Inhibitory effect of the coffee diterpene kahweol on carrageenan-induced inflammation in rats

Previous studies reported that kahweol, a coffee-specific diterpene, inhibits cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in cultured lipopolysaccharide-activated macrophages. The aim of this study was to confirm the anti-inflammatory effects of kahweol by examining its effect on the inflammatory response induced by carrageenan in a rat using an acute air pouch inflammation model. Kahweol significantly reduced the levels of the inflammatory process markers in the air pouch, such as the volume of exudates, the amount of protein and the number of leukocytes and neutrophils. The levels of nitrite, TNF-alpha and prostaglandin E2 (PGE2) were also markedly lower in the air pouch of the kahweol-treated animals than in the controls. Immunoblot analysis showed that kahweol reduced the COX-2 and iNOS expression level in the exudate cells. The histological examination showed that there was a lower inflammatory response in the pouch tissues from the kahweol-treated animals. In addition, kahweol significantly reduced the paw edema induced by carrageenan and also markedly reduced the level of PGE2 production in the inflamed paw. These results suggest that kahweol has significant anti-inflammatory effects in vivo, which might be due to the inhibition of iNOS and COX-2 expression in the inflammatory sites.     

Changes in Glutathione Peroxidase Activities and the Oxidative Burst of Leukocytes During Inflammation in the Mouse and Rat

The relationship between glutathione peroxidase (GSH-Px) activity and opsonized zymosan-induced chemiluminescence (CL) has been studied with exudate leukocytes obtained at different times after induction of inflammatory responses in the mouse peritoneal cavity with heat-killed Corynebacterium parvum and in the rat pleural cavity with I-carrageenin. GSH-Px activity in mouse peritoneal exudate cells fell markedly after 2–4h, returning to normal within 1–2 days. The lowered enzyme activity was associated with an increased ability of the cells to generate CL. Rat pleural exudate cells exhibited a slight fall in GSH-Px activity after 6h which increased to supranormal levels within 1–2 days. During this period the ability of the cells to generate CL continually increased. The data indicate that during the early phase of increased generation of reactive oxygen species (ROS) by inflammatory leukocytes, the intracellular protective mechanism, represented by GSH-Px, is compromised. Subsequently, GSH-Px activity increases to or above initial levels possibly due to the presence of mononuclear cells and/or as a response to the increased generation of ROS.     

Proinflammatory role of glucocorticoid-induced TNF receptor-related gene in acute lung inflammation

Glucocorticoid-induced TNFR-related gene (GITR) participates in the immune/inflammatory response. Because GITR expression has been described in cells other than T lymphocytes, we investigated whether it also modulates acute inflammatory response. Using GITR-deficient (GITR(-/-)) mice, we analyzed the role of GITR in the development of carrageenan-induced lung inflammation (pleurisy) by studying several proinflammatory markers 2-8 h after carrageenan injection. When compared with GITR(+/+), GITR(-/-) mice exhibited decreased production of turbid exudate containing a lower number of leukocytes. This was correlated with the reduction of inflammatory markers (including TNF-alpha, IL-1beta, myeloperoxidase, inducible NO synthase, and cyclooxygenase 2) in the pleural exudate and/or in the lung. Moreover, endothelial cells expressed lower levels of adhesion molecules. In lungs of GITR(+/+) mice, GITR ligand expression was not modulated during pleurisy, while that of GITR increased, as a consequence of increased infiltration by GITR-expressing cells and of GITR up-regulation in macrophages and endothelial cells. Finally, cotreatment of GITR(+/+) mice with carrageenan and Fc-GITR fusion protein decreased the number of inflammatory cells (pleural macrophages and lung neutrophils) as compared with carrageenan treatment alone, confirming that GITR plays a role in the modulation of pleurisy.     

Expression and function of cyclooxygenase-2 in mesothelial cells during late phase of rat carrageenin-induced pleurisy.

We have previously shown that the inducible isoform of the cyclooxygenases, COX-2, is strongly expressed in pleural exudate leukocytes early (between 3 and 7 hr after irritation) during rat carrageenin-induced pleurisy. The present study further examined COX-2 expression and disclosed that mesothelial cells expressed COX-2 later (12 to 24 hr after irritation) in this model. A COX-2 inhibitor, nimesulide, lowered the intrapleural level of 6-keto-PGF1alpha and inhibited hyperplasia of the pleural matrix, suggesting that COX-2 expressed in mesothelial cells may play a role in the synthesis of extracellular matrix through formation of PGI2.     

Oxidative Burst of Circulating Neutrophils Following Traumatic Brain Injury in Human

Besides secondary injury at the lesional site, Traumatic brain injury (TBI) can cause a systemic inflammatory response, which may cause damage to initially unaffected organs and potentially further exacerbate the original injury. Here we investigated plasma levels of important inflammatory mediators, oxidative activity of circulating leukocytes, particularly focusing on neutrophils, from TBI subjects and control subjects with general trauma from 6 hours to 2 weeks following injury, comparing with values from uninjured subjects. We observed increased plasma level of inflammatory cytokines/molecules TNF-α, IL-6 and CRP, dramatically increased circulating leukocyte counts and elevated expression of TNF-α and iNOS in circulating leukocytes from TBI patients, which suggests a systemic inflammatory response following TBI. Our data further showed increased free radical production in leukocyte homogenates and elevated expression of key oxidative enzymes iNOS, COX-2 and NADPH oxidase (gp91^phox) in circulating leukocytes, indicating an intense induction of oxidative burst following TBI, which is significantly greater than that in control subjects with general trauma. Furthermore, flow cytometry assay proved neutrophils as the largest population in circulation after TBI and showed significantly up-regulated oxidative activity and suppressed phagocytosis rate for circulating neutrophils following brain trauma. It suggests that the highly activated neutrophils might play an important role in the secondary damage, even outside the injured brain. Taken together, the potent systemic inflammatory response induced by TBI, especially the intensively increase oxidative activity of circulating leukocytes, mainly neutrophils, may lead to a systemic damage, dysfunction/damage of bystander tissues/organs and even further exacerbate secondary local damage. Controlling these pathophysiological processes may be a promising therapeutic strategy and will protect unaffected organs and the injured brain from the secondary damage.     

Roles of COX-2 and iNOS in the bony repair of the injured growth plate cartilage

Growth plate injuries often lead to bone growth defects, which primarily occur due to bony repair at injury sites. Bony repair is preceded by an injury-induced inflammatory response, which could play a role in regulating the repair process. Here, roles of two inflammatory mediators, cyclo-oxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS), in the injury responses were analysed by examining their gene expression and effects of blocking their activities, respectively, with celecoxib and aminoguanidine during 2 days prior to and until 7 days after injury in a rat tibial growth plate injury model. Quantitative RT-PCR assays revealed upregulated expression of COX-2 on days 1 and 4 and iNOS on day 1. Histological analysis of injury sites revealed significant reductions in inflammatory infiltrate (particularly neutrophils) on day 1 in treated groups compared to saline control. While bony tissue proportions at injury sites were unaffected by either treatment, mesenchymal tissue proportions were larger but cartilaginous tissue proportions were smaller on day 8 (though statistically insignificant), and bone remodelling appeared delayed with a smaller bone marrow proportion on day 14 in both treatment groups. These findings suggest that COX-2 and iNOS mediate injury-induced inflammatory response, and may play a role in enhancing mesenchymal cell differentiation to cartilaginous cells and in promoting bone remodelling during bony repair of growth plate injury sites. Furthermore, increased expression of cartilage-related (collagen-2, collagen-10, SOX-9) and bone-related molecules (osteocalcin, cbfalpha-1) suggest involvement of both endochondral and direct bone formation mechanisms during bony repair.     

Role of prostaglandin H synthase-2 in prostaglandin E2 formation in rat carrageenin-induced pleurisy

Rat carrageenin-induced pleurisy was used to clarify the role of prostaglandin H synthase (PGHS)-2 in acute inflammation. Intrapleural injection of 0.2 ml of 2% λ-carrageenin induced accumulation of exudate and infiltration of leukocytes into the pleural cavity. When PGHS-1 and -2 proteins in the pleural exudate cells were analyzed by Western blot analysis, PGHS-2 was detectable from 1 hr after carrageenin injection. Its level rose sharply, remained high from 3 to 7 hr after injection, and then fell to near the detection limit. PGHS-1 was also detected, but kept almost the same level throughout the course of the pleurisy. Levels of prostaglandin (PG) E₂ and thromboxane (TX) B₂ in the exudate increased from hour 3 to hour 7, and then declined. Thus, the changes of the level of PGE₂ were closely paralleled those of PGHS-2.The selective PGHS-2 inhibitors NS-398, nimesulide and SC-58125 suppressed the inflammatory reaction and caused a marked decrease in the level of PGE₂ but not in those of TXB₂ and 6-keto-PGF_1α. These results suggest that the PGHS-2 expressed in the pleural exudate cells may be involved in PGE₂ formation at the site of inflammation.     

Role of tumor cell apoptosis in tumor antigen migration to the draining lymph nodes

Establishment of an immune response against cancer may depend on the capacity of dendritic cells to transfer tumor Ags into T cell-rich areas. To check this possibility, we used a colon cancer cell variant that yields tumors undergoing complete T cell-dependent rejection when injected into syngeneic rats. We previously demonstrated that immunogenicity of these tumors depended on the early apoptosis of a part of these tumor cells. In this paper we show that fluorescent tumor cell proteins are released from FITC-labeled tumor cells and undergo engulfment by tumor-infiltrating monocytes without a phenotype of mature dendritic cells or macrophages. Fluorescence-labeled mononuclear cells with a phenotype of MHC class II+ dendritic cells are also found in the T cell areas of the draining lymph nodes. Interestingly, no fluorescent cell can be found in lymph nodes after a s.c. injection of Bcl2-transfected apoptosis-resistant tumor cells that yielded progressive tumors. Proliferation of tumor-immune T lymphocytes was induced by dendritic cells isolated from the draining lymph nodes recovered after a s.c. injection of apoptosis-sensitive, but not apoptosis-resistant, tumor cells. These results show that tumor cell apoptosis releases proteins that are engulfed by inflammatory cells in the tumor, then transported to lymph node T cell areas where they can induce a specific immune response leading to tumor rejection.     

Generation of inflammatory cytokines in zymosan-induced pleurisy in rats: TNF induces IL-6 and cytokine-induced neutrophil chemoattractant (CINC) in vivo

Levels of inflammatory cytokines tumour necrosis factor (TNF), interleukin 1 (IL-1), IL-6, and cytokine-induced neutrophil chemoattractant (CINC), which is a member of the alpha-chemokine family in rats, were measured in the pleural exudates during zymosan-induced pleurisy to examine the relationship between the local production of cytokines and the inflammatory reaction. All four cytokine levels in the pleural exudate began to increase after 1-2 h, preceding the influx of neutrophils, and peaked after 4-5 h. Thereafter, these cytokine levels declined after 24 h, whereas the exudate volume still continued to increase and leukocyte number reached a plateau. Concomitant injection of actinomycin D (10 microg) with zymosan markedly suppressed the neutrophil infiltration, parallel with CINC production in the pleural exudate at 4 h. A transient elevation of IL-6 level, peaking at 5 h, and subsequent rise in the level of an acute-phase protein, T-kininogen, were also observed in the plasma. When recombinant human TNF-alpha (rhTNF-alpha) (20 000 U) was intrapleurally injected a rapid increase in pleural CINC level, followed by neutrophil infiltration, and a sharp rise in IL-6 level in the plasma, followed by an increase in T-kininogen, were demonstrated. These results suggest that CINC produced in the pleural exudate may participate in neutrophil infiltration, that IL-6 induced in the plasma stimulates T-kininogen production, and that endogenous TNF may be partly involved in the induction of CINC and IL-6 in this zymosan inflammation.     

Cyclooxygenase-2-mediated prostaglandin E2 production in mesenteric lymph nodes and in cultured macrophages and dendritic cells after infection with Salmonella

Although numerous studies have demonstrated the ability of intestinal epithelial cells to produce PGs after infection with wild-type strains of Salmonella, few studies have focused on Salmonella-induced prostanoids in mucosal lymphoid tissues. This is surprising in view of the profound effects PGs can have on the host response. To begin to address PG production at mucosal sites, mice were orally inoculated with Salmonella, and at varying times postinfection cyclooxygenase-2 (COX-2) mRNA expression and PGE(2) synthesis were investigated. COX-2 mRNA expression was highly inducible in the mesenteric lymph nodes, whereas COX-1 mRNA levels were constitutive. PGE(2) production also increased significantly in the mesenteric lymph nodes following exposure to viable Salmonella, but not after exposure to killed bacteria. This increased PGE(2) response could be blocked by treatment of mice with the selective COX-2 inhibitor, celecoxib. Treatment of mice with celecoxib during salmonellosis resulted in increased viable bacteria in the mesenteric lymph nodes by day 3 postinfection. However, celecoxib treatment prolonged the survival of lethally infected animals. In vitro studies demonstrated Salmonella-induced up-regulation of COX-2 mRNA expression and PGE(2) secretion by both macrophages and dendritic cells, which could also be blocked in the presence of celecoxib. Interestingly, exposure of these cultured APCs to viable Salmonella was a much greater stimulus for induction of PGE(2) synthesis than exposure to Salmonella-derived LPS. The present study demonstrates induction of PGE(2) synthesis in mesenteric lymph nodes, macrophages, and dendritic cells after infection with wild-type salmonella.     

Demonstration of lysozyme, α1-antichymotrypsin, α1-antitrypsin,albumin, and transferrin with the immunoperoxidase method in lymph node cells

The immunoperoxidase method was used to investigate the presence of intracytoplasmic lysozyme, alpha 1-antichymotrypsin (alpha 1-ACT), alpha 1-antitrypsin (alpha 1-AT), transferrin, and albumin in hyperplastic and inflamed human lymph nodes. Lysozyme was demonstrated in eosinophils, neutrophils, histiocytes, in epithelioid cells, mast cells, and some lining cells of lymph node sinuses. alpha 1-ACT was detectable in many, but not all histiocytes that stained for lysozyme, and in sinus histiocytes, epithelioid cells, and mast cells, but not in neutrophils or eosinophils. alpha 1-AT was demonstrable in mast cells, neutrophils, and some epithelioid cells, but not in histiocytes. Transferrin was found in mast cells, but not in any of the other cell types investigated. Albumin was detectable in a few epithelioid cells and giant cells of the Langhans type. Lysozyme, alpha 1-ACT, alpha 1-AT, transferrin, and albumin were never demonstrable in interdigitating reticulum cells, dendritic reticulum cells, or lymphoid cells.     

Immunocytochemical localization of cytokines and inducible nitric oxide synthase (iNOS) in oral mucosa and lymph nodes of patients with paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is a deep mycosis caused by Paracoccidioides brasiliensis, with high incidence in Brazil. In order to examine the immune response in lesional tissue from patients with PCM, we analyzed cytokines as well as the phenotype of the cell infiltrate. Paraffin-embedded tissue from the oral mucosa of eight patients with the localized adult form (AF) of PCM and from the lymph nodes of 10 patients with the juvenile form (JF) of PCM was analyzed by immunohistochemistry to detect tumor necrosis factor-alpha (TNF-alpha), inducible nitric oxide synthase (iNOS), transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10). Most of the inflammatory cells in the lymph nodes were CD68+ (macrophages, epithelioid and giant cells), while a mixed infiltrate with macrophages, plasma cells and neutrophils was detected in the oral mucosa. TNF-alpha as well as iNOS expression was similar in lymph nodes and oral mucosa, whereas TGF-beta and IL-10 were observed in a larger number of macrophages, epithelioid and giant cells in the lymph nodes, where numerous yeast cells were visualized. The higher expression of anti-inflammatory cytokines (IL-10 and TGF-beta) in lesions of patients with the JF of PCM (lymph nodes) may represent a mechanism by which the fungus evades the host immune response, contributing to a more severe and disseminated form of the disease.     

Inhibitory effect of the saponins derived from roots of Platycodon grandiflorum on carrageenan-induced inflammation

Previous studies have reported that the saponins isolated from the roots of Platycodon grandiflorum A. DC (Campanulaceae), Changkil saponins (CKS), inhibited cyclooxygenase-2 (COX-2) expression in cultured lipopolysaccharide-activated macrophages. The aim of this presented study was to confirm the anti-inflammatory effects of CKS by examining their effect on the inflammatory response induced by carrageenan in a rat by using an acute air pouch inflammation model. CKS significantly reduced the levels of the inflammatory process markers in the air pouch, such as the volume of exudates, the amount of protein and the number of leukocytes and neutrophils. The levels of TNF-alpha and prostaglandin E2 (PGE2) were also markedly lower in the air pouch of the CKS-treated animals than in the controls. An immunoblot analysis showed that CKS reduced the COX-2 expression level in the exudate cells. In addition, CKS significantly reduced the paw edema induced by carrageenan and also markedly reduced the level of PGE2 production in the inflamed paw. These results suggest that CKS had significant anti-inflammatory effects in vivo.     

Production of hapten-specific T cell hybridomas and their use to study the effect of ultraviolet B irradiation on the development of contact hypersensitivity

We produced a series of T cell hybridomas that produce IL-2 when cultured with syngeneic APC coupled to FITC or TNP. These hybridomas are hapten specific and Ia restricted. The hybridomas were used to detect hapten-bearing APC in draining lymph nodes of mice sensitized with trinitrochlorobenzene or FITC in vivo. Hapten-bearing APC capable of stimulating the hybridomas were detectable in draining lymph nodes of hapten-painted mice within 3 h after sensitization. The ability of lymph node APC to stimulate the hybridomas peaked at 24 h and declined by 48 h. The dendritic cell subpopulation was the subpopulation of cells that were found in the regional lymph nodes of hapten-painted animals that were capable of stimulating the hybridomas to produce IL-2. Prior treatment of the skin with low dose UVB irradiation before epicutaneous application of contact sensitizers significantly reduced the capacity of hapten-bearing APC to stimulate the hybridomas. This observation was corroborated by results obtained from flow microfluorometry analysis of lymph node cells from FITC-sensitized mice. Lymph node dendritic cells obtained from FITC-painted mice contain a brightly staining group of cells by flow microfluorometry analysis. Lymph node dendritic cells from FITC-painted, UVB-irradiated mice did not contain this brightly staining population. These results indicate that low dose, local UVB irradiation may affect APC migration and/or function. We believe that these hybridomas will prove to be useful tools in the study of the development and regulation of contact hypersensitivity.     

Immunodetection of cyclooxygenase-2 (COX-2) is restricted to tissue macrophages in normal rat liver and to recruited mononuclear phagocytes in liver injury and cholangiocarcinoma

It has been suggested that cyclooxygenase-2 (COX-2)-mediated prostaglandin synthesis is associated with liver inflammation and carcinogenesis. The aim of this study is to identify the cellular source of COX-2 expression in different stages, from acute liver injury through liver fibrosis to cholangiocarcinoma (CC). We induced in rats acute and “chronic” liver injury (thioacetamide (TAA) or carbon tetrachloride (CCl₄)) and CC development (TAA) and assessed COX-2 gene expression in normal and damaged liver tissue by RT-PCR of total RNA. The cellular localization of COX-2 protein in liver tissue was analyzed by immunohistochemistry as well as in isolated rat liver cells by Western blotting. The findings were compared with those obtained in human cirrhotic liver tissue. The specificity of the antibodies was tested by 2-DE Western blot and mass spectrometric identification of the positive protein spots. RT-PCR analysis of total RNA revealed an increase of hepatic COX-2 gene expression in acutely as well as “chronically” damaged liver. COX-2-protein was detected in those ED1⁺/ED2⁺ cells located in the non-damaged tissue (resident tissue macrophages). In addition COX-2 positivity in inflammatory mononuclear phagocytes (ED1⁺/ED2⁻), which were also present within the tumoral tissue was detected. COX-2 protein was clearly detectable in isolated Kupffer cells as well as (at lower level) in isolated “inflammatory” macrophages. Similar results were obtained in human cirrhotic liver. COX-2 protein is constitutively detectable in liver tissue macrophages. Inflammatory mononuclear phagocytes contribute to the increase of COX-2 gene expression in acute and chronic liver damage induced by different toxins and in the CC microenvironment.     

Inducible nitric oxide synthase upregulates cyclooxygenase-2 in mouse cholangiocytes promoting cell growth

Both inducible nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) have been implicated in the biliary tract carcinogenesis. However, it is not known whether these inflammatory mediators are induced by interdependent or parallel pathways. Because iNOS activity has been associated with diverse gene expression, the aim of this study was to determine whether iNOS induces COX-2. To address this objective, immortalized, but nonmalignant, murine cholangiocytes, 603B cells were employed for these studies. Both iNOS and COX-2 protein and mRNA were expressed in these cells. However, iNOS inhibition with either N-[3-(aminomethyl) benzyl]acetamidine or stable transfection with an iNOS antisense construct inhibited COX-2 mRNA and protein expression, an effect that was reversed by NO donors. COX-2 mRNA expression in 603B cells was reduced by pharmacological inhibitors of the p38 MAPK and JNK1/2 pathways. In contrast, neither inhibitors of the soluble guanylyl cyclase inhibitor/protein kinase G nor p42/44 MAPK pathways attenuated COX-2 mRNA expression. Finally, 603B cells grew at a rate threefold greater than 603B-iNOS antisense cells. The low growth rate of 603B-iNOS antisense cells could be restored to near that of the parent cell line with exogenous PGE(2.) In conclusion, iNOS induces COX-2 expression in cholangiocytes, which promotes cell growth. COX-2 induction may contribute to iNOS-associated carcinogenesis.     

iNOS signaling interacts with COX-2 pathway in colonic fibroblasts

COX-2 and iNOS are two major inflammatory mediators implicated in colorectal inflammation and cancer. Previously, the role of colorectal fibroblasts involved in regulation of COX-2 and iNOS expression was largely ignored. In addition, the combined interaction of COX-2 and iNOS signalings and their significance in the progression of colorectal inflammation and cancer within the fibroblasts have received little investigation. To address those issues, we investigated the role of colonic fibroblasts in the regulation of COX-2 and iNOS gene expression, and explored possible mechanisms of interaction between COX-2 and iNOS signalings using a colonic CCD-18Co fibroblast line and LPS, a potential stimulator of COX-2 and iNOS. Our results clearly demonstrated that LPS activated COX-2 gene expression and enhanced PGE(2) production, stimulated iNOS gene expression and promoted NO production in the fibroblasts. Interestingly, activation of COX-2 signaling by LPS was not involved in activation of iNOS signaling, while activation of iNOS signaling by LPS contributed in part to activation of COX-2 signaling. Further analysis indicated that PKC plays a major role in the activation and interaction of COX-2 and iNOS signalings induced by LPS in the fibroblasts.     

Leukocyte-endothelial cell recognition: evidence of a common molecular mechanism shared by neutrophils, lymphocytes, and other leukocytes

The interaction of leukocytes with endothelial cells is intrinsic to the process of leukocyte extravasation, whether during the entry of blood polymorphonuclear leukocytes and monocytes into sites of acute and chronic inflammation, or during the homing of lymphocytes to lymphoid organs. A lymphocyte surface glycoprotein, defined by monoclonal antibody MEL-14, has been described that appears to mediate lymphocyte recognition of postcapillary venules in peripheral lymph nodes, and to control the migration of lymphocytes from the blood into these lymphoid organs. We now report that the antigenic determinant recognized by MEL-14 is present at high levels on other leukocytes as well, including neutrophils, monocytes, and eosinophils; and we demonstrate involvement of the MEL-14 antigen in neutrophil-endothelial cell interactions. MEL-14 immunoprecipitates a neutrophil surface protein of Mr approximately 100,000, similar in m.w. to the 80,000 to 90,000 dalton lymphocyte surface MEL-14 antigen, and it blocks the interaction of neutrophils with endothelial cells in an in vitro model of adhesion to postcapillary venules in lymph node frozen sections. Neutrophil binding to lymph node venules is also inhibited by PPME, a mannose-6-phosphate-rich yeast polysaccharide that is thought to mimic the endothelial cell ligand for the MEL-14-defined lymphocyte receptor. Interestingly, neither MEL-14 nor PPME exhibit a major effect on neutrophil binding to postcapillary venules in Peyer's patches, suggesting that as for lymphocytes, the neutrophil MEL-14 antigen is involved in recognition of tissue-specific endothelial determinants. Finally, we show that MEL-14 inhibits the capacity of neutrophils to migrate from the blood into sites of acute inflammation in the skin. These observations lead us to propose that receptors for tissue-specific endothelial determinants are utilized by neutrophils and lymphocytes and probably other leukocytes during the physiologic process of leukocyte extravasation in vivo.     

The leukocyte podosome

Myeloid leukocytes are the first line of host defence. When they sense perturbations in tissue homeostasis such as infection, inflammation and ischemia, they respond by trafficking. Whilst neutrophils and macrophages migrate to sites of infection, dendritic cells (DC) migrate from tissue-resident sites back into lymph nodes where they activate T and B lymphocytes. The directed migration of these leukocytes through peripheral tissues is thus crucial for their function. This article considers recent advances in our understanding of the adhesive and motile behaviour of macrophages and DC, with particular emphasis on the podosomes that appear to be required for normal migration through extracellular matrices.     

Dermal dendritic cells, and not Langerhans cells, play an essential role in inducing an immune response

Langerhans cells (LCs) serve as epidermal sentinels of the adaptive immune system. Conventional wisdom suggests that LCs encounter Ag in the skin and then migrate to the draining lymph nodes, where the Ag is presented to T cells, thus initiating an immune response. Platelet-activating factor (PAF) is a phospholipid mediator with potent biological effects. During inflammation, PAF mediates recruitment of leukocytes to inflammatory sites. We herein tested a hypothesis that PAF induces LC migration. Applying 2,4-dinitro-1-fluorobenzene (DNFB) to wild-type mice activated LC migration. In contrast, applying DNFB to PAF receptor-deficient mice or mice injected with PAF receptor antagonists failed to induce LC migration. Moreover, after FITC application the appearance of hapten-laden LCs (FITC+, CD11c+, Langerin+) in the lymph nodes of PAF receptor-deficient mice was significantly depressed compared with that found in wild-type mice. LC chimerism indicates that the PAF receptor on keratinocytes but not LCs is responsible for LC migration. Contrary to the diminution of LC migration in PAF receptor-deficient mice, we did not observe any difference in the migration of hapten-laden dermal dendritic cells (FITC+, CD11c+, Langerin-) into the lymph nodes of PAF receptor-deficient mice. Additionally, the contact hypersensitivity response generated in wild-type or PAF receptor-deficient mice was identical. Finally, dermal dendritic cells, but not LCs isolated from the draining lymph nodes after hapten application, activated T cell proliferation. These findings suggest that LC migration may not be responsible for the generation of contact hypersensitivity and that dermal dendritic cells may play a more important role.