Primary human alveolar type II epithelial cell CCL20 (macrophage inflammatory protein-3alpha)-induced dendritic cell migration

Inhalation of antigenic matter stimulates rapid recruitment of dendritic cells (DCs) into the lung. Recent studies propose that the chemokine CCL20 (macrophage inflammatory protein-3alpha) may play an important role in DC recruitment. We previously showed that primary human alveolar type II epithelial (ATII) cells are a rich source of chemokines and so hypothesized that the ATII cell produces CCL20 and might therefore be a key regulator of DC recruitment into the lung. Here, we show that primary human ATII cells, but not human alveolar macrophages, produce CCL20 both constitutively (403.5 +/- 85.4 pg/ml; 24 h) and in response to endotoxin (lipopolysaccharide) exposure (1,525.0 +/- 169.4 pg/ml; 1 mug/ml lipopolysaccharide; 24 h) in a time- and dose-dependent manner. In addition, we show that peripheral blood monocyte-derived CD1a+ DCs migrate in response to conditioned media from ATII cells but not those from alveolar macrophages; DC migration was significantly correlated with the amount of CCL20 (r(2) > 0.9; P < 0.05) detected in the media but not with any other chemokine measured. We therefore conclude that the alveolar epithelium is an important source of CCL20 in the lung and that the ATII cell may play a critical role in controlling the movement of DCs through the lung both under normal and inflammatory conditions.     

Serum amyloid A induces CCL20 secretion in mononuclear cells through MAPK (p38 and ERK1/2) signaling pathways

Although the serum levels of SAA had been reported to be upregulated during inflammatory/infectious process, the role of this acute-phase protein has not been completely elucidated. In previous studies, we demonstrated that SAA stimulated the production of TNF-alpha, IL-1beta, IL-8, NO, and ROS by neutrophils and/or mononuclear cells. Herein we demonstrate that SAA induces the expression and release of CCL20 from cultured human blood mononuclear cells. We also focus on the signaling pathways triggered by SAA. In THP-1 cells SAA promotes phosphorylation of p38 and ERK1/2. Furthermore, the addition of SB203580 (p38 inhibitor) and PD98059 (ERK 1/2 inhibitor) inhibits the expression and release of CCL20 in mononuclear cells treated with SAA. Our results point to SAA as an important link of innate to adaptive immunity, once it might act on the recruitment of mononuclear cells.     

Hypoxia induces expression of the chemokines monocyte chemoattractant protein-1 (MCP-1) and IL-8 in human dermal fibroblasts

Hypoxia is an important factor in the pathophysiology of vascular and inflammatory diseases. Leucocyte infiltration, as a consequence of adhesion molecule up-regulation and chemokine release, is a prominent feature of these diseases. The objective of our study was to investigate the potential role of resident fibroblasts in hypoxia-induced chemotactic responses. We show that MCP-1 and IL-8 mRNA are specifically induced by hypoxia in dermal fibroblasts. This response is paralleled by increased NF-kappaB p65/p50 binding activity, and it is inhibited by pretreatment with N-acetyl-L-cysteine. MCP-1 secreted by fibroblasts is chemotactic for monocytic cells and this activity is significantly increased by hypoxia. Chemotactic index correlates with MCP-1 protein levels and is significantly decreased by neutralizing anti-MCP-1 MoAb. These findings demonstrate the ability of resident fibroblasts to mediate chemotaxis of leucocytes through the release of chemokines in response to hypoxia. Our data point to MCP-1 as an important component in this response, and therefore it may be a potential target in inflammatory responses associated with hypoxia.     

Neutrophils produce biologically active macrophage inflammatory protein-3alpha (MIP-3alpha)/CCL20 and MIP-3beta/CCL19.

Macrophage inflammatory protein-3alpha (MIP-3alpha)/CCL20 and MIP-3beta/CCL19 are members of the CC chemokine subfamily which exert their effects through specific receptors, CCR6 and CCR7, respectively. Previously, we have reported that human neutrophils have the capacity to produce a number of chemokines, including IL-8/CXCL8, GROalpha/CXCL1, IP-10/CXCL10, and MIG/CXCL9. Herein, we show that neutrophils also have the ability to express and release MIP-3alpha/CCL20 and MIP-3beta/CCL19 when cultured with either LPS or TNF-alpha. We also report that MIP-3alpha/CCL20 and MIP-3beta/CCL19 production by LPS-stimulated neutrophils is negatively modulated by IL-10. Remarkably, we found that supernatants harvested from stimulated neutrophils not only induced chemotaxis of both immature and mature dendritic cells (DC), but also triggered rapid integrin-dependent adhesion of CCR6- and CCR7-expressing lymphocytes to purified VCAM-1 and ICAM-1, respectively. Importantly, both chemotaxis and rapid integrin-dependent adhesion were dramatically suppressed by anti-MIP-3alpha/CCL20 and anti-MIP-3beta/CCL19 neutralizing antibodies, indicating that MIP-3alpha/CCL20 and MIP-3beta/CCL19 present in the supernatants were both biologically active. As these chemokines are primarily chemotactic for DC and specific lymphocyte subsets, the ability of neutrophils to produce MIP-3alpha/CCL20 and MIP-3beta/CCL19 might be significant in orchestrating the recruitment of these cell types to the inflamed sites and therefore in contributing to the regulation of the immune response.     

CCL20/macrophage inflammatory protein-3alpha production in LPS-stimulated neutrophils is enhanced by the chemoattractant formyl-methionyl-leucyl-phenylalanine and IFN-gamma through independent mechanisms

We have recently demonstrated that polymorphonuclear neutrophils (PMN), when cultured with LPS or TNF-alpha, have the capacity to release CCL20, a chemokine primarily chemotactic for immature dendritic cells and specific lymphocyte subsets. Here, we report that the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP), as well as the immunoregulatory cytokine IFN-gamma, can significantly up-modulate the production of neutrophil-derived CCL20 through entirely unrelated mechanisms. We found that fMLP dramatically up-regulates CCL20 mRNA expression and synthesis in neutrophils stimulated with LPS for 2-3 h, and that its effect takes place through CCL20 mRNA stabilization. In contrast, IFN-gamma potentiates CCL20 gene expression and production only after 21 h of LPS treatment, its effect being mediated by endogenous TNF-alpha in an autocrine fashion, as revealed using neutralizing anti-TNF-alpha antibodies added to IFN-gamma plus LPS-treated PMN. Finally, we demonstrate that activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in mediating the production of CCL20 induced by LPS (with or without IFN-gamma), whereas activation of p42/44 extracellular signal-regulated kinases (ERK) is involved in the enhancing effect of fMLP. Taken together, these findings identify novel biological actions exerted by fMLP and IFN-gamma, potentially involved in the orchestration of inflammatory and immune responses within epithelial and mucosal tissue.     

CCL20/macrophage inflammatory protein 3alpha and tumor necrosis factor alpha production by primary uterine epithelial cells in response to treatment with lipopolysaccharide or Pam3Cys

Having previously shown that CCL20/macrophage inflammatory protein 3alpha and tumor necrosis factor alpha (TNF-alpha) are released by polarized primary rat uterine epithelial cells (UEC) in response to Escherichia coli but not to Lactobacillus rhamnosus, we sought to determine if epithelial cells are responsive to pathogen-associated molecular patterns (PAMP), including lipopolysaccharide (LPS), lipoteichoic acid (LTA), and Pam(3)Cys, a bacterial lipoprotein analog. Epithelial cells were grown to confluence on Nunc cell culture inserts prior to apical treatment with PAMPs. In response to LPS, LTA, and Pam(3)Cys (EMC Microcollection GmbH, Tubingen, Germany), CCL20 levels increased (4- to 10-fold) while PAMPs caused increased TNF-alpha (1- to 4-fold) in the medium collected after 24 h of incubation. Both apical and basolateral secretion of CCL20 and TNF-alpha increased in response to PAMPs, but treatments had no effect on cell viability and integrity, as measured by transepithelial resistance. Time course studies of CCL20 and TNF-alpha release in response to Pam(3)Cys and LPS indicated that CCL20 release peaked between 2 and 4 h after treatment, whereas TNF-alpha release was gradual over the length of the incubation. Freeze-thaw and cell lysis experiments, along with actinomycin D studies, suggested that CCL20 and TNF-alpha are synthesized in response to PAMP stimulation. Taken together, these studies demonstrate that E. coli and selected PAMPs have direct effects on the production of CCL20 and TNF-alpha without affecting cell integrity. Since CCL20 is known to be both chemotactic and antimicrobial, the increase in apical and basolateral release by UEC in response to PAMPs suggests a new mechanism of innate immune protection in the female reproductive tract.     

Hepatocyte nuclear factor 1 alpha (HNF-1 alpha) mutations in maturity-onset diabetes of the young

Maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes mellitus characterized by autosomal dominant inheritance, early age of onset (<25 years) and pancreatic beta-cell dysfunction. MODY is genetically heterogeneous with five different genes identified to date: hepatocyte nuclear factor-4 alpha (HNF-4 alpha) [MODY1]; glucokinase [MODY2]; hepatocyte nuclear factor-1 alpha (HNF-1 alpha) [MODY3]; insulin promoter factor-1 (IPF-1) [MODY4]; and hepatocyte nuclear factor-1 beta (HNF-1 beta) [MODY5]. Mutations in the HNF-1 alpha gene represent a common cause of MODY in the majority of populations studied. Sixty-five different mutations have been described in a total of 116 families. The most common mutation is a C-insertion (P291fsinsC) in the polyC tract of exon 4, which has been reported in 22 families. The identification of an HNF-1 alpha gene mutation in a patient with type 2 diabetes confirms the diagnosis of MODY and has important implications for clinical management.     

Substance P regulates macrophage inflammatory protein 3alpha/chemokine C-C ligand 20 (CCL20) with heme oxygenase-1 in human periodontal ligament cells

Although substance P (SP), a potent proinflammatory peptide, is involved in inflammation and immune responses, the effect of SP on the expression of macrophage inflammatory protein 3alpha[MIP-3alpha, chemokine C-C ligand 20 (CCL20)] in periodontal ligament (PDL) cells is unknown. Equally enigmatic is the link between SP, the stress protein heme oxygenase-1 (HO-1), and CCL20 production. We investigated whether SP induces the release of chemokine CCL20 from immortalized PDL (IPDL) cells, and further clarify SP-mediated pathways. We also examined the relationship between HO-1 and CCL20 by treating PDL cells with SP. Incubating IPDL cells with SP increased expression of CCL20 mRNA and CCL20 protein in a dose-time-dependent manner. Highly selective p38 and extracellular-regulated kinase 1/2 (ERK1/2) inhibitors abrogated SP-induced expression of CCL20 in IPDL cells. SP is also responsible for initiating phosphorylation of IkappaB, degradation of IkappaB and activation of nuclear factor (NF)-kappaB. SP induced expression of HO-1 in both a concentration- and time-dependent manner, and CCL20 reflected similar patterns. The inductive effects of SP on HO-1 and CCL20 were enhanced by HO-1 inducer hemin and the membrane-permeable guanosine 3',5'-monophosphate (cGMP) analogue 8-bromo-cGMP. Conversely, this pathway was inhibited by the HO-1 inhibitor zinc protoporphyrin IX (ZnPP IX) and the selective inhibitor of guanylate cyclase, 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ). We report herein the pathway that connects SP along with other modulators of neuroimmunoregulation to the induction of HO-1 and the inflammatory mediator macrophage inflammatory protein (MIP)-3alpha/CCL20 in IPDL cells, which play an important role in the development of periodontitis or inflammation during orthodontic tooth movement.     

Production of macrophage inflammatory protein 3alpha (MIP-3alpha) (CCL20) and MIP-3beta (CCL19) by human peripheral blood neutrophils in response to microbial pathogens

Effects of bacterial pathogens on the production of macrophage inflammatory protein 3alpha (MIP-3alpha) and MIP-3beta from human peripheral blood neutrophils were investigated. Neutrophils produced both chemokines by coincubation with either gram-positive or gram-negative bacteria. Neutrophils may initiate antigen-specific immune responses through the release of these chemokines that are capable of promoting selective recruitment of dendritic cells and T-cell subsets.     

Modulation of surface CD11/CD18 glycoproteins (Mo1, LFA-1, p150,95) by human mononuclear phagocytes

Mo1, LFA-1, and p150,95 are structurally related glycoproteins of the CD11/CD18 complex that are expressed on the membrane of human leukocytes. In the neutrophil, the surface expression of the CD11/CD18 complex is up-modulated (Mo1 greater than p150,95 much greater than LFA-1) by stimulatory factors that include calcium ionophore A23187, phorbol myristate acetate (PMA), and N-L-formyl-L-leucyl-L-phenylalanine (fMLP). Here, in an immunofluorescence analysis, we have examined CD11/CD18 glycoprotein expression by human monocytes, pulmonary alveolar macrophages (PAM, obtained by bronchoalveolar lavage), and breast milk macrophages (BMM) as compared to neutrophils before and after exposure to A23187 (1 microM), fMLP (0.1 microM), or PMA (0.1 microgram/ml) for 15 min at 37 degrees C. Unstimulated monocytes within unfractionated blood mononuclear cells kept at 4 degrees C (n = 13) expressed all three CD11/CD18 glycoproteins, and exposure to A23187 resulted in significant increases in the surface expression of Mo1 (median of 5.7-fold), LFA-1 (median of 2.1-fold), and p150,95 (median of 7.2-fold). Exposure to fMLP- or PMA-stimulated increases of lesser magnitude. CD11/CD18 expression by PAM (n = 9) was barely detectable and was unaffected by exposure to A23187. In contrast, BMM (n = 11) expressed all three CD11/CD18 glycoproteins (with considerable variability among specimens), but no increase was stimulated by A23187. These results demonstrate that monocytes, like neutrophils, have the capacity to respond to activating factors with an increase in CD11/CD18 glycoprotein expression; macrophage differentiation is accompanied by a loss (PAM) or retention (BMM) of CD11/CD18 expression that is unmodulated in response to activation.     

Insulin-like growth factor (IGF)-I and IGF binding protein-3 concentrations in fluid from human stimulated follicles

Insulin-like growth factor-I (IGF-I) and IGF binding protein-3 (IGFBP- 3) play an important role in regulating follicle growth and maturation. We have evaluated whether responsiveness to gonadotrophins during an in- vitro fertilization (IVF) treatment is related to follicular fluid IGF- I and IGFBP-3 concentrations. We also investigated if a difference is present in IGF-I and IGFBP-3 concentrations between patients treated with human menopausal gonadotrophin (HMG) and patients treated with highly purified follicle stimulating hormone (FSH). We have measured IGF-I and IGFBP-3 in follicular fluid from pre-ovulatory follicles in an IVF programme. All 70 patients were stimulated after being down- regulated with a gonadotrophin-releasing hormone (GnRH) analogue. IGF-I concentrations in follicular fluid were significantly inversely correlated with the number of ampoules FSH administered and number of days of FSH administration, and significantly correlated with the number of follicles aspirated. IGFBP-3 concentrations were not correlated with any other parameter measured nor were IGF-I and IGFBP-3 concentrations correlated. IGFBP-3 concentrations were significantly higher in patients receiving highly purified FSH compared with patients receiving HMG (P < 0.005). These results are new evidence that IGF-I concentration in follicular fluid is higher in women who respond better to follicular stimulation, i.e. women who grow many follicles, women who need a shorter duration of stimulation and women who need fewer ampoules FSH before oocyte retrieval.      

IgA specific helper factor (alpha HF) in human colostrum.

Induction of immunoglobulin secretion by human colostrum was investigated using human peripheral blood lymphocytes (PBL) and Epstein-Barr virus transformed human B lymphoblastoid cells. Stimulation of the cells with colostrum induced IgA plaque forming cells but neither IgG nor IgM plaque forming cells, indicating the occurrence of IgA specific helper factor (alpha HF) in human colostrum. alpha HF activity was eluted into fractions with an apparent molecular weight of about 80 kD by gel filtration, and with a PI range of 5.8 to 6.2 by chromatofocusing. IgA secreted by PBL stimulated with alpha HF had a similar molecular weight distribution to that of IgA in human colostrum. From these results a hypothesis is proposed; IgA-committed B cells in the mammary gland differentiate to plasma cells producing dimeric IgA after stimulation by alpha HF so that the dominant immunoglobulin in human colostrum is IgA.