OBJECTIVE

To investigate the expression of adhesion molecules belonging to the immunoglobulin superfamily on human primary articular chondrocytes and to determine their response pattern to cytokines with respect to the adhesion of lymphocytes.

METHODS

The expression of adhesion molecules was studied by flow cytometry (cultured cells), immunohistochemistry (cartilage), reverse transcription-polymerase chain reaction, and Northern blotting. Adhesion of T cells to chondrocytes was measured using the Jurkat T cell line.

RESULTS

Vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) were found to be constitutively expressed on large percentages of unstimulated chondrocytes in culture and in cartilage ex vivo. ICAM-2, ICAM-3, and very late activation antigen 4 (VLA-4; alpha4beta1 integrin), the ligand for VCAM-1, were not detected. Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) further induced VCAM-1 and ICAM-1 messenger RNA and protein expression. Transforming growth factor beta (TGFbeta) had no effect on ICAM-1 and decreased the expression of VCAM-1. Another adhesion molecule, VLA-2 alpha2beta1 integrin) that was also expressed on unstimulated chondrocytes, was differentially regulated by cytokines. While neither IL-1beta nor TNFalpha had any effect on expression of VLA-2, TGFbeta markedly increased the alpha2 subunit of VLA-2. Adhesion of Jurkat T cells to chondrocytes was further induced by IL-1beta and TNFalpha. Pretreatment of chondrocytes with monoclonal antibodies to VCAM-1 and ICAM-1 inhibited adhesion of T cells to chondrocytes.

CONCLUSIONS

VCAM-1, ICAM-1, and VLA-2 are constitutively expressed by human articular chondrocytes. Expression is regulated by cytokines. As shown for other chondrocyte genes, IL-1beta/TNFalpha and TGFbeta antagonistically modulate the expression of adhesion molecules. VCAM-1 and ICAM-1 contribute to adhesion of T lymphocytes to chondrocytes, and may thus participate in host defense mechanisms during inflammatory joint conditions such as rheumatoid arthritis and after cartilage transplantation.

OBJECTIVE

To evaluate the inflammatory status and the cartilage regenerative potential of pathological synovial fibroblasts from patients with osteoarthritis (OA) compared with non-inflamed synovium (NS)-derived cells from patients with chondropathy.

METHODS

The inflammatory cell phenotype was investigated based on the constitutive and inducible surface expression and secretion of various effector molecules using flow cytometry or ELISA assays. The capacity of cells to produce cartilage-like extracellular matrix was assessed using acid Alcian blue staining and type II collagen immunostaining after treatment with transforming growth factor beta1 (TGF-beta1).

RESULTS

OA and NS fibroblasts consistently expressed CD29, CD44, CD49e, CD54, CD90 and CD106. Expression of high-affinity receptors for IL-4, IL-15, CXCL8 and CXCL12 was also detected but only intracellularly. All types of fibroblasts spontaneously released abundant amounts of CXCL12, CCL2, IL-6 and tissue inhibitor of metalloproteinase 1, while the production of IL-11, TGF-beta1, matrix metalloproteinase 1 (MMP-1) and MMP-9 was detected at moderate levels. Several other secreted factors remained undetectable. No statistically significant differences were noted between the two groups of fibroblasts. Treatment with the proinflammatory cytokine tumour necrosis factor alpha (TNF-alpha) up-regulated the same set of surface and secreted molecules, including CD54, CD106, membrane IL-15, CCL2 and CCL5. Under TGF-beta1 treatment and adipogenic culture conditions, both OA and NS fibroblasts displayed chondrogenic and adipocytic activities that were reduced in OA compared with NS cells.

CONCLUSIONS

OA synovial fibroblasts did not display a distinct activated inflammatory phenotype compared with NS cells. However, they did differ in their reduced ability to produce cartilage-like matrix. This difference may be an additional important factor contributing to OA pathogenesis.

Dendritic cells (DCs) consist of a heterogeneous population of hematopoietic cells characterized by their unique dendritic morphology, their efficient antigen-presenting capability to activate naïve CD4+ and CD8+ T cells, as well as their lack of lineage-specific markers. Functional properties comparing umbilical cord blood monocyte-derived and umbilical cord blood stem cell-derived DCs have not yet been investigated. Human umbilical cord blood CD14+ monocytes and CD34+ stem cells were induced to differentiate into dendritic cells using 100 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 25 ng/mL interleukin (II)-4, 2.5 ng/mL tumor necrosis factor alpha (TNF-alpha) and 100 ng/mL GM-CSF, 25 ng/mL stem cell factor, and 2.5 ng/mL TNF-alpha, respectively. Differentiated dendritic cells were CD80+, CD86+, CD83+, CD54+, CD1a+, CD11b+, CD11c+, HLA-DR+, CD34-, CD3-, CD19-, CD14-, and CD16-. Reverse transcription polymerase chain reaction revealed that differentiating monocytes initially expressed CD86 mRNA while CD80 mRNA appeared on Day 2. Differentiating stem cells expressed both CD80 and CD86 mRNA on Day 2 of culture. Mixed lymphocyte reaction was employed to evaluate the two types of lineage-derived DCs. Monoclonal antibodies (mabs) to CD80 and CD86 were employed to assess their costimulatory roles. CD14 and CD34 derived DCs prior to the functional assay were stimulated for 18 h with 0.1 and 1.0 mg/mL Escherichia coli lipopolyssacharide, respectively. A decrease in stimulation as depicted by decreased T-cell activation was significant with mabs to both CD80 and CD86 on monocyte-derived DCs while only mabs to CD86 induced decreased T-cell activation by stem cell-derived DCs. The varied functional role of CD80 and CD86 costimulatory molecules is associated with DC differentiation from distinct cord blood-isolated hematopoietic lineages. These studies demonstrate that DC association with distinct hematopoietic lineages is of relevance in transplantation and vaccine therapies.

We examined the expression of co-stimulatory molecules on leukaemic cells of 52 adult patients with acute myeloid leukaemia (AML) (34 men and 18 women) and analysed the relationship between these expressions and the patient's prognosis. B7-1 was not expressed in any of the 23 patients investigated, whereas B7-2 was expressed in 26/52 patients (50.0%). B7-2 was expressed in all AML patients with monocytic morphology (M4 or M5) and in 16/42 cases without monocytic morphology. CD54 was expressed in 28/ 37 patients examined (75.7%), and CD58 was expressed in all of the AML patients except one (M 7). The overall survival of the 26 B7-2-positive leukaemia patients (1-24 months, median survival 11.5 months) was significantly shorter than that of the 26 B7-2-negative leukaemia patients (1-71+ months, median 35.1 months) (P=0.0080). In addition, the B7-2-positive patients exhibited significantly shorter disease-free survival periods compared to the B7-2-negative patients (P=0.021). There was no significant difference in age, sex, haematological data and complete remission rate between the B7-2-positive and B7-2-negative patients. Our results indicated that B7-2 is one of the most crucial factors in the prognosis of adult acute leukaemia and can be expected to have an important role in tumour immunity.

IL-12 promotes Th1 cell differentiation and cell-mediated immunity. In the present study, the potential role of IL-12 was analyzed in an experimental colitis model in scid mice reconstituted with syngeneic CD45RBhighCD4+ T cells. Real-time reverse transcription-PCR studies demonstrated that IL-12 p40 mRNA in inflamed colon is induced shortly after T cell transfer and maintained at a stable level after week 4, at the time when wasting disease starts. Administration of anti-IL-12 on days 0,14, and 28 (early treatment) or on days 28, 42, and 56 (delayed treatment) after T cell transfer, effectively prevented or, respectively cured wasting disease and colitis in scid recipients. Anti-IL-12 treatment abrogated mucosal inflammation with significantly diminished leukocyte infiltration (CD4 cells, macrophages) and CD54 expression, and down-regulated proinflammatory cytokines IFN-gamma and IL-2. Of note, although splenic CD4+ T cells are unable to induce disease as a result of the presence of regulatory CD45RBlow cells, splenic CD4+ T cells, preactivated by IL-12 and anti-CD3 in vitro, were highly pathogenic in inducing severe mucosal inflammation, suggesting that IL-12 and anti-CD3 abrogated regulatory T cell function. These findings indicate that IL-12 is important for the induction of experimental colitis through effects on proinflammatory cytokine production and on regulatory T cell function.

The replacement of endothelium by endothelial progenitor cells (EPCs) for therapeutic use in order to ameliorate the vascular status of ischemic organs is now in the focus of vascular research. The aim of our studies was to investigate whether EPCs derived from peripheral blood mononuclear cells (PBMNCs-derived EPCs) or EPCs propagated from CD34(+) hematopoietic stem cells (HSCs-derived EPCs), both isolated from human cord blood, are able to differentiate into early mature endothelial cells (ECs) under certain in vitro conditions. We characterized both cell populations by flow cytometry, phase contrast microscopy, fluorescence microscopy and confocal laser scanning microscopy as well as ultrastructurally using transmission and scanning electron microscopy. While PBMNCs gave rise to clusters of spindle-like EPCs after few days but did not further mature under in vitro conditions, mature ECs could only be successfully propagated from a starting population of isolated HSCs. Both, PBMNCs- and HSCs-derived EPCs, took up Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL) and could be positively stained for CD31, CD105, the vascular endothelial growth factor receptor 2 (VEGFR-2, KDR) and ulex europaeus agglutinin 1 (UEA-1) at the cell surface. EPC showed surface expression of CD54 and CD106. However, only a small portion of HSCs-derived EPCs was positive for CD54 but negative for CD106. Intracellular staining for von Willebrand factor (vWF) provided a homogenous stain in PBMNC-derived EPCs while in HSCs-derived EPCs, during cultivation for 2-3 weeks, more and more a typical punctuated staining pattern related to Weibel-Palade bodies (WPBs) was visible. By phase contrast and scanning electron microscopy, an arrangement of PBMNCs-derived EPCs in cord-like structures could be demonstrated. In these formations, cells showed parallel alignment but exhibited only few cell contacts. Well-developed WPBs could never be found in PBMNCs-derived EPCs. In contrast, differentiating HSCs-derived EPCs developed adherence junctions, interdigitating junctions as well as syndesmos. During maturation, spindle-like cell types appeared with abundant WPBs as well as cobblestone-like cell types with a fewer content of these organelles. WPBs, in the spindle-like cell types displayed conspicuous shapes and were concentrated in close proximity to mitochondria-rich areas. HSCs-derived EPCs exhibited signs of high synthetic activity such as a well-developed rough endoplasmic reticulum (RER) and multiple Golgi complexes. In the trans-Golgi network (TGN), close to the Golgi complex, a new formation of WPBs could be observed. These morphological features correlated well with a high growing capacity. Although it was not possible to demonstrate the complete differentiation line from HSCs to early matured ECs by immunologic markers because of the limited number of cells available for such investigations, distinct morphologic maturation stages could be shown at light and electron microscopical levels. In conclusion, the study presented here characterizes not only the different cell populations involved in the differentiation of early EPCs into mature ECs but also the transition stage where the maturation step takes place by demonstration of the new formation of WPBs. In this respect, these investigations provide new insights into the in vitro differentiation which could have some in vivo correlation.

Frozen tissue sections obtained from human glioblastomas, brain tumor metastases and normal brain were examined for the expression of molecules known to be involved in lymphocyte activation and/or adhesion and migration. The molecules studied included CD3, CD45R, UCHL-1 (CD45RO), lymphocyte function-associated antigen 1 (LFA-1) (CD11a, CD18), intercellular adhesion molecule 1 (ICAM-1) (CD54), 4B4 (CD29), CD44, CD2, and LFA-3 (CD58). CD3+ lymphocytes infiltrating human glioblastomas and brain tumor metastases expressed LFA-1 alpha and beta. Many cells were also UCHL-1+ whereas only a small percentage were CD45R+. CD2+ lymphocytes were also present. Tumor-infiltrating lymphocytes (TIL) were found to be negative for CD29, which was, however, expressed on intratumoral vessels in addition to vessels found in normal brain. Glioblastoma cells and intratumoral vessels expressed ICAM-1 whereas no ICAM-1 was found on TIL or on normal brain. Glioblastoma cells also expressed high levels of both CD44 and LFA-3 whereas TIL were negative for these antigens. CD44 was also expressed on certain regions of normal brain. Antibodies to LFA-1 alpha and -beta and ICAM-1 could significantly block the binding of lymphokine-activated killer (LAK) cells or TIL to human glioblastoma cells suggesting that these molecules play a role in the binding and subsequent migration of lymphocytes into brain tumor tissue.

The decrease in NK cell activity and the loss of gammadelta T cells in active pulmonary tuberculosis patients have been reported. In this study, we observed that the proliferating response of gammadelta T cells to the heat-treated Ags of Mycobacterium tuberculosis from different individuals was noted to be dependent on the content or function of NK cells in PBMC in a population study. We also found that NK cells were directly rapidly activated by the heat-treated Ags from M. tuberculosis (H37Ra) in vitro; in turn, the activated NK cells improved gammadelta T cell proliferation both by CD54-mediated cell-cell contact through the forming immune synapse and by soluble factors TNF-alpha, GM-CSF, and IL-12, but not IFN-gamma. Our results demonstrated that an interaction between NK cells and gammadelta T cells existed in antituberculosis immunity. Up-regulating the function of NK cells might be beneficial to the prevention and control of pulmonary tuberculosis.

OBJECTIVE

To establish a method of isolating and culturing adult human blood-derived stem cells(MSCs) and to investigate their osteogenic potential in vitro.

METHODS

Thirty peripheral blood collected from 30 adult volunteers (15 ml per person). Adult human MSCs derived from peripheral blood from the lymphocyte separation fluid fraction of mononuclear cells, cultured in alpha-Modified Eagle's Medium glucose containing 20% fetal bovine serum, and proliferated through a process of subculturing. The phenotype was analyzed with flow cytometry. For in vitro osteogenic differentiation, MSCs from the second passage presence of osteogenic supplements (100 nmol/L dexamethasone,10 mmol/L beta-glycerophosphate, 50 micromol/L and 10 nmol/L 1,25-2-hydroxide vitamin D3). In the fifth passage cells, the activity of alkaline phosphatase, expression level of collagen type I, osteocalcin and osteonectin were determined. And the calcium tubercle would be examined after the continual one-month culture of the fifth passage.

RESULTS

MSCs existed in blood of adult human. And the clone forming efficiency of blood-derived MSCs was 0.27 +/- 0.22/10(6) mononuclear The MSCs expressed CD44,CD54,CD105,and CD166, but did not CD14, CD34, CD45,and CD31. Under osteogenic supplements, the MSCs were found to be higher activity of alkaline phosphatase and higher expression of collagen type I , osteocalcin and osteonectin. And the calcium tubercle formation was examined through fluorescence labeling method.

CONCLUSIONS

The isolation and culture conditions established for adult human select a distinct population of peripheral blood-derived adherent cells. Adult human blood-derived osteogenic potential in vitro, and may be used as seed cells for bone tissue engineering.

Exosomes (EXOs) derived from tumor cells have been used to stimulate antitumor immune responses. It has been demonstrated that EXO released by tumor cells engineered to express cytokines are of enhanced stimulatory effect on CD8(+) cytotoxic T-lymphocyte (CTL) responses and antitumor immunity. J558 is a mouse myeloma cell line expressing tumor antigen P1A. In this study, we purified EXO(TNF-a), EXO(IL-2), and EXO(IFN-gamma) released by three cytokine-gene (TNF-alpha, IL-2 and IFN-gamma)-engineered J558 (J558(TNF-a), J558(IL-2) and J558(IFN-gamma)) tumor cell lines from their culture supernatants, respectively, by differential ultracentrifugation. These EXOs showed a "saucer" or round shape with a diameter between 50 and 90 nm by electron microscopy and contained EXO-associated proteins, such as LAMP-1 and AIP1, but not lysate-associated protein galectin, by Western blot analysis. EXO displayed expression of molecules (H-2K(d), CD54, and P1A) similarly to, but to a lesser extent to, J558 tumor cells. We then compared the stimulatory effect of these EXOs on P1A-specific CD8(+) CTL responses and antitumor immunity 6 days subsequent to intravenous (i.v.) EXO immunization (30 microg/each BALB/c mouse). We demonstrated that EXO(TNF-alpha) immunization was able to induce more efficient P1A-specific CD8(+) T-cell response accounting for 0.62% of the total CD8(+) T-cell population, using PE-H-2K(d)/P1A peptide and FITC-CD8 staining by flow cytometric analysis then EXO(IL-2) (0.31%) and EXO(IFN-gamma) (0.22%) immunization (P < 0.05), respectively, at day 6 after immunization. EXO(IL-2) and EXO(IFN-gamma) vaccine (i.v. 30 microg/each mouse) only protected 3 of 8 (38%) and 2 of 8 (25%) mice from tumor growth after subcutaneous (s.c.) challenging of immunized mice with J558 tumor cells (0.5 x 10(6) cells/each mouse), whereas EXO(TNF-alpha) immunization protected all 8 of 8 (100%) mice from tumor growth (P < 0.05). Taken together, we demonstrate that EXO(TNF-a) released by engineered J558(TNF-a) tumor cells more efficiently stimulate tumor antigen P1A-specific CD8(+) CTL responses and antitumor immunity than EXO(IL-2) and EXO(IFN-gamma) released by engineered J558(IL-2) and J558(IFN-gamma) tumor cells. Therefore, TNF-alpha-expressing tumor cell-released EXO may represent a more effective EXO-based vaccine in the induction of antitumor immunity.

Human osteoblasts isolated from bone tissue samples have their own specific antigen profile but also share expression of antigens that are characteristic of other immunocompetent cells. Given that these findings come from studies performed in primary cultures of human osteoblasts, it was decided to test whether these antigen profiles and functional characteristics are retained in a characterized osteoblast cell line (MG-63). We show that some of these characteristics are also found in the MG-63 osteosarcoma cell line. We have demonstrated, using monoclonal antibodies and cytometry, that these cells expressed CD10, CD13, CD44, and CD54 antigens but were negative for CD69 and HLA-DR antigens. Functionally, 100% of MG-63 cells showed phagocytic capacity with a high phagocytic index. This study corroborates that osteoblastic cells have an immunological profile.

Adhesion and activation molecules as well as cytokines play an important role in an immune scenario. In acute pancreatitis, we have studied some of these in order to evaluate dysregulation. For this we took peripheral blood mononuclear cells and pancreatitis tissue cells. We analysed activation markers like CD69, CD25 and HLA-DR and found a marked elevation of CD69 as well as CD25 in both peripheral blood cells and tissue mononuclear cells when compared to controls. In PBMC-CD69: P<0.01 and CD25: P<0.01; in tissue-CD69: P<0.001 and CD25: P<0.001. The HLA-DR levels, however, were reduced in the disease state (in acute pancreatitis patient blood (P<0.01) and tissue cells (P<0.001)). The adhesion molecules showed unanimous rise in the blood and the tissue samples. In blood samples, CD11a: P<0.05 and CD11b: P<0.05 and tissue samples CD11a: P<0.01 and CD11b: P<0.01and CD54 in peripheral blood (P<0.05) and tissue (P<0.01) of AP was high as compared to controls. By simultaneous flowcytometric analysis, we determined the co-expression of a surface marker (CD4/CD8/CD14) and intracellular cytokine (TNF-alpha and IFN-gamma) in individual cells. The IFN-gamma producing CD8+T cells were elevated in pancreatic tissue (P<0.01). TNF-alpha producing cell numbers were significantly higher in tissue cells than in blood and also in CD8+ T cells (P<0.001). We conclude that monocyte function is affected in AP as shown by reduced HLA-DR numbers and lowered TNF-alpha producing cells. Moreover, the CD8+T cells appear to play an important role in cytokine synthesis at the effector site.

Streptococcus suis serotype 2 is known to be a major pathogen of swine, causing mainly meningitis. It is also a zoonotic agent leading predominantly to meningitis in humans working in close contact with pigs. In this study, we investigated the ability of S. suis to up-regulate the expression of adhesion molecules involved in inflammation, using an enzyme-linked immunosorbent assay. S. suis serotype 2 stimulated the up-regulation of the expression of intercellular adhesion molecule-1 (ICAM-1, CD54), CD11a/CD18 and CD11c/CD18 on human THP-1 monocytes, but did not change that of ICAM-1, vascular cell adhesion molecule-1 (VCAM-1, CD106) and E-selectin (CD62E) on human endothelial cells. The up-regulation of adhesion molecules was time- and bacterial concentration-dependent, and cell wall components were largely responsible for such stimulation. To a lesser extent, purified haemolysin of S. suis also stimulated adhesion molecule expression. Stimulation of monocytes with strains of different origin showed that there was no clear tendency for human strains to induce a higher expression of adhesion molecules than strains from diseased pigs. Finally, monocytes stimulated with S. suis also showed an increase in adherence to endothelial cells. Hence, S. suis is capable of up-regulating important adhesion molecules involved in inflammation, which may result in an increased leucocyte recruitment into sites of infection, thus providing a possible mechanism for some of the inflammatory features of meningitis caused by this pathogen.

OBJECTIVE

To determine if the cell surface antigen, the intercellular adhesion molecule 1 (ICAM-1), is expressed on head and neck (H&N) squamous cell carcinoma (SCC) cell lines, and if treatment with interferon gamma (IFN-gamma) enhances the expression of the antigen. Intercellular adhesion molecule 1 mediates effector cell adhesion, activation, and function in inflammatory and immunologic reactions, and it may be important in the generation of antitumor immune surveillance and cytotoxicity against H&N SCC.

METHODS

Four human SCC cell lines, JHU-011-SCC, JHU-020-SCC, JHU-022-SCC, and FaDu, established by explant technique from tumors of the upper aerodigestive tract, were utilized for these experiments. The cell lines were maintained and tested under standard tissue culture conditions.

METHODS

Fluorescence-activated cell sorting and enzyme-linked immunosorbent assay were performed to identify the presence of ICAM-1 on the H&N SCC cell lines after staining with an anti-ICAM-1 monoclonal antibody (CD54). The SCC cell lines were treated with either fresh media or varying dosages (1 to 1000 U/mL) of recombinant human interferon gamma (rHuIFN-gamma) to determine constitutive and enhanced antigen expression. The kinetics of the response to rHuIFN-gamma were determined for the JHU-022-SCC cell line. The effect of the cytokines interleukin 1, interleukin 2, tumor necrosis factor alpha, and interferon alfa on ICAM-1 expression on JHU-022-SCC was also tested.

METHODS

Constitutive and enhanced ICAM-1 expression.

RESULTS

Low levels of constitutive expression of ICAM-1 were identified on all four H&N SCC cell lines, with significantly enhanced expression seen after rHuIFN-gamma treatment (P = .0001). Maximally enhanced expression of the antigen on JHU-022-SCC occurred after treatment for 48 hours with 100 U/mL of rHuIFN-gamma (P = .0001). Induction of ICAM-1 expression was detectable after treatment with as little as 10 U/mL of rHuIFN-gamma (P < .001). Induction was also present after treatment with interleukin 1 and tumor necrosis factor alpha, but not with interleukin 2 or interferon alfa.

CONCLUSIONS

Intercellular adhesion molecule 1 is constitutively expressed on H&N SCC cell lines, with enhanced expression seen after treatment with interferon gamma and other cytokines. This suggests that the antigen may be involved in the generation of an immune response against SCC of the H&N.

Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-α) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-α exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-α exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-α exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-α exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-α exposure, which might influence MSC differentiation stage and capacity.

Oral epithelium may play a regulatory role in local immune responses when interacting with bacteria. The present study was undertaken to investigate the effects of selected bacterial pathogens found in periodontal and endodontic infections on oral epithelial cells. Expression of cell surface molecules (major histocompatibility complex (MHC) Class II, CD54, CD70, CD80 and CD86) and secretion of inflammatory cytokines (interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha) in response to selected bacterial challenge were examined on an immortalized oral epithelial cell line, HOK-18A and a skin epithelial cell line, HaCaT. Actinomyces viscosus, Actinomyces israelii, Fusobacterium nucleatum lipopolysaccharide (LPS) or primary human periradicular exudate from a granuloma were co-cultured with epithelial cells for 4 or 24 h. Subsequently, cell surface expression of MHC Class II, CD54, CD70, CD80 and CD86, along with pro-inflammatory cytokine levels were determined using flow cytometry, ELISA and RT-PCR. Results indicated that the selected oral bacteria have greater effects on oral versus skin epithelial cells. F. nucleatum increased MHC Class II and CD54 (ICAM-1) cell surface expression on HOK-18A and HaCaT cells. A. israelii also had enhancing effects on the expression of CD54 and MHC Class II. A. israelii and LPS induced a 2.8-fold (P < 0.001) and 4.4-fold (P < 0.005) TNF-alpha secretion, respectively, while F. nucleatum and LPS induced a 10-fold (P < 0.0004) and 6-fold (P < 0.01) IL-1beta secretion, respectively by HOK-18A. Interestingly, CD70, CD80, and CD86 were generally decreased upon bacteria and LPS challenge on HOK-18A. The effects of increased MHC Class II and decreased CD70 were also evident with challenge of human periradicular exudate on HOK-18A. The implications of the study are unique in that oral epithelial cells may play both activating and inhibitory roles in the host immune response towards infection by oral bacteria. We introduce a concept of 'dormancy' where the differential expression of key cell surface antigens on oral epithelial cells may keep the recruited immune effector cells in a state of unresponsiveness, thus contributing to the long term quiescent period observed in many periodontal and endodontic lesions.

Arthritis is one of the most common complications of human active brucellosis, but its pathogenic mechanisms have not been completely elucidated. In this paper, we describe the role of synoviocytes in the pathogenesis of brucellar arthritis. Our results indicate that Brucella abortus infection inhibited synoviocyte apoptosis through the upregulation of antiapoptotic factors (cIAP-2, clusterin, livin, and P21/CIP/CDNK1A). In contrast, infection did not change the expression of proteins that have been involved in apoptosis induction such as Bad, Bax, cleaved procaspase 3, CytC, and TRAIL, among others; or their expression was reduced, as occurs in the case of P-p53(S15). In addition, B. abortus infection induced upregulation of adhesion molecules (CD54 and CD106), and the adhesion of monocytes and neutrophils to infected synoviocytes was significantly higher than to uninfected cells. Despite this increased adhesion, B. abortus-infected synoviocytes were able to inhibit apoptosis induced by supernatants from B. abortus-infected monocytes and neutrophils. Moreover, B. abortus infection increased soluble and membrane RANKL expression in synoviocytes that further induced monocytes to undergo osteoclastogenesis. The results presented here shed light on how the interactions of B. abortus with synovial fibroblasts may have an important role in the pathogenesis of brucellar arthritis.

Canine cutaneous histiocytoma (CCH) is a common, benign neoplasm of the dog. Histiocytomas most commonly occur as solitary lesions that undergo spontaneous regression. The age-specific incidence rate for histiocytomas drops precipitously after 3 years, although histiocytomas occur in dogs of all ages. Langerhans cells (LCs) in humans and dogs express abundant major histocompatibility complex class II molecules and a variety of leukocyte antigens characteristic of dendritic cell differentiation including CD1a, CD1b, CD1c, and CD11c. The immunophenotype of CCH resembled that of cutaneous LCs by virtue of the expression of CD1 molecules (CD1a, -b, and -c), CD11c, and major histocompatibility complex class II. Furthermore, histiocytoma cells had a tropism for epidermis, which was also consistent with an epidermal LC lineage. The expression of adhesion molecules such as CD11b (variable), CD44, CD54 (ICAM-1), and CD49d (VLA-4) in CCH indicated that the infiltrating cells had some of the characteristics of activated LCs, as these molecules are not expressed by normal, resting canine epidermal LCs. CCH did not express Thy-1 or CD4. Thy-1 expression is a characteristic of human and canine dermal dendrocytes, which are perivascular dendritic antigen-presenting cells closely related to epidermal LCs. CD4 expression is prevalent in human LC histiocytosis, and in this respect CCH differed from human LC histiocytosis. Here we demonstrate that CCH is a localized form of self-limiting LC histiocytosis, which predominantly expresses an epidermal LC phenotype. CCH occurs as solitary or, less commonly, as multiple cutaneous nodules or plaques, which rarely may extend beyond the skin to local lymph nodes. Regression of CCH occurs spontaneously in the vast majority of cases in primary and secondary sites, and is mediated by CD8+ alpha beta T cells. The high frequency of CCH within the general canine population offers the potential that the dog may provide an interesting model system to further the understanding of LC proliferative disorders, particularly the self-limiting, cutaneous form of human LC histiocytosis.

We previously developed the human cell-line activation test (h-CLAT) in vitro skin sensitisation test, based on our reported finding that a 24-hour exposure of THP-1 cells (a human monocytic leukaemia cell line) to sensitisers is sufficient to induce the augmented expression of CD86 and CD54. The aim of this study is to confirm the predictive value of h-CLAT for skin sensitisation activity by employing a larger number of test chemicals. One hundred chemicals were selected, according to their categorisation in the local lymph node assay (LLNA), as being: extreme, strong, moderate and weak sensitisers, and non-sensitisers. The correlation of the h-CLAT results with the LLNA results was 84%. There were some false negatives (e.g. benzoyl peroxide, hexyl cinnamic aldehyde) and some false positives (e.g. 1-bromobutane, diethylphthalate). Eight out of the 9 false negatives (89%) were water-insoluble chemicals. The h-CLAT could positively predict not only extreme and strong sensitisers, but also moderate and weak sensitisers, though the detection rates of weak sensitisers and non-sensitisers were comparatively low. Some sensitisers enhanced both CD86 and CD54 levels, and some enhanced the level of only one of them. The use of the combination of CD86 and CD54 induction as a positive indicator, improved the accuracy of the test. In conclusion, the h-CLAT is expected to be a useful cell-based in vitro method for predicting skin sensitisation potential.

A review of recent information on the expression and the ATRA-driven modulation of cell surface adhesion molecules of acute myelogenous leukemia blast cells is presented. Cytofluorometric studies on fresh blast cells have demonstrated that CD11a, CD11b CD11c, CD15, CD45RO and CD54 expression is significantly lower in acute promyelocytic leukemia (APL) than is acute myeloid leukemia of other subtypes (AML). In vitro treatment with ATRA dramatically modifies the adhesion phenotype of APL blast cells, promoting a consistently striking up-regulation of CD11b, CD11c, CD15, CD65, CD54, and CD38. Which is in general, poorly demonstrable in AML. The behaviour of CD15s is variable and fully independent from CD15 and CD65 in induction experiments, suggesting a differential enzyme regulation within the selectin ligand system. ATRA is capable, in both APL and AML, of producing a switch from the high- (RA) to the low- (RO) molecular weight isoform of CD54, Moreover, treatment with this retinoid exerts a negative regulation of the membrane expression of CD49e, CD58 and CD11a in APL as well as in AML. Of particular interest is the fact that the negative effect on CD1 1a expression generates an asynchronous phenotype in APL (CD11a-, CD11b+, CD15+), undetectable on normal maturing myeloid cells. In the last part of this review the possible implications of adhesion molecule modulation in the pathogenesis of ATRA syndrome are discussed.

Inflammatory immune activation has been frequently associated with the development of major depression. This association was confirmed in patients receiving long-term treatment with pro-inflammatory interferon-α (IFN-α). Microglia, the resident immune cells in the brain, might serve as an important interface in this immune system-to-brain communication. The aim of the present study was to investigate the role of microglia in an IFN-α mouse model of immune-mediated depression. Male BALB/c mice were treated with daily injections of IFN-α for two weeks. Depressive-like behavior was analyzed in the forced swim and tail suspension test. Activation of microglia was measured by flow cytometry. Pro-inflammatory M1 type (MHC-II, CD40, CD54, CD80, CD86, CCR7), anti-inflammatory M2 type (CD206, CD200R), and maturation markers (CD11c, CCR7) were tested, as well as the chemokine receptor CCR2. IFN-α led to a significant increase in depressive-like behavior and expression of the pro-inflammatory surface markers MHC-II, CD86, and CD54, indicating M1 polarization. Because IFN-α-treated mice showed great individual variance in the behavioral response to IFN-α, they were further divided into vulnerable and non-vulnerable subgroups. Only IFN-α vulnerable mice (characterized by their development of depressive-like behavior in response to IFN-α) showed an increased expression of MHC-II and CD86, while CD54 was similarly enhanced in both subgroups. Thus, IFN-α-induced activation of microglia was specifically associated with depressive-like behavior.

The innate ability of B lymphoma cells to escape control by tumor-reactive T cells must be overcome to develop effective immunotherapies for these diseases. Because signals from both the innate and adaptive immune systems direct the acquisition of strong immunogenicity by professional APCs, the effects of IL-2 and the TLR-7 agonist, S28690, on the immunogenic properties of chronic lymphocytic leukemia (CLL) B cells were studied. IL-2 with S28690 caused CLL cells to proliferate and increased their expression of B7-family members, production of TNF-alpha and IL-10, and levels of tyrosine-phosphorylated STAT-1 and STAT-3 proteins. S28690 increased CD25 expression on CLL cells and sensitized them to IL-2 signaling. However, IL-2 did not change TLR-7 expression or signaling in CLL cells. The ability to stimulate T cell proliferation required additional activation of protein kinase C, which inhibited tumor cell proliferation, "switched off" IL-10 production, and caused essentially all CLL cells (regardless of clinical stage) to acquire a CD83(high)CD80(high)CD86(high)CD54(high) surface phenotype marked by the activation of STAT-1 without STAT-3. These findings suggest that TLR-7 "licenses" human B cells to respond to cytokines of the adaptive immune system (such as IL-2) and provide a strategy to increase the immunogenicity of lymphoma cells for therapeutic purposes.

Cytokines released from CD4+ T lymphocytes contribute to the pathogenesis of asthma by influencing the differentiation and function of eosinophils, the primary effector cells that cause airway epithelial damage. Using a model of ovalbumin (OA)-induced, eosinophil-rich chronic lung inflammation in sensitized mice, we have defined the role of T lymphocytes further by using three-color flow cytometry to characterize the adhesion and activation antigens that may be associated with the migration of these cells into the lung and airway lumen. OA inhalation in OA-sensitized C57BL/6 mice resulted in an early (6 to 24 h) influx of neutrophils into the bronchial lumen as enumerated by bronchoalveolar lavage (BAL), which was followed by a marked accumulation of lymphocytes and eosinophils between 24 to 72 h. Phenotypic analysis of BAL or lung tissue T cells showed that most Thy-1 CD3+ T cells were CD4+ (CD4: CD8 ratio of 3 to 4:1). The majority (90%) of the T cells in lung or BAL fluid expressed alpha beta T-cell receptors (TCR). Only 3 to 7% of the T cells were gamma delta TCR+ even though almost 25% of the T cells were CD4- CD8-. There were very few natural killer (NK) or B cells in BAL fluid compared with 15% B cells in dissagregated lung tissue. In contrast to T cells in spleen, almost all the lung and BAL T cells were of the memory phenotype, as ascertained by the expression of high levels of CD44 and by the absence of L-selectin and CD45RB on the cell surface. Fifty to ninety percent of lung and BAL T cells from vehicle-sensitized or OA-sensitized and challenged mice expressed the adhesion molecules CD11a (LFA-1), CD54 (ICAM-1), and CD49d (VLA-4). The early T-cell activation marker CD69 was upregulated on 30% of the lung and BAL T cells in OA-sensitized mice after antigen inhalation. When BAL fluid T cells from OA-sensitized and challenged mice were analyzed for their coexpression of adhesion and/or activation molecules, 75% of the cells that expressed one of three adhesion molecules, CD54, CD49d, or CD11a, also expressed at least one of the other two antigens. At least 15% of BAL T cells had all three of these molecules on their cell surfaces. The OA-dependent, temporally regulated emigration of T cells into the bronchial lumen after exposure to aerosolized antigen may be correlated with the accumulation of cells that express the memory phenotype with enhanced expression of adhesion molecules.

This study investigated whether there are marked differences in surface markers between rabbit and human mesenchymal stem cells (MSCs). Murine and rabbit MSCs have been reported to be CD90-negative. Rat MSCs have been reported to be CD71-negative. Our previous study also shows that rabbit MSCs are CD29-negative. However, human MSCs are generally considered to be CD29-, CD71-, and CD90-positive. Therefore, the surface markers of human MSCs might differ from those of other species. Rabbit bone marrow MSCs were obtained that had a multi-differentiation potential. The phenotype of these cells was studied using flow cytometry antibodies for 25 rabbit surface markers, namely, CD13, CD14, CD29, CD31, CD34, CD44, CD45, CD49d, CD49f, CD51, CD54, CD59, CD71, CD73, CD90, CD105, CD106, CD133, CD166, MHC I, MHC II, α-smooth muscle actin (α-SMA), cytokeratin, desmin, and vimentin. The phenotype of commercially available human MSCs was similarly studied using antibodies for human surface markers. CD14, CD31, CD34, CD45, CD49d, CD49f, CD51, CD54, CD71, CD106, CD133, MHC II, and cytokeratin were absent from both rabbit and human MSCs, while CD44, α-SMA, and vimentin were present on both cell lines. CD13, CD29, CD59, CD73, CD90, CD105, CD166, and MHC I were present on human MSCs, but not on rabbit MSCs. However, desmin was present on rabbit MSCs, but not on human MSCs. In total, the surface expression of nine markers differed between human and rabbit MSCs, whereas the surface expression of 16 markers was the same in the two cell lines.