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.     

Control of Salmonella dissemination in vivo by macrophage inflammatory protein (MIP)-3alpha/CCL20

While chemokines are clearly important in the generation of protective immunity, the role of individual chemokines in the control of bacterial infection is still poorly understood. In this study, we investigated the role of macrophage inflammatory protein (MIP)-3alpha/CCL20, a chemokine that attracts activated T and B lymphocytes and immature dendritic cells, in host responses to bacterial infection. CCL20 production was induced in subcutaneous tissue in the BALB/c mouse in response to Salmonella enteritidis, Staphylococcus aureus and zymosan, with S. enteritidis being the most potent. S. enteritidis induced CCL20 production in the spleen following either oral administration or injection into the peritoneal cavity. In contrast, no increase was observed in the Peyer's patches. In this model, following intraperitoneal injection, dose-dependent colonization of the spleen and Peyer's patches by S. enteritidis, expression of IFNgamma and IL-4, and production of antibodies against the S. enteritidis surface antigen SefA were observed. Prior treatment with neutralizing antibodies against CCL20 enhanced bacterial dissemination to the spleen and Peyer's patches and strongly biased the IFNgamma/IL-4 ratio towards a type 2 profile in the spleen, while the humoral response was unaffected. In contrast, treatment with neutralizing anti-MIP-1alpha/CCL3 antibodies enhanced the bacterial burden in the Peyer's patches but not in the spleen, had no significant effect on the cytokine ratio, but significantly inhibited anti-SefA production. Together, these results demonstrate an important role for CCL20 in the control of bacterial infection and more specifically in the regulation of cell-mediated immunity against intracellular bacteria such as S. enteritidis.     

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.     

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.     

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.     

Effects of estradiol on lipopolysaccharide and Pam3Cys stimulation of CCL20/macrophage inflammatory protein 3 alpha and tumor necrosis factor alpha production by uterine epithelial cells in culture

We have previously demonstrated that rat uterine epithelial cells (UEC) produce CCL20/macrophage inflammatory protein 3 alpha (MIP3alpha) and tumor necrosis factor alpha (TNF-alpha) in response to live and heat-killed Escherichia coli and to the pathogen-associated molecular patterns (PAMP) lipopolysaccharide (LPS) and Pam3Cys. To determine whether estradiol (E2) modulates PAMP-induced CCL20/MIP3alpha and TNF-alpha secretion, primary cultures of rat UEC were incubated with E2 for 24 h and then treated with LPS or Pam3Cys or not treated for an additional 12 h. E2 inhibited the constitutive secretion of TNF-alpha and CCL20/MIP3alpha into culture media. Interestingly, E2 pretreatment enhanced CCL20/MIP3alpha secretion due to LPS and Pam3Cys administration. In contrast, and at the same time, E2 lowered the TNF-alpha response to both PAMP. To determine whether estrogen receptors (ER) mediated the effects of E2, epithelial cells were incubated with E2 and/or ICI 182,780, a known ER antagonist. ICI 182,780 had no effect on E2 inhibition of constitutive TNF-alpha and CCL20/MIP3alpha secretion. In contrast, ICI 182,780 reversed the stimulatory effect of E2 on LPS- and/or Pam3Cys-induced CCL20/MIP3alpha secretion as well as partially reversed the inhibitory effect of E2 on TNF-alpha production by epithelial cells. Overall, these results indicate that E2 regulates the production of TNF-alpha and CCL20/MIP3alpha by UEC in the absence as well as presence of PAMP. Since CCL20/MIP3alpha has antimicrobial activity and is chemotactic for immune cells, these studies suggest that regulation of CCL20/MIP3alpha and TNF-alpha by E2 and PAMP may have profound effects on innate and adaptive immune responses to microbial challenge in the female reproductive tract.     

Role of C-C chemokine receptors 1 and 5 and CCL3/macrophage inflammatory protein-1alpha in the cutaneous Arthus reaction: possible attenuation of their inhibitory effects by compensatory chemokine production

The deposition of immune complexes induces an acute inflammatory response with tissue injury. Immune complex-induced tissue injury is mediated by inflammatory cell infiltration that is highly regulated by multiple chemokines. To assess the role of the chemokine receptors CCR1 and CCR5, and a ligand for these receptors CCL3/macrophage inflammatory protein-1alpha, in this pathogenic process, the reverse passive cutaneous Arthus reaction was induced in mice lacking CCR1, CCR5, or CCL3. Edema was significantly attenuated in CCR1-deficient (CCR1(-/-)) and CCL3(-/-) mice but not CCR5(-/-) mice, compared with wild-type mice. Numbers of infiltrating neutrophils and mast cells were reduced in CCL3(-/-) and CCR1(-/-) mice, respectively, compared with wild-type mice. CCR1 and CCR5 were expressed on neutrophils and mast cells. Remarkably, the intradermal mRNA expression of CCL5/RANTES, another ligand for CCR1 and CCR5, was increased in CCR5(-/-) and CCL3(-/-) mice, compared with wild-type mice, while the cutaneous CCL3 mRNA expression was augmented in CCR1(-/-) and CCR5(-/-) mice. These results indicate that CCR1, CCR5, and CCL3 cooperatively contribute to the cutaneous Arthus reaction, and also suggest that enhanced expression of CCL3 and CCL5 compensates for the loss of CCR1, CCR5, and CCL3 in the reaction.     

Interleukin-4 and IFN-gamma differentially stimulate macrophage chemoattractant protein-1 (MCP-1) and eotaxin production by intestinal epithelial cells.

When the intestine becomes infected by pathogenic organisms, intestinal epithelial cells (IEC) respond with the production of chemokines, which then attract and activate specific subsets of leukocytes. During chronic inflammation, the panel of IEC chemokines produced likely represents the net effect of a plethora of mediators present in the milieu, including cytokines from activated T lymphocytes. To explore the influence of T lymphocyte cytokines, we treated IEC-18 cells with interferon-y (IFN-gamma) and interleukin-4 (IL-4) and measured the effect on production of the CC chemokines, monocyte chemoattractant protein-1 (MCP-1) and eotaxin, and the CXC chemokine, macrophage inflammatory protein-2 (MIP-2). Both IFN-gamma and IL-4 enhanced MCP-1 mRNA levels but with different kinetics. IFN-gamma stimulated a transient increase in MCP-1 mRNA levels, which peaked at 2 h, whereas IL-4-stimulated MCP-1 mRNA levels were markedly increased at 1 h and remained elevated at all time points studied. With each stimulus, the increase in MCP-1 mRNA levels was accompanied by a steady time-dependent increase in MCP-1 secretion. In addition, treatment with IFN-gamma or IL-4 enhanced IL-1beta-stimulated MCP-1 mRNA production and protein secretion. Eotaxin mRNA was detectable in unstimulated IEC-18 cells, and IL-4 but not IFN-gamma caused a rapid enhancement in levels, which remained elevated for 24 h after treatment. Finally, IL-1beta but not IFN-gamma or IL-4 enhanced MIP-2 mRNA levels. Knowledge gained from studying the outcome of T lymphocyte-derived stimuli will help understand the complex sequence of events during chronic intestinal inflammation.     

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.     

Monocyte chemoattractant protein-1 or macrophage inflammatory protein-1alpha deficiency does not affect angiotensin II-induced intimal hyperplasia in carotid artery ligation model.

BACKGROUND: Angiotensin II (Ang II) promotes atherosclerotic vascular diseases, in which proinflammatory and proliferative effects play a major pathogenic role. Ang II up-regulates chemokines, such as monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha, which are important pro-inflammatory factors mediating infiltration of inflammatory cells into atherosclerotic lesion. The aim of the present study was to determine whether the presence of MCP-1 or MIP-1alpha is essential in Ang II-induced intimal hyperplasia in the carotid artery ligation model. METHODS: Six-month-old male C57BL/6-, MCP-1-, or MIP-1alpha-deficient mice underwent ligation of the common left carotid artery and were randomly assigned to receive either vehicle or Ang II (1.4 mg kg(-1) day(-1)) via a subcutaneously implanted osmotic infusion pump (model 2004, Alzet) for 4 weeks. RESULTS: Ang II not only increased MCP-1 and MIP-1alpha production but also enhanced neo-intimal formation, media thickness, and adventitia development in the ligated carotid arteries in C57BL/6 mice. However, MCP-1 or MIP-1alpha deficiency failed to affect intimal hyperplasia in vascular remodeling. CONCLUSION: These results indicate that MCP-1 or MIP-1alpha may not be essential in mediating the proliferative effects of Ang II, a major pathological changes in intimal hyperplasia in the carotid artery ligation model.     

Effect of oestradiol on PAMP-mediated CCL20/MIP-3 alpha production by mouse uterine epithelial cells in culture

The present study was undertaken to establish whether mouse uterine epithelial cells produce CCL20/macrophage inflammatory protein 3 alpha (CCL20/MIP-3 alpha) and to determine whether secretion is under hormonal control and influenced by pathogen-associated molecular patterns (PAMPs). In the absence of PAMPs, polarized uterine epithelial cells grown to confluence on cell culture inserts constitutively secreted CCL20/MIP-3 alpha with preferential accumulation into the apical compartment. When epithelial cells were treated with the Toll-like receptor (TLR) agonists Pam3Cys (TLR2/1), peptidoglycan (TLR2/6) or lipopolysaccharide (LPS; TLR4), CCL20/MIP-3 alpha increased rapidly (4 hr) in both apical and basolateral secretions. Time-course studies indicated that responses to PAMPs added to the apical surface persisted for 12-72 hr. Stimulation with loxoribin (TLR7) and DNA CpG motif (TLR9) increased basolateral but not apical secretion of CCL20/MIP-3 alpha. In contrast, the viral agonist Poly(I:C) (TLR3) had no effect on either apical or basolateral secretion. In other studies, we found that oestradiol added to the culture media decreased the constitutive release of CCL20/MIP-3 alpha. Moreover, when added to the culture media along with LPS, oestradiol inhibited LPS-induced increases in CCL20/MIP-3 alpha secretion into both the apical and basolateral compartments. In summary, these results indicate that CCL20/MIP-3 alpha is produced in response to PAMPs. Since CCL20/MIP-3 alpha is chemotactic for immature dendritic cells, B cells and memory T cells and has antimicrobial properties, these studies suggest that CCL20/MIP-3 alpha production by epithelial cells, an important part of the innate immune defence in the female reproductive tract, is under hormonal control and is responsive to microbial challenge.     

The production and clinical evaluation of macrophage colony-stimulating factor and macrophage chemoattractant protein-1 in human follicular fluids

PROBLEM: In order to investigate the role of macrophage colony-stimulating factor (M-CSF) and macrophage chemoattractant protein-1 (MCP-1) in human ovulation, we measured the concentrations of M-CSF and MCP-1 in human follicular fluids (FFs) and correlated them with oocyte maturation. METHOD OF STUDY: The oocytes were obtained from the FFs of 46 women undergoing in vitro fertilization and embryo transfer (IVF ET). The concentrations of M-CSF and MCP-1 in the FFs were measured by enzyme-linked immunosorbent assay. In addition, granulosa cells obtained from the FFs of IVF patients were cultured and treated with forskolin and 12-O-tetradecanoylphorbol 13-acetate (TPA) for 24 48 hr. RESULTS: Concentrations of M-CSF and MCP-1 were significantly higher in the FFs than in the serum (P < 0.01). M-CSF concentrations tended to be higher, while MCP-1 concentrations were significantly higher in the FFs containing mature oocytes than in FFs containing immature oocytes (P < 0.05). The production of M-CSF was markedly increased over the basal level after treatment with forskolin (10 microM) for 24 (P < 0.02) and 48 hr (P < 0.01); however, the production of MCP-1 was unchanged. CONCLUSIONS: Our data suggest that M-CSF and MCP-1 may play an important role in human preovulatory processes and that M-CSF, in particular, may be regulated by cyclic adenosine monophosphate. M-CSF and MCP-1 may also be valuable biochemical markers in the evaluation of oocyte maturation.     

同型半胱氨酸对单核细胞NF-κB活化及巨噬细胞炎性蛋白-1α表达的影响

目的　探讨同型半胱氨酸对体外培养的人单核细胞株THP -1核因子-κB(NF -κB)、抑制因子(IκB- α)活性的影响及其与巨噬细胞炎性蛋白-1α(MIP- 1α)表达上调的关系。方法　THP -1单核细胞分别用同型半胱氨酸和NF- κB抑制剂PDTC预处理后,应用Northernblot和流式细胞术分别检测MIP- 1αmRNA和蛋白的表达,并应用Western蛋白印迹进一步检测核蛋白NF -κB蛋白含量和胞质IκB -α含量。结果　与未加任何处理因素的对照组比较,MIP- 1αmRNA和蛋白在0. 1mmol/L同型半胱氨酸处理后明显增加,分别为对照组的3 .69倍和1 .16倍(P<0 .01),同时NF κBP65亚基核转位亦增加。而加入100μmol/LNF κB抑制剂PDTC预处理30min后,再用同样浓度的同型半胱氨酸刺激,则MIP -1αmRNA和蛋白表达受到明显的抑制,NF- κBP65核转位增加。单独加入PDTC对MIP -1αmRNA、蛋白表达及NF -κBP65核转位无明显影响。此外,同型半胱氨酸( 0 .1mmol/L)处理THP- 1可引起IκB -α蛋白水平显著降低, 120min后有所回升。结论　同型半胱氨酸在病理浓度可促进NF -κB活化,发生核转位,进而促进THP -1细胞表达MIP 1αmRNA和蛋白,这种作用与IκB -α蛋白磷酸化降解有关。