3 was paired with either IL-1 or IL-6, and this was significantly enhanced as additional factors were included. A remarkable frequency of as much as 1 colony per 1.7 input Sca+ cells was achieved when IL-1, IL-3, IL-6, and colony-stimulating factors were used together. These differences in factor requirements presumably reflect the need for multiple factor signaling in the more primitive stem cell population. In most other aspects of colony formation, Sca+ and Sca- cells were very similar. They generated colonies that had equivalent distributions in size and cellular composition. One notable difference was found in the kinetics of their response. Whereas nearly all Sca- cells formed colonies within 7 days, a significant fraction of Sca+ cells delayed colony formation for greater than 1 week. During this quiescent period, cell survival was absolutely dependent on the presence of factors in the medium.

In vivo administration of antibody to interleukin-5 inhibits increased generation of eosinophils and their progenitors in bone marrow of parasitized mice.

Bone marrow of mice parasitized with Nippostrongylus brasiliensis showed increased numbers of eosinophils as early as 4 days after infection. By day 7, their bone marrow also contained elevated numbers of progenitors that form small eosinophil colonies (20 to 50 cells) in soft agar cultures supplemented with interleukin-5 (IL-5). However, when mice were infused with anti-IL-5 antibodies at the time of infection, the number of recognizable eosinophils present in bone marrow remained low and eventually dropped below normal levels. The antibody treatment also prevented increased generation of IL-5-responsive precursors capable of differentiating into mature eosinophils in liquid culture and inhibited the generation of progenitor cells capable of forming small eosinophil colonies or clusters in soft agar cultures. The results of these in vivo experiments directly show that IL-5 is an essential regulatory molecule required for the bone marrow-dependent phase of a parasite-induced eosinophilia.

Antibody to interleukin-5 inhibits helminth-induced eosinophilia in mice.

When rodents are infected with the nematode Nippostrongylus brasiliensis, large numbers of eosinophils appear in their blood and lungs and their serum immunoglobulin E (IgE) is increased. Injection of a monoclonal antibody to interleukin-5 completely suppressed the blood eosinophilia and the infiltration of eosinophils in the lungs of parasitized mice but had no effect on serum IgE. In contrast, an antibody to interleukin-4 inhibited parasite-induced IgE but not the eosinophilia. These results show that interleukin-5 is important in eosinophil production in vivo and that IgE and eosinophil production are regulated by different cytokines produced by the TH2 subset of CD4-expressing T cells.

Interleukin-6 interacts with interleukin-4 and other hematopoietic growth factors to selectively enhance the growth of megakaryocytic, erythroid, myeloid, and multipotential progenitor cells.

The growth-promoting activities of interleukin-6 (IL-6) in combination with different factors were assessed in bone marrow (BM) cultures prepared from normal mice and from mice treated with 5-fluorouracil (5-FU). Effects on hematopoietic colony formation with respect to number, size, and cellular composition were evaluated. In agreement with previous reports, IL-6 acts synergistically with IL-3 to stimulate increased numbers of granulocyte/macrophage (GM) and multilineage colonies in day-2 and day-4 post-5-FU BM cultures. Furthermore, day 4 but not day 2 post-5-FU BM showed enhanced GM colony formation when stimulated with IL-6 plus interleukin-4 (IL-4) or granulocyte colony-stimulating factor (G-CSF). In contrast, IL-6 did not increase the number of colonies supported by M-CSF or GM-CSF. Nevertheless IL-6 interacted with all factors, including M-CSF and GM-CSF, to stimulate an increase in colony size. Many of these myeloid colonies attained a diameter of greater than or equal to 0.5 mm, suggesting they derive from high proliferative potential cells (HPP-CFC). The response of normal and day-8 post-5-FU BM containing high numbers of more mature progenitors was also assessed. We found IL-6 enhanced colony formation by lineage-restricted megakaryocytic and erythroid progenitors in the presence of IL-3 and IL-4 plus erythropoietin (Epo), respectively. The sum of these results shows that IL-6 interacts with a variety of factors to regulate the growth of progenitor cells at different stages of lineage commitment and maturation.

Characterization of B-cell populations bearing Fc epsilon receptor II.

We have characterized the B-cell population that expresses low affinity Fc receptors for IgE (Fc epsilon RII). Fc epsilon RII+ B cells from normal adult BALB/c mice expressed high levels of surface IgD and low/medium levels of surface IgM and constituted the majority of mature splenic B cells. Fc epsilon RII+ splenic B cells expressed high levels of class II MHC antigens and medium to high levels of B220, and consisted of approximately equal numbers of J11dhigh and J11dlow cells. CD5+ B cells did not appear to be Fc epsilon RII+, and interleukin 4 did not induce Fc epsilon RII expression on CD5+ B cells. Fc epsilon RII was not expressed by B cells that had differentiated to secrete immunoglobulin but was expressed on activated B cells. These data suggest that Fc epsilon RII is a differentiation marker expressed on mature and activated B lymphocytes in the major B-cell lineage.