Macrophage inflammatory protein 3alpha is involved in the constitutive trafficking of epidermal langerhans cells

Certain types of dendritic cells (DCs) appear in inflammatory lesions of various etiologies, whereas other DCs, e.g., Langerhans cells (LCs), populate peripheral organs constitutively. Until now, the molecular mechanism behind such differential behavior has not been elucidated. Here, we show that CD1a(+) LC precursors respond selectively and specifically to the CC chemokine macrophage inflammatory protein (MIP)-3alpha. In contrast, CD14(+) precursors of DC and monocytes are not attracted by MIP-3alpha. LCs lose the migratory responsiveness to MIP-3alpha during their maturation, and non-LC DCs do not acquire MIP-3alpha sensitivity. The notion that MIP-3alpha may be responsible for selective LC recruitment into the epidermis is further supported by the following observations: (a) MIP-3alpha is expressed by keratinocytes and venular endothelial cells in clinically normal appearing human skin; (b) LCs express CC chemokine receptor (CCR)6, the sole MIP-3alpha receptor both in situ and in vitro; and (c) non-LC DCs that are not found in normal epidermis lack CCR6. The mature forms of LCs and non-LC DCs display comparable sensitivity for MIP-3beta, a CCR7 ligand, suggesting that DC subtype-specific chemokine responses are restricted to the committed precursor stage. Although LC precursors express primarily CCR6, non-LC DC precursors display a broad chemokine receptor repertoire. These findings reflect a scenario where the differential expression of chemokine receptors by two different subpopulations of DCs determines their functional behavior. One type, the LC, responds to MIP-3alpha and enters skin to screen the epidermis constitutively, whereas the other type, the "inflammatory" DC, migrates in response to a wide array of different chemokines and is involved in the amplification and modulation of the inflammatory tissue response.     

Macrophage inflammatory protein 3alpha transgene attracts dendritic cells to established murine tumors and suppresses tumor growth

Dendritic cells (DCs) are powerful antigen-presenting cells that function as the principal activators of T cells. Since the human CC chemokine, macrophage inflammatory protein 3alpha (MIP-3alpha), is chemotactic for DCs in vitro, we hypothesized that adenovirus-mediated gene transfer of MIP-3alpha (AdMIP-3alpha) to tumors might induce local accumulation of DCs and inhibit growth of preexisting tumors. AdMIP-3alpha directed expression of mRNA and protein in vitro, and the supernatant of A549 cells infected with AdMIP-3alpha was chemotactic for DCs. In vivo, injection of AdMIP-3alpha into subcutaneous tumors resulted in local expression of the MIP-3alpha cDNA and in the local accumulation of DCs. In four syngeneic tumor models, growth of established tumors was significantly inhibited compared with untreated tumors or tumors injected with control vector, and in all but the poorly immunogenic LLC carcinoma model, this treatment increased survival advantage of the preexisting tumors. In all four tumor models, intratumoral injection of AdMIP-3alpha induced the local accumulation of CD8b. 2(+) cells and elicited tumor-specific cytotoxic T-lymphocyte activity, and adoptive transfer of splenocytes of animals receiving this treatment protected against a subsequent challenge with the identical tumor cells. In wild-type but not in CD8-deficient mice, AdMIP-3alpha inhibited the growth of tumors. Finally, AdMIP-3alpha also inhibited the growth of distant tumors. This strategy may be useful for enlisting the help of DCs to boost anti-tumor immunity against local and metastatic tumors without the necessity of ex vivo isolation and manipulation of DCs.     

Hepatocyte growth factor is the most potent endogenous stimulant of rabbit gastric epithelial cell proliferation and migration in primary culture.

Various growth factors are suggested to be involved in gastric mucosal repair. Our previous studies have shown that exogenous hepatocyte growth factor (HGF) has a proliferative effect on gastric epithelial cells. In the present study, comparison of the maximum proliferative effects and the optimum concentrations of several growth factors revealed that HGF was the most potent mitogen for gastric epithelial cells, as is the case for hepatocytes. Restitution of gastric epithelial cell monolayers was assessed using a round wound restitution model. HGF was the most effective agent for facilitating gastric epithelial restitution among those tested. A binding assay revealed specific binding of HGF to its receptor on gastric epithelial cells. Northern blot analysis confirmed the expression of specific HGF receptor mRNA (c-met) by gastric epithelial cells but not by gastric fibroblasts. To investigate endogenous HGF production, we determined the effect of gastric fibroblast-conditioned medium on epithelial proliferation and restitution. The conditioned medium produced similar effects to HGF and its activity was neutralized by an anti-HGF antibody. In addition, expression of HGF mRNA was detected in gastric fibroblasts but not in gastric epithelial cells. Our immunohistochemical study confirmed these in vitro data by means of demonstrating the existence and localization of HGF at human native gastric mucosa. HGF was localized at fibroblasts under the epithelial cell layer around gastric ulcers. These results suggest that HGF may be a potent endogenous promotor of gastric epithelial cell proliferation and migration, and may contribute to gastric mucosal repair through a paracrine mechanism.     

CXC chemokine receptor 1 (CXCR1) is expressed mainly by neutrophils in inflamed gut and stomach tissues

CXC chemokine receptor 1 (CXCR1) is one of the important receptors for CXC chemokines with ELR motif, of which interleukin 8 (IL-8; CXCL8) is representative. To identify the cell type(s) of CXCR1-expressing cells in inflamed stomach and gut tissues, we performed immunoperoxidase method using pre-fixed frozen sections. In chronic gastritis associated with Helicobacter pylori infection (7 cases), CXCR1 was positive in neutrophils (polymorphonuclear leucocytes) in the lamina propria near the neck region and those in pit abscess. In ulcerative colitis (6 cases) and Crohn's disease (5 cases), CXCR1 was sporadically expressed by neutrophils in the mucosa, and particularly CXCR1+ neutrophils were abundantly distributed in inflammatory granulation tissue in ulcer base. Double staining confirmed co-localization of CXCR1 and neutrophil elastase. Neither CD3+ T lymphocytes nor CD68+ macrophages were positive for CXCR1. Immunoelectron microscopy confirmed the cell surface localization of CXCR1. Neutrophils protect the host from microbial pathogens. However, they also cause damages to host tissues in chronic inflammation. Therefore, our study underscores the importance of CXCR1 expression in inflammatory processes.     

Macrophage inflammatory protein 1 alpha, interleukin 3 and diffusible marrow stromal factors maintain human hematopoietic stem cells for at least eight weeks in vitro

Factors that induce proliferation of the human hematopoietic stem cell are ill-defined. Primitive hematopoietic progenitors can be maintained and differentiate in stroma-dependent, long-term bone marrow cultures (LTBMC), originally described by Dexter et al. (Dexter, T. M., L. H. Coutinho, E. Spooncer, C. M. Heyworth, C. P. Daniel, R. Schiro, J. Chang, and T. D. Allen. 1990. Molecular Control of Haemopoiesis). However, 70-80% of primitive progenitors capable of reinitiating secondary stromal cultures (LTBMC-initiating cells [IC]) are lost over a period of 5 wk in such cultures. We have recently described a novel "stroma-noncontact" culture system, in which hematopoietic progenitors are separated from the stromal layer by a 0.4-micron microporous filter membrane. Primitive progenitors in such cultures can not only differentiate into committed progenitors, but are also maintained to a greater extent than in "Dexter" cultures. However, still only 50% of the originally seeded LTBMC-IC are recovered at week 5. Since maintenance of primitive progenitors may depend not only on growth- promoting factors but also on factors that inhibit differentiation and/or proliferation, we evaluated the effect of macrophage inflammatory protein 1 alpha (MIP-1 alpha) or "stem cell inhibitor" in combination with the growth-inducing factor interleukin 3 (IL-3) on the recovery of LTBMC-IC from stroma-noncontact cultures. We demonstrate that addition of MIP-1 alpha alone to stroma-noncontact cultures does not change the number of LTBMC-IC present after 8 wk, indicating that this factor may not directly inhibit or stimulate proliferation of primitive progenitors. Addition of the growth stimulatory cytokine, IL- 3, alone results in exhaustion of LTBMC-IC after 8 wk of culture, possibly as a result of their terminal differentiation. However, LTBMC- IC can be maintained for at least 8 wk when grown in stroma-noncontact cultures supplemented with both MIP-1 alpha plus IL-3. This effect depends on soluble (ill-defined) stromal factors, and results from a direct interaction of these cytokines with the progenitor population or its progeny, but not the stroma.     

Type I interferons regulate inflammatory cell trafficking and macrophage inflammatory protein 1alpha delivery to the liver

Macrophage inflammatory protein 1alpha (MIP-1alpha, CCL3) is critical for liver NK cell inflammation and delivery of IFN-gamma to mediate downstream protective responses against murine cytomegalovirus (MCMV) infections. This system was used to evaluate the upstream contribution of the type 1 IFNs, IFN-alpha/beta, in promotion of MIP-1alpha production. Mice deficient in IFN-alpha/beta functions, as a result of mutation in the receptor for these cytokines (IFN-alpha/betaR(-)), were profoundly deficient in MIP-1alpha expression and accumulation of NK cells and macrophages in the liver and had increased sensitivity to MCMV infection. The cytokines themselves were responsible for the immunoregulatory effects, since administration of recombinant IFN-alpha (rIFN-alpha) to immunocompetent mice also induced these changes. IFN-alpha/beta was required for NK cell accumulation during infection, and MIP-1alpha was required for NK cell accumulation in response to administered rIFN-alpha. In vivo trafficking assays demonstrated a requirement for IFN-alpha/betaR signaling for leukocyte localization in, and delivery of MIP-1alpha-producing macrophages to, the liver. These results extend characterization of the cytokine and chemokine cascade required for protection against viral infections in tissues by defining IFN-alpha/beta-dependent mechanisms promoting MIP-1alpha production and the resulting hepatic accumulation of NK cells.     

The unfolded protein response sensor IRE1alpha is required at 2 distinct steps in B cell lymphopoiesis

B lymphocyte differentiation is coordinated with the induction of high-level Ig secretion and expansion of the secretory pathway. Upon accumulation of unfolded proteins in the lumen of the ER, cells activate an intracellular signaling pathway termed the unfolded protein response (UPR). Two major proximal sensors of the UPR are inositol-requiring enzyme 1alpha (IRE1alpha), an ER transmembrane protein kinase/endoribonuclease, and ER-resident eukaryotic translation initiation factor 2alpha (eIF2alpha) kinase (PERK). To elucidate whether the UPR plays an important role in lymphopoiesis, we carried out reconstitution of recombinase-activating gene 2-deficient (rag2-/-) mice with hematopoietic cells defective in either IRE1alpha- or PERK-mediated signaling. IRE1alpha-deficient (ire1alpha-/-) HSCs can proliferate and give rise to pro-B cells that home to bone marrow. However, IRE1alpha, but not its catalytic activities, is required for Ig gene rearrangement and production of B cell receptors (BCRs). Analysis of rag2-/- mice transplanted with IRE1alpha trans-dominant-negative bone marrow cells demonstrated an additional requirement for IRE1alpha in B lymphopoiesis: both the IRE1alpha kinase and RNase catalytic activities are required to splice the mRNA encoding X-box-binding protein 1 (XBP1) for terminal differentiation of mature B cells into antibody-secreting plasma cells. Furthermore, UPR-mediated translational control through eIF2alpha phosphorylation is not required for B lymphocyte maturation and/or plasma cell differentiation. These results suggest specific requirements of the IRE1alpha-mediated UPR subpathway in the early and late stages of B lymphopoiesis.     

Glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1) mucin is expressed by lactating mammary gland epithelial cells and is present in milk.

Glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1) is a mucinlike endothelial glycoprotein that acts as an adhesive ligand for L selectin by presenting one or more O-linked carbohydrates to the lectin domain of this leukocyte cell surface selectin. The GlyCAM 1 glycoprotein has been previously shown to be expressed specifically by the endothelial cells of peripheral and mesenteric lymph nodes and in an unknown site in lung. Here we report that this protein is also expressed during lactation by mammary epithelial cells. Northern blot analysis has shown that the mRNA for GlyCAM 1 appears to be induced during pregnancy in a manner similar to that previously described for hormonally induced milk proteins. In situ hybridization analysis reveals that the site of GlyCAM 1 synthesis in the mammary gland is in the epithelial cells that produce these same milk proteins. Immunohistochemistry of mammary glands using antisera directed against GlyCAM 1 peptides demonstrates that these epithelial cells contain GlyCAM 1 protein, and that this protein is also found lumenally in the milk of the secreting mammary gland. Analysis of murine milk shows that immunoreactive GlyCAM 1 is found in the soluble whey fraction. Finally, labeling analysis of milk GlyCAM 1 has demonstrated that this form of the glycoprotein lacks the sulfate-modified carbohydrate that has recently been shown to be required for the ligand binding activity to L selectin. The nonsulfated mammary GlyCAM 1 is unable to interact with L selectin, consistent with the hypothesis that milk GlyCAM 1 has a different function than endothelial GlyCAM 1. These data thus suggest that milk GlyCAM 1 is a hormonally regulated milk protein that is part of the milk mucin complex. In addition, the finding that the mammary form of GlyCAM 1 contains different carbohydrate modifications than the endothelial form suggests that this glycoprotein may be a scaffold for carbohydrates that mediate functions in addition to cell adhesion.     

Fine idiotype analysis of B cell precursors in the T-dependent and T- independent responses to alpha 1-3 dextran in BALB/c mice

In this study BALB/c B cell precursors responsive to the T-independent (TI) type 2 (TI-2) antigen, dextran B1355S (DEX), and the T-dependent (TD) derivative, dextran-Limulus hemocyanin (DEX-Hy) were examined for isotype and idiotope expression using the splenic focus assay. The predominant isotype detected in the TI assay was IgM, while IgA was the predominant isotype expressed in the TD assay. There was also a fourfold increase in the number of foci secreting more than one isotype in the TD assay vs. the TI assay without an overall change in anti-DEX precursor frequency, suggesting that carrier-primed T cells enhance the expression of non-IgM isotypes possibly by increasing the frequency of isotype switching by individual B cell precursors. A panel of distinct monoclonal antiidiotype antibodies (MAIDs) was then used to examine idiotope expression by antibodies secreted in splenic foci responding to DEX and DEX-Hy. This analysis revealed considerable diversity in the idiotope profiles expressed by all isotypes tested. There appeared to be no differences in idiotope diversity among the various isotypes. A similar diversity of idiotope profiles was obtained from both TI and TD splenic foci, indicating that a comparable degree of diversity was associated with the antibodies generated by TI and TD precursors. Idiotype analysis of IgM-IgA-secreting foci with a panel of monoclonal antiidiotope antibodies revealed slight idiotypic differences between the two isotypes secreted in the same focus in about half the cases. These results suggest that somatic variation occurs during the antigen-driven maturation of B cell precursors, within the 15-d time frame of the splenic focus assay, and may be associated with isotype switching.     

Epigenetic augmentation of the macrophage inflammatory protein 2/C-X-C chemokine receptor type 2 axis through histone H3 acetylation in injured peripheral nerves elicits neuropathic pain

Although there is growing evidence showing that the involvement of chemokines in the pathogenesis of neuropathic pain is associated with neuroinflammation, the details are unclear. We investigated the C-X-C chemokine ligand type 2 [macrophage inflammatory protein 2 (MIP-2)]/C-X-C chemokine receptor type 2 (CXCR2) axis and epigenetic regulation of these molecules in neuropathic pain after peripheral nerve injury. Expression of MIP-2 and CXCR2 were up-regulated and localized on accumulated neutrophils and macrophages in the injured sciatic nerve (SCN) after partial sciatic nerve ligation (PSL). Perineural injection of MIP-2-neutralizing antibody (anti-MIP-2) or the CXCR2 antagonist N-(2-bromophenyl)-N'-(2-hydroxy-4-nitrophenyl)urea (SB225002) prevented PSL-induced tactile allodynia and thermal hyperalgesia. Perineural injection of recombinant MIP-2 elicited neuropathic pain-like behaviors. Anti-MIP-2 suppressed neutrophil accumulation in the SCN after PSL. Neutrophil depletion by intraperitoneal injection of Ly6G antibody attenuated PSL-induced neuropathic pain. Both anti-MIP-2 and SB225002 suppressed up-regulation of inflammatory cytokines and chemokines in the injured SCN. In addition, acetylation of histone H3 [lysine (Lys9)-acetylated histone H3 (AcK9-H3)] on the promoter region of MIP-2 and CXCR2 was increased in the injured SCN after PSL. Expression of AcK9-H3 was observed in the nuclei of neutrophils and macrophages surrounding the epineurium. Administration of the histone acetyltransferase inhibitor anacardic acid suppressed the up-regulation of MIP-2 and CXCR2 in the SCN after PSL and resulted in the prevention of PSL-induced neuropathic pain. Taken together, these results show that augmentation of the MIP-2/CXCR2 axis by hyperacetylation of histone H3 on the promoter region of MIP-2 and CXCR2 located in the injured peripheral nerve elicits chronic neuroinflammation through neutrophil accumulation, leading to neuropathic pain.     

The phosphatidylinositol 3-kinase inhibitor, PX-866, is a potent inhibitor of cancer cell motility and growth in three-dimensional cultures

The phosphatidylinositol 3-kinase (PI3K) pathway is activated in many human tumors and mediates processes such as cell proliferation, survival, adhesion, and motility. The natural product, wortmannin, has been widely used to study the functional consequences of PI3K inhibition in both normal and transformed cells in culture but is not a suitable cancer chemotherapeutic agent due to stability and toxicity issues. PX-866, an improved wortmannin analogue, displays significant antitumor activity in xenograft models. Here, we directly compare PX-866 and wortmannin in human cancer cell lines cultured in monolayer or as three-dimensional spheroids. Both PI3K inhibitors failed to inhibit monolayer cell growth at concentrations up to 100 nmol/L but strongly suppressed spheroid growth at low nanomolar concentrations, with PX-866 showing greater potency than wortmannin. Relative to wortmannin, PX-866 treatment results in a more sustained loss of Akt phosphorylation, suggesting that the increased potency of PX-866 is related to a more durable inhibition of PI3K signaling. PX-866 and wortmannin both inhibit spheroid growth without causing cytotoxicity, similar to known cytostatic agents, such as rapamycin. PX-866 also inhibits cancer cell motility at subnanomolar concentrations. These findings suggest that the antitumor activities of PX-866 stem from prolonged inhibition of the PI3K pathway and inhibition of cell motility. In addition, we propose that the use of three-dimensional tumor models is more predictive of in vivo growth inhibition by PI3K inhibitors in cancer cell lines lacking phosphatase and tensin homologue activity or expression.     

Involvement of the interleukin 2 pathway in the rearrangement and expression of both alpha/beta and gamma/delta T cell receptor genes in human T cell precursors

In this report, we have undertaken the phenotypic, functional and molecular characterization of a minor (less than 5%) subpopulation of adult thymocytes regarded as the earliest intrathymic T-cell precursors. Pro-T cells were immunoselected and shown to express different hematopoietic cell markers (CD45, CD38, CD7, CD5) and some activation-related molecules (4F2, Tr, HLA class II), but lack conventional T cell antigens (CD2-1-3-4-8-). TCR-gamma RNA messages are already expressed at this early ontogenic stage, while alpha and beta chain TCR genes remain in germline configuration. In vitro analyses of the growth requirements of pro-T cells demonstrated the involvement of the IL-2 pathway in promoting their proliferation and differentiation into CD3+ CD4+ or CD8+ mature thymocytes. Moreover, during the IL-2-mediated maturation process rearrangements and expression of both alpha and beta chain TCR genes occurred, and resulted in the acquisition of alpha/beta as well as gamma/delta (either disulphide-linked or non-disulphide-linked) heterodimeric TCR among the pro-T cell progeny.     

Osteopontin is involved in the initiation of cutaneous contact hypersensitivity by inducing Langerhans and dendritic cell migration to lymph nodes.

Osteopontin (OPN) is a chemotactic protein that attracts immune cells, to inflammatory sites. The sensitization phase of allergic cutaneous contact hypersensitivity (CHS) requires the migration of Langerhans cells/dendritic cells (LCs/DCs) from skin to draining lymph nodes. Characterizing OPN function for LC/DC migration we found upregulated OPN expression in hapten sensitized skin and draining lymph nodes. OPN induces chemotactic LC/DC migration, initiates their emigration from the epidermis, and attracts LCs/DCs to draining lymph nodes by interacting with CD44 and alphav integrin. Furthermore, OPN-deficient mice have a significantly reduced CHS response that correlates with an impaired ability of OPN-deficient mice to attract LCs/DCs to draining lymph nodes. In conclusion, OPN is an important factor in the initiation of CHS by guiding LCs/DCs from skin into lymphatic organs.     

Interleukin 3 and interleukin 6 synergistically promote the proliferation and differentiation of malignant plasma cell precursors in multiple myeloma

PBMC from 11 patients with multiple myeloma (MM) were cultured in vitro in presence of IL-3 and IL-6. After 3 d, actively proliferating immunoblast-like B cells (20-62%) were apparent. After 6 d, a population of morphologically evident plasma cells was observed (30-50%) that expressed, in each individual case, the same light and heavy chain produced by bone marrow malignant plasma cells. We conclude that in MM the malignant plasma cell precursors are circulating and their growth and terminal differentiation are under the synergistic control of IL-3 and IL-6.