O desenvolvimento da doença periodontal (DP) é influenciado pela resposta imunológica do hospedeiro frente ao desafio bacteriano. Apesar da função protetora de tal resposta frente à infecção, a mesma leva à destruição dos tecidos periodontais. Neste estudo, analisamos o papel de MIP-1 na imunomodulação da DP experimental em camundongos. Camundongos C57Bl/6 (WT) infectados com A. actinomycetemcomitans desenvolveram uma intensa reação inflamatória e severa reabsorção óssea alveolar, associada a uma alta expressão de MIP-1 e uma intensa migração de células CCR5+ e CCR1+ para os tecidos periodontais. Além disso, verificamos que uma intensa expressão de citocinas inflamatórias (TNF-) e do tipo Th1 (IFN-), RANKL e MMPs estava associada à progressão da doença. Entretanto, camundongos geneticamente modificados para não expressar MIP-1 (MIP-1KO), quando infectados com A. actinomycetemcomitans, apresentaram um padrão de resposta muito semelhante aos animais WT. De fato, a ausência de MIP-1 não interferiu na migração de células inflamatórias para os tecidos periodontais e na reabsorção óssea alveolar nos camundongos infectados. Semelhantemente, a expressão de citocinas (TNF-, IFN- e IL-10), dos fatores osteoclastogênicos (RANKL e OPG) e das MMPs e seus inibidores (MMP-1, MMP-2, MMP-3, TIMP-1 e TIMP-3) foram similares entre os camundogos das linhagens WT e MIP-1KO. Além disso, a ausência de MIP-1 não interferiu no controle da infecção por A. actinomycetemcomitans como demonstrado pela similaridade na quantidade de carga bacteriana presente nos tecidos e nos níveis de MPO e iNOS. Os resultados obtidos podem ser explicados pela relativa redundância no sistema quimiocinas/receptores de quimiocinas, onde mais de uma quimiocina pode ligar-se ao mesmo receptor. De fato quimiocinas como MIP-1 e RANTES, que utilizam os mesmos receptores de MIP-1 (CCR5 e CCR1), apresentaram-se intensamente expressas nos tecidos periodontais dos camundongos infectados com A. actinomycetemcomitans, independentemente da ausência de MIP-1. De fato, camundongos tratados com Met-RANTES, um antagonista específico dos CCR5 e CCR1, resultou em uma redução significativa no influxo de células inflamatórias e perda óssea alveolar quando comparados com os camundongos não tratados. Nossos resultados demonstraram que a ausência de MIP-1 não afeta o desenvolvimento da doença periodontal experimental em camundongos, provavelmente pela presença de quimiocinas homólogas que suprem sua ausência. / Periodontal disease (PD) development is highly influenced by the host immune response to the bacterial challenge. Despite the protective role of this response against infectious agents it leads to periodontal tissues destruction. In this study, we analyzed the role of chemokine MIP-1 on the immunomodulation of experimental PD in mice. C57Bl/6 (WT) mice infected with A. actinomycetemcomitans developed an intense inflammatory reaction and severe alveolar bone resorption, associated with a high expression of MIP-1 and the migration of CCR5+ and CCR1+ cells to the periodontal tissues. In addition, an intense expression of Th1 (IFN-) and inflammatory (TNF-) cytokines, RANKL and MMPs are associated with the disease progression. However, mice genetically deficient of MIP-1 (MIP-1KO), when infected with A. actinomycetemcomitans, developed a very similar response pattern to that observed for WT strain. Indeed, the absence of MIP-1 does not interfere in inflammatory cells migration to periodontal tissues and alveolar bone resorption in response to A. actinomycetemcomitans infection. Similarly, the expression of cytokines (TNF-, IFN- and IL-10), osteoclastogenic factors (RANKL and OPG) and MMPs and its inhibitors (MMP-1, MMP-2, MMP-3, TIMP-1 and TIMP-3) was similar between WT and MIP-1KO strains. Furthermore, the lack of MIP-1 does not interfere in the control of A. actinomycetemcomitans infection as demonstrated by the similar bacterial load, and similar levels of the antimicrobial MPO and iNOS. Such results can be explained by the relative redundancy of chemokine system, where more than one chemokine can bind to the same receptor. Indeed, chemokines, such as MIP-1 and RANTES, which use the same receptors used by MIP-1 (CCR1 and CCR5), are intensely expressed in periodontal tissues of mice infected with A. actinomycetemcomitans, regardless of the absence of MIP-1. In accordance, the treatment of mice with Met-RANTES, a specific antagonist of the receptors CCR5 and CCR1, resulted in a significant reduction in the influx of inflammatory cells and alveolar bone loss when compared with untreated mice. Our results demonstrate that the absence of MIP-1 does not affect the development of experimental periodontitis in mice, probably due to the presence of homologous chemokines that overcome the absence of this chemokine.

Fibroblastos são as células mais numerosas encontradas nos tecidos orais como gengiva, ligamento periodontal e polpa dental. Além de exercerem função estrutural, estas células também desempenham papel importante na resposta imune destes tecidos através do reconhecimento de antígenos e produção de mediadores inflamatórios e citocinas. Evidências apontam ainda para o fato de que fibroblastos não constituem um grupo único de células. Sendo assim, os objetivos deste estudo foram: (I) avaliar a produção diferencial de fibroblastos de gengiva, ligamento periodontal e polpa dental de dentes permanentes e decíduos quanto à produção das quimiocinas CCL3 e CXCL12; (II) avaliar a produção de pró-colágeno I pelas células de polpa e (III) avaliar a expressão diferencial dos fibroblastos quanto a microRNAs. Dentes recentemente extraídos (terceiros molares hígidos) e fragmentos de gengivas saudáveis de três pacientes adultos foram obtidos no Laboratório de Farmacologia e Fisiologia Clínica da Faculdade de Odontologia de Bauru. Caninos decíduos de dois pacientes com indicação para extração por motivos ortodônticos foram obtidos na Clínica de Odontopediatria da mesma unidade. Culturas primárias de fibroblastos de gengiva (n=3), ligamento periodontal (n=3) e polpa de dente permanente (n=3) e polpa dental de dente decíduo (n=2) foram estabelecidas a partir de tecidos humanos por meio de técnica de explant. Após a quarta passagem, a produção de CCL3 e de CXCL12 foi avaliada após estímulo com concentrações crescentes (0 10 µg/mL) de ácido lipoteicóico de Enterococcus faecalis (EfLTA), lipopolissacarídeo de Porphyromonas gingivalis (PgLPS) ou LPS de Escherichia coli (EcLPS) por ELISA após 1, 6 e 24 h. O RNAm para as quimiocinas no grupo estimulado com EcLPS por 24 h foi avaliado por transcrição reversa seguida de reação em cadeia da polimerase quantitativa. A produção de pró-colágeno I por células de polpa estimuladas com EfLTA e PgLPS foi avaliada por imunofluorescência. O perfil de expressão de microRNAs foi investigado por ensaio de microarranjo. A produção de CCL3 foi aumentada (p< 0,05) pelos antígenos empregados, porém de maneira mais evidente para EcLPS em células de gengiva. A quimiocina CXCL12 foi detectada em níveis basais em todos os grupos de células, porém em maiores quantidades em fibroblastos de gengiva seguidos pelos de ligamento periodontal. A adição dos antígenos diminuiu a produção de CXCL12 de maneira distinta entre células e entre antígenos (p< 0,05). Fibroblastos de polpa decídua não apresentaram qualquer alteração na produção desta quimiocina pelos antígenos (p> 0,05). No período experimental de 24 h, a expressão do RNAm para CXCL12 não foi alterada enquanto a de CCL3 não foi detectada. A produção de pró-colágeno I se mostrou aumentada (p< 0,05) na presença do desafio antigênico em células de polpa com exceção para fibroblastos de polpa permanente que apresentaram diminuição na produção desta proteína quando estimulados com EfLTA. Em condições basais, fibroblastos do mesmo doador apresentaram perfil distinto de expressão de microRNAs envolvidos com a produção das proteínas-alvo deste estudo. A expressão de imunomiRs por EcLPS também se mostrou modificada de maneira distinta entre os fibroblastos, em especial os de ligamento periodontal. Com base nestes resultados, pode-se concluir que fibroblastos de diferentes tecidos orais apresentam comportamento diferencial frente a antígenos bacterianos comumente relacionados a patologias que afetam a cavidade oral. / Fibroblasts are the dominant cells within oral tissues such as gingiva, periodontal ligament and dental pulp. Besides the architectural maintenance of the connective tissues, fibroblasts are also involved in connective tissue immune response through antigen recognition and production of inflammatory mediators and cytokines. Recent studies also demonstrated that fibroblasts do not constitute a unique group of cells. Taken this togeter, the objectives of the present study were: (I) to evaluate the production of the chemokines CCL3 and CXCL12 by human gingival, periodontal ligament as well as permanent and deciduous dental pulp fibroblasts; (II) to evaluate the production of procollagen I by dental pulp fibroblasts and (III) to evaluate the differential pattern of expression of microRNAs by the oral fibroblasts. Recently extracted teeth (non-carious third molars) and fragments of healthy gingiva from three adults were obtained at the Laboratory for Clinical Pharmacology and Physiology at Dental School of Bauru. Deciduous canines from two patients with orthodontic indication for extraction were obtained at Pediatrics Clinics of Dental School of Bauru. Primary cultures of fibroblasts from gingiva (n=3), periodontal ligament (n=3) as well as permanent pulp (n=3) and deciduous pulp (n=2) were established through an explant technique. After the fourth passage, fibroblasts were challenged with increasing concentrations (0 10 µg/mL) of Enterococcus faecalis lipoteichoic acid (EfLTA), Porphyromonas gingivalis lipopolysaccharide (PgLPS) or Escherichia coli LPS (EcLPS) for 1, 6 and 24 h. The chemokines were assessed through ELISA while the mRNA for CCL3 and CXCL12 (EcLPS at 24 h) were assessed through reverse transcription followed by quantitative polymerase chain reaction. The expression of microRNAs was screened through a microarray assay. The production of CCL3 on cell supernatants was detected in all cellular groups, with higher amounts at gingival fibroblasts. EcLPS induced more important chemokine differences compared to the other antigens. CXCL12 basal levels were higher for gingival fibroblasts followed by periodontal ligament ones, but also detected in dental pulp fibroblasts. The production of this chemokine was decreased by stimulation in a different fashion for each antigen and cell type. Deciduous pulp fibroblasts did not display any differences in CXCL12 synthesis even in the presence of the microbial challenge. No differences were detected at mRNA level for CXCL12, while no expression for CCL3 could be detected at 24 h. Increased production of procollagen type I was observed for dental pulp cells in general, with the only exception for permanent pulp cells which displayed decreased production of the protein with EfLTA. Microarray analysis showed differential expression pattern of microRNAs comparing unstimulated cells from the same donnor. EcLPS was able to alter immunomiRs expression in some of the cellular groups, in particular periodontal ligament fibroblasts. In conclusion, our results showed that fibroblasts from distinct oral tissues display differential behavior against bacterial antigens commonly related to the diseases that affect the oral cavity.

A nefropatia diabética é uma doença crônica caracterizada por falência renal, que torna necessária a hemodiálise. A diálise peritoneal é uma alternativa para a hemodiálise, porém causa peritonite e morte, principalmente devido à infecção com Staphylococcus aureus, especialmente em pacientes imunodeprimidos, como pacientes diabéticos. Nossa hipótese é que a insulina possa modular a peritonite causada por S. aureus. Para tanto, investigamos sua intervenção, após a indução de diabetes mellitus, na infecção peritoneal por cepas diferentes de S. aureus, analisando os mecanismos moleculares (produção/liberação de citocinas, expressão de moléculas de adesão) e a atividade microbicida dos macrófagos peritoneais envolvidos. Ratos Wistar, machos, diabéticos (aloxana, 42 mg/kg, i.v., 10 dias) e respectivos controles (salina, i.v.) foram submetidos à injeção intraperitoneal de uma suspensão de S. aureus (5x109 CFU/mL) ou volume equivalente de PBS estéril. Os animais foram submetidos a dois tratamentos com insulina NPH: 1) dose única (1UI e 4UI respectivamente, controle e diabético), administrada por via subcutânea; ou, 2) com 4 doses sendo a primeira administrada 2 horas antes da infecção, seguida de metade desta dose às 17 horas e no mesmo horário pelos próximos 2 dias (dose inicial 4UI e 1UI, grupo diabético e grupo controle, respectivamente), passadas 16 horas da última dose de insulina, a glicemia foi determinada e, em seguida, foi realizada eutanásia e coleta de amostras. Avaliamos: a) número de células no lavado peritoneal (LPe), leucograma e glicemia (monitor de glicose); b) níveis séricos de corticosterona e insulina (ELISA); c) concentrações de citocinas (IL-1&#946;, TNF-&#945;, IL-6, IFN-&#947;, IL-4, IL-10, IL-12) e quimiocinas (CINC-1, CINC-2, CINC-3) no sobrenadante do LPe (ELISA); d) expressão de moléculas de adesão (P-selectina, PECAM-1, ICAM-1) no endotélio vascular (imunoistoquímica); e) atividade microbicida. Após a infecção com a cepa ATCC 25923, comparados aos não infectados, ratos diabéticos apresentaram aumento no número de leucócitos (350%) e nas concentrações de CINC-1 (1900%), IL-1&#946; (1300%), IFN-&#947; (280%), IL-4 (800%). O tratamento destes animais com dose única de insulina diminuiu as concentrações de CINC-1 (17%) e IFN-&#947; (30%) e o leucócitos (55%); e aumentou a concentração de IL-4 (260%); enquanto o tratamento com 4 doses diminuiu o número de leucócitos (82%) e as concentrações de CINC-1 (96%) e CINC-2 (45%); e, aumentou as concentrações de TNF-&#945; (270%), IFN-&#947; (220%), IL-1&#946; (42%), IL-6 (760%) e a expressão de ICAM-1 (1360%); enquanto as concentrações de CINC-3, IL-10 e IL-12 não foram alteradas pelos tratamentos com insulina. Após a infecção com a cepa N315 HLA+, comparados aos não infectados, ratos diabéticos apresentaram aumento do número de leucócitos (200%), nas concentrações de CINC-1 (1000%), IL-4 (860%), IFN-&#947; (200%) e na expressão de PECAM-1 (800%) e diminuição de CINC-2 (92%). O tratamento destes animais com dose única de insulina diminuiu a concentração de CINC-1 (85%); e aumentou a concentração de CINC-2 (2030%), IL-1&#946; (370%) e IL-4 (250%); enquanto o tratamento com 4 doses diminuiu a concentração de CINC-1 (92%); e, aumentou número de leucócitos (66%), as concentrações de CINC-2 (100%), IL-1&#946; (490%), IL-6 (1870%) e IFN-&#947; (330%), e os outros parâmetros não foram modificados pelos diferentes tratamentos com insulina. Estes dados sugerem que a insulina possa modular a peritonite induzida por cepas diferentes de S. aureus, controlando pelo menos em parte, o infiltrado inflamatório, a produção das citocinas CINC-1, CINC-2, IL-4, IL-6, IFN-&#947;, TNF-&#945; e IL-1&#946;; e, consequentemente a expressão P-selectina e PECAM-1 no endotélio vascular do mesentério. / Diabetic nephropathy is a chronic disease characterized by kidney failure, so hemodialysis is necessary. Peritoneal dialysis is an alternative to hemodialysis, but causes peritonitis and death primarily due to infection by Staphylococcus aureus, especially in immunocompromised patients, such as diabetics. Our hypothesis is that insulin can modulate peritonitis caused by S. aureus, therefore, we investigated its action, after diabetes induction, in peritoneal infection with different S. aureus strains, analyzing the molecular mechanisms (cytokines production/release, adhesion molecules expression) and the microbicidal activity of peritoneal macrophages involved. Wistar male diabetic (alloxan, 42 mg/kg, iv, 10 days) and their respective controls (saline,iv) were subjected to intraperitoneal injection of S. aureus suspension (5x109CFU/mL) or an equivalent volume of PBS sterile. Animals were submitted to two treatments with NPH insulin administered subcutaneously : single dose (1IU and 4IU respectively , control and diabetic) 8 hours prior to euthanasia ; or 4 doses : first dose 2 hours before infection (4IU and 1IU , diabetic and control group, respectively), then half this dose of 17 pm and in the same time for the next 2 days, after 16 hours of the last dose, blood glucose was determined, and then it was carried out euthanasia and sampling. We evaluated: a) number of cells in peritoneal wash (PW), white blood cell count and blood sugar (glucose monitor) ; b) serum insulin and corticosterone (ELISA); c) concentrations of cytokines (IL-1&#946;, TNF-&#945;, IL-6, IFN-&#947;, IL-4, IL-10, IL-12) and chemokines (CINC -1, CINC-2, CINC-3 ) in supernatant of the SBA assay (ELISA); d) expression of adhesion molecules (P- selectin, ICAM-1, PECAM-1) in vascular endothelium ( immunohistochemistry); e) microbicidal activity. After infection with ATCC 25923 strain, compared to uninfected, diabetic rats showed an increase in leukocytes number (350%) and in concentrations of CINC-1 (1900%), IL-1&#946; (1300%), IFN-&#947; (280%), IL-4 (800%). Treatment of these animals with a single dose of insulin decreased concentrations of CINC-1 (17%) and IFN-&#947; (30%) and leukocytes number (55%); and increased IL-4 concentrations (260%); while treatment with 4 doses decreased leukocytes number (82%) and concentrations of CINC-1 (96%) and CINC-2 (45%); and increased concentrations of TNF-&#945; (270%), IFN-&#947; (220%), IL-1&#946; (42%), IL-6 (760%) and expression of ICAM-1 (1360%); while the concentrations of CINC-3, IL-10 and IL-12 were not affected by treatments with insulin. After infection with N315 HLA+ strain, compared to uninfected, diabetic rats showed an increase in leukocytes (200%), concentrations of CINC-1 (1000%), IL-4 (860%), IFN-&#947; (200%) and in the expression of PECAM-1 (800%); and CINC-2 decrease (92%). Treatment of these animals with a single dose of insulin decreased the concentration of CINC-1 (85%); and increased concentrations of CINC-2 (2030%), IL-1&#946; (370%) and IL-4 (250%); while treatment with 4 doses decreased the concentration of CINC-1 (92%); and increased leukocytes number (66%), concentrations of CINC-2 (100%), IL-1&#946; (490%), IL-6 (1870%) and IFN-&#947; (330%), and other parameters were not modified by treatments with insulin. These results suggest that both S. aureus strains activate differently the innate response during peritonitis, and insulin was not always able to modulate this response.

Les chimiokines (CK) et leurs récepteurs (RCK) régulent l’homéostasie leucocytaire et sont également des partenaires actifs dans la physiopathologie du syndrome WHIM et du lupus systémique (LS).Le syndrome WHIM est un déficit immuno-hématologique rare qui se caractérise notamment par une profonde lympho-neutropénie périphérique. Ce déficit est expliqué par un gain de fonction du RCK CXCR4, qui entraîne un défaut de désensibilisation du récepteur et une hyper-sensibilité à son ligand CXCL12. Malgré la lymphopénie caractéristique, les patients parviennent à générer une réponse immune humorale mais celle-ci n’est pas maintenue dans le temps. Afin de mieux comprendre la physiopathologie du syndrome WHIM, notre laboratoire a développé le modèle murin Cxcr4+/1013 qui présente une mutation gain de fonction de Cxcr4 similaire à celle observée chez certains patients. La première partie de ma thèse s’est intéressée à l’impact de ce gain de fonction de Cxcr4 sur la réponse immune. L’immunisation des souris Cxcr4+/1013 a permis de montrer que la désensibilisation de Cxcr4 limitait la différenciation des cellules productrices d’anticorps (Ac) appelées plasmocytes (PC). De plus, le gain de fonction de Cxcr4 est associé à une migration aberrante des PC et un défaut de leur maintien au long cours dans la moelle osseuse, pouvant ainsi expliquer la réponse vaccinale défectueuse observée chez les patients.La seconde partie de ma thèse a été consacrée à la caractérisation du profil migratoire des PC dans le LS. Le LS est une maladie auto-immune systémique, qui évolue par poussées et rémissions, et qui affecte plusieurs organes (peau, rein, système nerveux central…). L’initiation et l’exacerbation du LS sont médiées par les auto-Ac sécrétés par les PC autoréactifs. A ce jour, de par l’absence d’identification de marqueur spécifique de cette population, aucune thérapie ne parvient à cibler spécifiquement ces cellules. Une stratégie alternative consiste en la dérégulation de la domiciliation des PC autoréactifs dans les tissus enflammés, à l’origine même des dommages tissulaires. Ce projet s’est intéressé à l’identification du code migratoire des PC circulants de patients lupiques. J’ai cherché à savoir si les PC circulants de patients lupiques présentaient un profil d’expression de RCK distinct de ceux d’individus sains, pouvant ainsi expliquer le recrutement aberrant de ces cellules dans les tissus enflammés. Ces études ont été effectuées chez des patients en phases de poussée et de rémission mais également dans le modèle murin lupique NZB/W F1. Ces travaux suggèrent que les patients peuvent être stratifiés selon les RCK exprimés par les PC circulants. / The chemokines (CK) and their receptors (CKR) are essential for leukocyte homeostasis. They are also involved in the pathophysiology of several diseases including the WHIM syndrome and systemic lupus erythematosus (SLE).The WHIM syndrome is a rare immunodeficiency characterised by a severe peripheral lympho-neutropenia. It is caused by a gain-of-function of the CKR CXCR4, leading to a defect in desensitization of this receptor and a hyper-responsiveness to its ligand CXCL12. To better understand the pathophysiology of the WHIM syndrome, our laboratory developed the mouse model Cxcr4+/1013 harboring a natural mutation observed in some patients. Despite the lymphopenia, WHIM patients can mount a potent humoral immune response but that is not sustained over time. The maintenance of long-term antibody (Ab) titers is guaranteed by plasma cells (PCs) in the bone marrow (BM). The first part of my thesis was thus dedicated to the analysis of the role of a gain-of-function of Cxcr4 on PC differentiation and migration. The analysis of humoral response of Cxcr4+/1013 mice revealed a defect in the persistence of specific antibody titres in the absence of Cxcr4 desensitization. Furthermore, this was associated with an abnormal accumulation of a population of immature PCs in the BM.The second part of my work aimed to characterize the migratory potential of circulating PCs from SLE patients. SLE is a systemic autoimmune disease which can affect several organs like the skin, the kidneys or the central nervous system. SLE evolves in flare and remission phases and auto-Ab secreted by autoreactive PCs contribute to its pathophysiology and severity. Today, no specific treatment against autoreactive PCs exist. Targeting their migratory capacity to the inflamed tissues could be an alternative strategy. The objective of this project was to identify the migratory potential of SLE PCs. These studies were processed on samples from SLE patients during flare or remission, and on the lupus mouse model, the NZB/W F1 mice. We observed that the expression of different combinations of surface CKR may stratify several groups of SLE patients.

Chronic peritonitis contributes to technique failure in peritoneal dialysis (PD), an effective replacement therapy for chronic kidney failure. Staphylococcus aureus infection is one of the most common causes of peritonitis in PD. Interestingly, mice deficient in the cell surface heparan sulfate proteoglycan, syndecan-1, were reported to clear S. aureus corneal infection more effectively than wild-type mice. The objectives of this study were to examine the protein expression and role of syndecan-1 in leukocyte recruitment, chemokine presentation and S. aureus infection in the microcirculation underlying the parietal peritoneum in wild-type and syndecan-1-/- mice. Immunofluorescence intravital microscopy (IVM) of the parietal peritoneum microcirculation revealed that syndecan-1 was localized to the subendothelial region of venules and the mesothelial layer but does not regulate leukocyte recruitment and is not necessary for presentation of the chemokine MIP-2 in peritoneal venules. IVM was also used to study the effects of a conventional PD solution injected through a peritoneal catheter in a mouse PD model. After 6 weeks of dialysis, the peritoneal catheter implant increased leukocyte rolling and extravasation, fibrosis and angiogenesis in the parietal peritoneum independently from the dialysis solution treatment. Furthermore, the role of syndecan-1 was examined using a 4 week PD model. Four hours after infection with S. aureus through the dialysis catheter or intraperitoneal injection, the dialyzed syndecan-1-/- mice were more susceptible to S. aureus infection than undialyzed syndecan-1-/- controls and wild-type animals. IVM showed that in S. aureus infection, syndecan-1 was removed from the subendothelial surface of peritoneal venules but syndecan-1 deficiency did not affect leukocyte recruitment during S. aureus infection. This study indicates that syndecan-1 in the peritoneum and microcirculation is not a regulator of inflammatory responses but is crucial for providing a barrier to S. aureus infection, which may have important implications for susceptibility to S. aureus infections in PD. / Dissertation / Doctor of Philosophy (Medical Science)

CXCL16 is an interferon-inducible chemokine of the CXC-subfamily and functions as an adhesion molecule, when membrane bound, and a chemoattractant when soluble. Upregulation of cell associated CXCL16 (cCXCL16) in colorectal cancer is associated with increased tumor infiltrating lymphocytes and good prognosis. ADAM10 and ADAM17 are metalloproteinases responsible for cleaving CXCL16, releasing soluble CXCL16 (sCXCL16) and contributing to proliferation and migration of mesangial cells, in kidney inflammatory disease. We hypothesize that cCXCL16 is a substrate for ADAM10 and ADAM17 cleavage in colorectal cancer, releasing sCXCL16, which mediates cell proliferation. To this end, we first identified CXCL16 in the human colon carcinoma cell line, RKO, by immunohistochemistry. cCXCL16 was found in the membrane, cytoplasm and nucleus. We treated RKO, in vitro, with an inflammatory cytokine mix containing 1.4 nM rhIFN[gamma], 2.0 nM rhTNF[alpha] and 2.0 nM rhIL1[beta] to increase the cleavage of cCXCL16 to sCXCL16. Overnight incubation with the cytokine mix significantly (P=.004) increased the release of sCXCL16 compared to normal conditions. To confirm that a metalloproteinase is responsible for the cleavage of cCXCL16, we used a broad spectrum metalloproteinase inhibitor, GM6001, in combination with inflammatory stimulation, in cell culture. We assayed the supernatant using ELISA for sCXCL16. GM6001 at 100 [mu]M decreased sCXCL16 to levels indistinguishable from the background. Using siRNA, we knocked down the expression of ADAM10 and ADAM17, independently, to determine if the activity of each on cCXCL16 was mediated by inflammatory stimulation. It was shown that ADAM10 constitutively cleaved cCXCL16, and ADAM17 cleavage activity was induced by inflammatory stimulation. To determine if sCXCL16 increased colorectal cancer cell (CRC) proliferation through ligand-receptor binding, we treated cells with a range of rhCXCL16 from 3.125-100 ng/mL. rhCXCL16 did not increase RKO proliferation at doses up to 100 ng/mL. We used GM6001, to inhibit the cleavage of cCXCL16 into sCXCL16 then performed an ATPase assay and 6 day cell cycle analysis, under inflammatory stimulation. Increased cleavage of cCXCL16 induced by inflammatory stimulation with the cytokine mix containing 1.4 nM rhIFN[gamma], 2.0 nM rhTNF[alpha] and 2.0 nM rhIL1[beta], increased RKO proliferation and reduced apoptosis. We conclude that ADAM10 and ADAM17 cleavage of cCXCL16 to sCXCL16 is increased by ADAM17 activation with inflammatory stimulation. The cleavage of the extracellular portion from cCXCL16 is associated with increased proliferation and decreased apoptosis of colorectal cancer cells.

No description available.

No description available.

No description available.

Opioid receptor modulation of pro-inflammatory cytokine production is vital for host defense and the inflammatory response. Previous results have shown the mu-opioid receptor (MOR) selective agonist, DAMGO, has the capacity to increase the expression of the pro-inflammatory chemokines, CCL2/MCP-1, CCL5/RANTES and CXCL10/IP-10 in peripheral blood mononuclear cells (PBMCs). We have shown that MOR activation is able to induce the expression of TGF-β, and TGF-β appears to be required for induction of CCL5 following MOR activation. This work suggests a novel role for TGF-β in the inflammatory response. NF-κB is a transcription factor that plays a pivotal role in inflammation and the immune response. We have found that NF-kB inhibitors can prevent the MOR-induced activation of CCL2 and CCL5, and that the NF-kB subunit, p65, is phosphorylated at serine residues 311 and 536 in response to μ-opioid receptor activation. In vivo, DAMGO administration can induce binding of p. 65 to the enhancer region of the CCL2 promoter. Furthermore, we demonstrate that PKCζ is phosphorylated following DAMGO-induced MOR activation and, is essential for NF-kB activity as well as CCL2 expression and transcriptional activity. In conclusion, these data suggest a pro-inflammatory role for MOR which involves NF-κB activation and PKCζ as well as a novel role for TGF-β as a regulator of pro-inflammatory chemokines. / Molecular Biology and Genetics