The role of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule 1 (ICAM-1, CD54) interactions in human T cell and B cell collaboration was examined by studying the effect of mAb to these determinants on B cell proliferation and differentiation stimulated by culturing resting B cells with CD4+ T cells activated with immobilized mAb to the CD3 molecular complex. In this model system, mAb to either the alpha (CD11a) or beta (CD18) chain of LFA-1 or ICAM-1 (CD54) inhibited B cell responses significantly. The mAb did not directly inhibit B cell function, inasmuch as T cell-independent activation induced by formalinized Staphylococcus aureus and IL-2 was not suppressed. Moreover, DNA synthesis and IL-2 production by immobilized anti-CD3-stimulated CD4+ T cells were not suppressed by the mAb to LFA-1 or ICAM-1. Although the mAb to LFA-1 inhibited enhancement of IL-2 production by co-culture of immobilized anti-CD3-stimulated CD4+ T cells with B cells, addition of exogenous IL-2 or supernatants of mitogen-activated T cells could not abrogate the inhibitory effects of the mAb to LFA-1 or ICAM-1 on B cell responses. Inhibition was most marked when the mAb were present during the initial 24 h in culture. Immobilized anti-CD3-stimulated LFA-1-negative CD4+ T cell clones from a child with leukocyte adhesion deficiency could induce B cell responses, which were inhibited by mAb to LFA-1 or ICAM-1. These results indicate that the interactions between LFA-1 and ICAM-1 play an important role in mediating the collaboration between activated CD4+ T cells and B cells necessary for the induction of B cell proliferation and differentiation, and for enhancement of IL-2 production by CD4+ T cells. Moreover, the data are consistent with a model of T cell-B cell collaboration in which interactions between LFA-1 on resting B cells and ICAM-1 on activated CD4+ T cells play a critical role in initial T cell-dependent B cell activation.

Epithelial-mesenchymal transition (EMT) is an important process in tumor metastasis. The EMT-related events associated with metastasis of NPC in the absence of EBV have not been elucidated. We established an EBV-negative NPC cell line from a bone marrow biopsy of an NPC patient. Using a Matrigel system we isolated an invasive and non-invasive sublines, designated NPC-BM29 and NPC-BM00. NPC-BM29 acquired an invasive-like phenotype characterized by EMT, marked by down-regulation of E-cadherin and beta-catenin with concomitant increased expression of Ets1. NPC-BM29 cells expressed>>or= 10-fold higher of MMP-9 than NPC-BM00 cells. NPC-BM29 cells grew better in 2% serum than NPC-BM00 cells, with a population doubling-time of 26.8 h and 30.7 h, respectively. A marked reduction in colony-formation ability of NPC-BM00 cells compared to NPC-BM29 was observed. Wound-healing assay revealed that NPC-BM29 cells displayed higher motility than NPC-BM00 and the motility was further enhanced by cell treatment with TPA, a PKC activator. Cell surface markers and tumor-associated molecules, AE3, MAK6 and sialyl-Tn, were up-regulated in NPC-BM29 cells, whereas the expression of HLA-DR and CD54 was significantly increased in NPC-BM00 cells. NPC-BM29 consistently released higher levels of IL-8 and IL-10 than NPC-BM00, with low levels of IL-1alpha expression in both cell lines. Higher level of VEGF production was detected in NPC-BM00 than NPC-BM29 cells. These data show that EBV is not required for exhibiting multiple metastatic phenotypes associated with EMT. More studies that target right molecules/signalings associated with the EMT may offer new therapeutic intervention options for NPC invasion and metastasis.

OBJECTIVE

Osteoblasts are classically considered to play an important role during bone tissue development, and to be involved in the formation of mineralized bone matrix. Recent reports have suggested that they can also exert some activities directly associated with the immune system (cytokine synthesis and antigen presentation). Moreover, some authors have found antigens on osteoblast-like cells normally expressed by other cells with a common origin in bone marrow.

METHODS

We isolated and cultured human osteoblast-like lines and studied their antigenic phenotype with flow cytometry using monoclonal antibodies against antigens associated with hematopoietic cells.

RESULTS

Cultured cells expressed CD34, but were negative for CD45. B cell antigens CD20 and CD23 and myelomonocytic antigens CD11b, CD13, and CD16 were detected. Expression of CD3, CD14, CD15 and CD68 was negative, whereas CD25 expression was positive. CD56, an antigen expressed on NK cells, was positive. These cells were CD10, CD44, CD54, CD80, CD86 and HLA-DR positive, as previously described. An antigen specific to follicular dendritic cells was also observed on cultured osteoblast-like cells.

CONCLUSIONS

The antigenic phenotypes of human osteoblast-like cells and FDC are similar. These data suggest that osteoblasts may be functionally related to certain dendritic cells and may play an additional role in bone tissue to that classically assigned.

Costimulatory blockade is one of the most promising therapeutic targets in autoimmune diseases as well as in transplant recipients, and inhibition of the cluster of differentiation (CD)40-CD154 interaction, which is required for T cell activation and development of an effective immune response, is particularly promising in islet transplant recipients. Here, we report the ability of several small-molecule organic dyes to concentration dependently inhibit this interaction with IC(50) values in the low-micromolar range. They were found to be considerably more active in inhibiting this interaction than the tumor necrosis factor (TNF)-R1-TNF-alpha or B cell-activating factor (BAFF)-R-BAFF interaction, which are members of the same family. They specifically inhibited CD154-induced cell responses in human B cells as well as in THP-1 myeloid cells, which can serve as surrogate dendritic cells, at concentrations well below their cytotoxic concentrations determined in the same cells. Flow cytometry experiments confirmed their ability to inhibit the CD154-induced, but not the Staphylococcus aureus Cowan I- or phorbol 12-myristate 13-acetate-induced increase in the surface expression of CD54, CD40, and major histocompatibility complex class II. Accordingly, these compounds can be useful not only for experimental investigations involving the inhibition of the CD40-CD154 costimulatory interaction but can also provide important structure-activity relationship information and can serve as the starting point of a targeted drug discovery program.

Dendritic cells (DCs), efficient-antigen presenting cells play an important role in initiating and regulating immune responses. DC maturation following exposure to nickel or DNCB induced an up-regulation of phenotypic markers and inflammatory cytokine secretion. Early intracellular mechanisms involved in DC maturation required to be precise. To address this purpose, DCs derived from human monocytes were treated with sensitizers (nickel, DNCB or thimerosal) in comparison with an irritant (SDS). Our data confirming the up-regulation of CD86, CD54 and cytokine secretion (IL-8 and TNFalpha) induced by sensitizers but not by SDS, signalling transduction involved in DC maturation was investigated using these chemicals. Kinase activity measurement was assessed using two new sensitive procedures (Facetrade mark and CBA) requiring few cells. SDS did not induce changes in signalling pathways whereas NiSO(4), DNCB and thimerosal markedly activated p38 MAPK and JNK, in contrast Erk1/2 phosphorylation was completely inhibited by DNCB or thimerosal and only activated by nickel. A pre-treatment with p38 MAPK inhibitor (SB203580) suppressed Erk1/2 inhibition induced by DNCB or thimerosal demonstrating a direct interaction between p38 MAPK and Erk1/2. A pre-treatment with an antioxidant, N-acetyl-L-cysteine (NAC) markedly reduced Erk1/2 inhibition and p38 MAPK phosphorylation induced by DNCB and thimerosal, suggesting a direct activation of p38 MAPK via an oxidative stress and a regulation of MAPK signalling pathways depending on chemicals. Because of a high sensitivity of kinase activity measurements, these procedures will be suitable for weak or moderate sensitizer screening.

We have previously demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed the induction of the costimulatory molecule CD86 (B7-2) on B220+ and Mac-1+ spleen cells following the injection of allogeneic P815 tumor cells. In this study, TCDD exposure was shown to suppress CD54 and major histocompatibility complex (MHC) class II expression on B220+, Mac-1+, and CD11c+ splenic antigen presenting cells (APC). Furthermore, interleukin-12 (IL-12) production by spleen cells from P815-immunized mice was significantly decreased following exposure to TCDD. To determine if exogenous costimulation could enhance the activation of APC, vehicle- and TCDD-treated mice were injected with an agonistic antibody to murine CD40. Stimulation with anti-CD40 increased the expression of CD86, CD54, and MHC class II on splenic APC and greatly enhanced the production of interleukin-12. TCDD treatment had minimal effects on the anti-CD40-induced expression of accessory molecules on splenic APC. TCDD exposure had no effect on anti-CD40-induced IL-12 in the plasma but suppressed its production from cultured spleen cells. Surprisingly, although stimulation via CD40 increased the activation of APC, allograft effector functions were not restored in TCDD-treated mice, perhaps due to persistent defects in antigen processing and presentation, cytokine production, T cell function, or CD40-independent pathways of APC activation.

Human mast cells have been purified from uterine tissues, and their surface marker profile and function have been evaluated as part of ongoing studies of mast cell heterogeneity. Using a panel of antibodies, purified uterine mast cells (UMC; 81% +/- 7% purity, n = 10) were analyzed by immunofluorescence and flow cytometry for surface expression of various antigens. Consistent with previous analyses of mast cells from other tissues, UMC expressed HLA class I, IgE, c-kit receptor, CD9, CD33, CD43, CD45, and CD54, while CD11a, CD11b, CD14, CD16, CD23, and CD64 were not detected. Unlike other mast cells, UMC expressed CD11c/CD18 (p150,95) and CD32 (Fc gamma RII). Additional antigens not previously studied on mast cells included the selectin LECAM-1 (Leu-8) and several beta 1 and beta 3 integrins; expression of very late activation antigen-4 (VLA-4) (CD49d/CD29), VLA-5 (CD49e/CD29), and the vitronectin receptor (CD51/CD61) was seen. Functional studies showed that treatment of human umbilical vein endothelial cells with interleukin-1 (5 ng/mL for 4 hours) resulted in a twofold to threefold increase in adhesiveness for UMC. Purification procedures did not alter histamine release responses to anti-IgE or the calcium ionophore A23187, and treatment of UMC with an anti-CD32 monoclonal antibody (IV.3) did not induce histamine release or alter anti-IgE-induced release. These data suggest that UMC may possess unique phenotypic characteristics, and support the concept of mast cell heterogeneity.

We have previously shown that tolerance can be induced against acute experimental autoimmune encephalomyelitis (EAE) in Lewis rats by bone marrow-derived dendritic cells (DC) that have been pulsed in vitro with encephalitogenic myelin basic protein peptide 68-86 (MBP 68-86), and injected subcutaneously into healthy rats prior to immunization with MBP 68-86 plus complete Freund's adjuvant. To elucidate better the properties of tolerogenic DC, we here compared plastic-adherent DC with floating, non-adherent DC, which were cultured for 7 days in the presence of granulocyte-macrophage colony-stimulating factor plus interleukin-4 (IL-4). Adherent DC expressed high levels of IL-10 mRNA and protein, and low levels of IL-12 mRNA and showed high expression of CD54 compared with floating DC. Proliferation, nitrite concentration and capacity for antigen presentation were lower in adherent DC than in floating DC. There were no differences between adherent and floating DC regarding expression of CD11c, OX62, major histocompatibility complex class II, CD80, or CD86. Most importantly, we observed that adherent DC induced tolerance to EAE in vivo when injected subcutaneously into Lewis rats prior to immunization, while floating DC did not. Adherent DC-mediated tolerance to EAE was associated with augmented proliferation, nitric oxide production and frequency of apoptotic cells as well as with up-regulation of transforming growth factor-beta (TGF-beta) -expressing cells in T-cell areas of lymph nodes. Tolerance induction by adherent DC seems to be related to a nitric oxide-apoptosis pathway and to up-regulation of TGF-beta-expressing cells.

The initial steps of Leishmania infection in humans are largely unknown. There is limited information on the Leishmania infected human monocytes, the first cells that the parasite lives in, particularly related to costimulatory molecules. We show here that Leishmania (L.) chagasi infection avoids inducing proinflammatory molecules and has striking down modulating effects on human monocytes or macrophages. It does not induce CD54, interleukin (IL)-12 or tumour necrosis factor-alpha, potent proinflammatory cytokines and down modulates CD11b expression in monocytes. Lipopolysaccharide stimulated IL-12 (p40) levels, CD54 and HLA-DR expression are diminished in infected monocytes as well as interferon-gamma stimulated HLA-DR and HLA-ABC expression in infected macrophages. There is a negative correlation between CD54 and CD86 expression in both monocytes and macrophages. The depressed expression of class I and II molecules, absence of key proinflammatory cytokines and impaired expression of costimulatory molecules induced by L. chagasi could leave the immune system, at least in its initial phases in anergy or ignorance.

Human intraepithelial lymphocytes (IEL), predominantly CD8+ T lymphocytes located between intestinal epithelial cells, may represent the first-line host defense against colon cancers. This study evaluates their lymphokine-activated killer (LAK) activity and compares it to that of lamina propria lymphocytes (LPL). The phenotypes of the precursor or effector LAK cells were determined by depleting IEL or LPL of specific subsets using antibody and complement lysis either before or after a 7-day culture with interleukin-2 (IL-2), respectively, and then measuring changes in lytic activity. The precursor and effector LAK cells in the IEL were mixed populations with some but not all expressing CD2, CD3, CD8, and NKHl. The LAK activity by LPL was due to CD2+CD3+CD4-CD8- precursor cells and CD8+ effector cells. Since the IEL LAK cells were heterogeneous, their lytic activity against two different target cell types was analyzed. The LAK activity against DLD-1 colonic adenocarcinoma cells was reduced by antibodies to CD2, CD11a, and HML-1, whereas LAK activity against K-562 cells was reduced by antibodies to CD2, CD11a, and CD54, but not HML-1, indicating a target-cell-specific mechanism. Cold competition experiments, however, showed that the same cytotoxic IEL bound to both target cells. In conclusion, LAK activity by IEL is due to a mixed population, whereas that by LPL is due to CD8- precursor cells and CD8+ effector cells. The same effector LAK IEL kill both DLD-1 and K-562 targets, but different mechanisms are involved.

BACKGROUND

During healthy pregnancy, T helper (Th) 1-type and inflammatory-type responses are down-regulated, and Th2-type and proinflammatory-type responses predominate. In Plasmodium falciparum-infected females, these responses induce enhanced production of tumor necrosis factor- alpha and interferon- gamma.

METHODS

To assess the respective implication of monocytes and T cells in this placental immunomodulation, we cocultured cells from delivering females living in an area where malaria is endemic. Monocytes and T cells from both peripheral and intervillous blood were crossed in in vitro cultures, to compare the proliferative response to several antigens. Moreover, monocyte cell-surface molecules were quantified by flow cytometry.

RESULTS

Coculture results confirmed placental immunomodulation and suggested that the most affected cells are not the intervillous monocytes, which are as able to present the antigen as the peripheral monocytes, but the intervillous T cells. Monocyte staining showed significant increases in human leukocyte antigen D-related, CD54, CD80, and CD86 surface markers in intervillous blood, compared with peripheral blood, which suggests a relative activation of monocytes in the placenta.

CONCLUSIONS

A state of T cell deactivation and monocyte activation is present at delivery. The T cell deactivation in reaction to purified protein derivative could be explained by the presence of local T cell immunoregulatory factors.

In vitro effects of human recombinant IL-6 (1-1000 U/ml) on highly enriched human NK CD3-CD56+ cells (94% +/- 2; mean +/- SEM; n = 8), obtained from PBL were studied. IL-6 induced low levels of NK cell proliferation (7- to 30-fold during 6-day incubation), which was IL-2-independent, because IL-6 did not induce detectable IL-2 production by NK cells. Two-color flow cytometry analysis demonstrated that incubation of NK cells with IL-6 at the optimal concentration of 250 U/ml for 6 days significantly increased the proportion of NK cells expressing the following activation Ag: CD25 (26% +/- 17, mean +/- SEM vs 4% +/- 1 in control, n = 5), CD54 (44% +/- 17 vs 9% +/- 3), HLA-DR (29% +/- 13 vs 12% +/- 4), CD69 (45% +/- 7 vs 12% +/- 3), and CD71 (34% +/- 17 vs 6% +/- 2). The mean fluorescence intensity of these activation Ag was increased as well. IL-6 induced expression of CD49b (alpha-chain of VLA-2, 20% +/- 11 vs 2% +/- 1) and CD49c (alpha-chain of VLA-3, 43% +/- 17 vs 5% +/- 3), which are not expressed on resting NK cells. IL-6 also enhanced the fluorescence intensity of beta 1 integrins, CD49d, CD49e, and CD49f, expressed on NK cells. IL-6-stimulated NK cells showed significantly increased integrin-mediated adhesion to fibronectin- or laminin-coated plates (26 +/- 3 mean % cells adhering +/- SEM vs 15 +/- 4 in control for FN and 19 +/- 1 vs 11 +/- 1 for LM, p < 0.05 for both) as determined in a 3 h binding assay. As assessed by inhibition of adhesion using mAb to the VLA-2, -3, -4, -5, and -6, NK cell adhesion to fibronectin was mediated by VLA-4 and 5, and their adhesion to laminin by VLA-3 and -6. NK cells incubated in the presence of IL-6 were found to produce a factor cytostatic to WEHI-164 clone 13 target cells. This effect was partly, although significantly, blocked by neutralizing antibodies to TNF-alpha or TNF-beta. Our data demonstrate that IL-6 can directly activate human NK cells, but is a less potent NK cell activator, for all activation and functional parameters studied, than IL-2.

Exposure of RPMI 8226, Karpas 707 and U266 human myeloma-like lines to low doses of As(2)O(3) was followed by a marked increase in lymphokine activated killers (LAK)-mediated killing and up- modulation of CD38 and CD54, two molecules involved in cell-cell interactions. Moreover, simultaneous exposure of effectors and targets to As(2)O(3) yielded the most effective condition for lysis. The expression of CD31 (CD38 ligand) and CD11a (CD54 ligand) was also up-regulated by LAK, suggesting that increased adhesion was responsible for the improved killing. Similar results were obtained using freshly isolated myeloma cells. These findings indicate that As(2)O(3) may be useful to boost the immune system against myelomas.

A pair of correspondent adhesion molecules: LFA-1 (CD11aCD18) and ICAM-1 (CD54) was shown to be involved in autoimmune insulitis in animal models. Anti-LFA-1 or anti-ICAM-1 monoclonal antibodies administered in vivo had a very strong preventive effect on the development of spontaneous diabetes with a marked reduction of insulitis. On the other hand elevated levels of the soluble form of ICAM-1 (sICAM-1) were documented in subjects at risk for type 1 diabetes. Recently sICAM-1 was shown to play an immunoregulatory role as an inhibitor of islet insulitis. The aim of the present study was to evaluate CD11a + mononuclear cells (lymphocytes and monocytes) and soluble sICAM-1 levels in the peripheral blood of subjects with preclinical and overt type 1 diabetes to assess their role in the development of the autoimmune process and their possible associations with the humoral autoimmune markers. The study was carried out in three groups of subjects: 26 first degree relatives of type 1 diabetes patients (prediabetics) with the combinations of autoantibodies against pancreatic B cells (ICA, GADA, IA-2A, IAA), 22 patients with a recent onset of type 1 diabetes and age and sex-matched 24 healthy volunteers (control group). We observed an increased fluorescence intensity of CD11a on mononuclear cells in overt diabetes subjects and a positive correlation between CD11a fluorescence intensity on monocytes and ICA titre. The highest sICAM-1 levels we obtained in the peripheral blood in the prediabetics in comparison to patients with clinical diabetes and the healthy controls. We found a positive correlation between slCAM-1 and values of ICA, GADA and a total number of antibodies present. In conclusion our study suggests that LFA-1 and sCAM-1 play an important role in the pathogenesis of type 1 diabetes. The assessment of the CD11a bearing monocytes and sICAM-1 levels are potential markers of the preclinical stage of the autoimmune diabetes, but further prospective studies in high risk diabetes type 1 subjects are needed.

Membrane-associated intercellular adhesion molecule-1 (mICAM-1; CD54) is constitutively expressed on the surface of type I alveolar epithelial cells (AEC). Soluble ICAM-1 (sICAM-1) may be produced by proteolytic cleavage of mICAM-1 or by alternative splicing of ICAM-1 mRNA. In contrast to inducible expression seen in most cell types, sICAM-1 is constitutively released by type I AEC and is present in normal alveolar lining fluid. Therefore, we compared the mechanism of sICAM-1 production in primary cultures of two closely juxtaposed cells in the alveolar wall, AEC and pulmonary microvascular endothelial cells (PVEC). AEC, but not PVEC, demonstrated high-level baseline expression of sICAM-1. Stimulation of AEC with TNFalpha or LPS resulted in minimal increase in AEC sICAM-1, whereas PVEC sICAM-1 was briskly induced in response to these signals. AEC sICAM-1 shedding was significantly reduced by treatment with a serine protease inhibitor, but not by cysteine, metalloprotease, or aspartic protease inhibitors. In contrast, none of these inhibitors effected sICAM-1 expression in PVEC. RT-PCR, followed by gel analysis of total RNA, suggests that alternatively spliced fragments are present in both cell types. However, a 16-mer oligopeptide corresponding to the juxtamembrane region of mICAM-1 completely abrogated sICAM-1 shedding in AEC but reduced stimulated PVEC sICAM-1 release by only 20%. Based on these data, we conclude that the predominant mechanism of sICAM-1 production likely differs in the two cell types from opposite sides of the alveolar wall.

Phenotypic changes in CD4(+) T cells undergoing antigen-dependent activation were compared in vivo and in vitro. The most obvious difference was in expression of CD25, the alpha chain of the high affinity receptor for IL-2. High level expression of CD25 in vivo is restricted to a small fraction of the cells at the leading edge of the cell division profile, whereas all activated cells express high levels of CD25 in cultures responding to antigen. Because IL-2 is known to upregulate expression of CD25 in preactivated T cells, this suggests a difference in IL-2 exposure in the two responses. A number of other markers, including CD54, show a similar difference in the pattern of expression in vivo and in vitro. Using 6-colour flow cytometry, it was demonstrated that the small percentage of cells expressing CD25 in vivo coexpresses a very high level of a number of other activation markers, including CD38, CD44 and Ly-6A/E, suggesting that these may also be upregulated by autocrine IL-2.

The up-regulation of different adhesion molecules such as VLA-4 (CD49d/CD29) and ICAM-1 (CD54) on the pulmonary endothelium and leukocytes, is a key event in sepsis-induced lung injury leading to inflammatory tissue alterations. The value of VLA-4 and ICAM-1 as micromorphological post-mortem markers for the detection of sepsis-induced lung injury, was evaluated in a semiquantitative immunohistochemical study. VLA-4 was strongly expressed on intravascular, interstitial and intra-alveolar leukocytes in sepsis-associated fatalities, whereas in non-septic fatalities an irregular weak immunoreactivity was observed on interstitial leukocytes and no positive immunohistochemical expression was detected on intravascular or intra-alveolar leukocytes. ICAM-1 was strongly expressed on endothelial cells of the pulmonary microvasculature and on pulmonary macrophages and lymphocytes in sepsis-associated fatalities. In contrast, an infrequent weak immunohistochemical reaction for ICAM-1 was found on pulmonary endothelium and on perivascular leukocytes in non-septic fatalities. Based on the results of the present preliminary study, VLA-4 and ICAM-1 can be considered as useful immunohistochemical post-mortem markers of sepsis.

Transition from hormone-sensitive to hormone-refractory metastatic tumor types poses a major challenge for prostate cancer treatment. Tumor antigens that are differentially expressed during this transition are likely to play important roles in imparting prostate cancer cells with the ability to grow in a hormone-deprived environment and to metastasize to distal sites such as the bone and thus, are likely targets for therapeutic intervention. To identify those molecules and particularly cell surface antigens that accompany this transition, we studied the changes in cell surface antigenic profiles between a hormone-sensitive prostate cancer line LNCaP and its hormone-refractory derivative C4-2B, using an antibody library-based affinity proteomic approach. We selected a naive phage antibody display library to identify human single-chain antibodies that bind specifically to C4-2B but not LNCaP. Using mass spectrometry, we identified one of the antibody-targeted antigens as the ICAM-1/CD54/human rhinovirus receptor. Recombinant IgG1 derived from this single-chain antibody binds to a neutralizing epitope of ICAM-1 and blocks C4-2B cell invasion through extracellular matrix in vitro. ICAM-1 is thus differentially expressed during the transition of the hormone-sensitive prostate cancer cell line LNCaP to its hormone-refractory derivative C4-2B, plays an important role in imparting the C4-2B line with the ability to invade, and may therefore be a target for therapeutic intervention.

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

BACKGROUND

Allogeneic tolerance can be reliably obtained with monoclonal antibody therapy targeting CD45RB. Although regulatory T cells play an important role in the mechanism, we have recently demonstrated the active participation of host B lymphocytes. After anti-CD45RB therapy, B lymphocytes demonstrate phenotypic alterations that include up-regulation of CD54 (intercellular adhesion molecule [ICAM]-1). We have investigated the hypothesis that alteration in ICAM-1 expression is required for tolerance induction.

METHODS

Recipients of heterotopic allogeneic cardiac grafts (C3H donors into B6 recipients) were treated with anti-CD45RB, anti-ICAM, anti-lymphocyte function-associated antigen-1 (LFA), or the combination of these agents. These data were extended by performing allogeneic cardiac transplants into ICAM or LFA recipients treated with a 5-day course of anti-CD45RB. Finally, B-cell-deficient animals were reconstituted with ICAM splenocytes to create a recipient with a selective deficiency of ICAM-1 restricted to the B-cell compartment.

RESULTS

Anti-CD45RB alone or the combination of anti-LFA/anti-ICAM reliably induced transplantation tolerance. However, the triple combination was routinely unsuccessful and induced long-term graft survival in no recipients. ICAM-deficient or LFA-deficient recipients were also resistant to tolerance induced by anti-CD45RB. Finally, transfer of control splenocytes to B-cell-deficient recipients permitted anti-CD45RB-induced tolerance, whereas transfer of ICAM cells was unable to support tolerance induction.

CONCLUSIONS

Expression of ICAM-1 by B lymphocytes and interaction with LFA-1 form a central aspect of transplantation tolerance induced by anti-CD45RB therapy. These data further elucidate the cellular mechanisms used by B lymphocytes in the induction of transplantation tolerance.

BACKGROUND

Alveolar macrophages (AM) are the major population in bronchoalveolar lavage (BAL) cells; we assessed their role in human lung allograft recipients by correlating the expression of adhesion molecules and inflammatory cytokines with clinical outcome of allograft.

METHODS

We obtained BAL samples from patients and enriched them for AM in plastic petri dish for 2 hours at 37 degrees C in 5% CO(2). Expression of intercellular adhesion molecule-1 (ICAM-1, CD54), platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31), and CD11c was assessed by flow cytometry using monoclonal antibodies. We assessed cytokine profile using Multi-Probe RNase protection assay.

RESULTS

Alveolar macrophages that express CD11c, CD31 and CD54 were increased in patients with either rejection or infection compared with those without rejection and infection. The difference in the percentage of AM expressing CD11c and CD31 between the rejection group and patients without rejection and infection group was statistically significant (CD11c, p < 0.01; CD31, p < 0.03). Interleukin (IL)-1 alpha, IL-1 beta, IL-1 receptor antagonist (IL-1Ra), and IL-6 expression was higher in the rejection group than in patients without rejection. Five out of 9 patients in the rejection group expressed high levels of IL-15 and tumor necrosis factor-alpha compared with patients without rejection and infection. The increased number of AM expressing adhesion molecules and elevated expression of cytokines observed during acute rejection declined to basal levels after successful treatment and resolution of rejection. This study demonstrates that lung allograft rejection is associated with increased expression of adhesion molecules and inflammatory cytokines by AM, which could facilitate mononuclear cell adhesion and extravasation contributing to the allograft injury in lung transplant recipients.

OBJECTIVE

Compelling evidence implicates inflammation in the pathogenesis of type 1 diabetes mellitus (T1DM) and associated vascular complications. Obesity is also characterized by low-grade systemic inflammation. In this study, we characterized the inflammatory response in diabetes by analyzing the expression of a panel of activation markers on the surface of peripheral blood monocytes in recently-diagnosed T1DM patients. The potential effects of glycemic control and body mass index (BMI) on monocyte phenotype were also investigated.

METHODS

Using flow cytometry, we analyzed the expression of CD11b, CD49d, <em>CD54</em>, CD62L and CD64 antigens on monocytes in a cohort of 51 T1DM patients (</= 2 months after diagnosis). To test whether phenotype change in monocytes was associated with abnormal cellular function, we studied the adhesive capacity of monocytes to human umbilical vein endothelial cells (HUVEC).

RESULTS

We found that circulating monocytes from T1DM patients tested at the clinical onset of the disease (i.e. within 1 week of diagnosis) had higher CD11b expression compared to patients analyzed 2 months after diagnosis (p = 0.02). The highest CD11b levels were detected in patients with HbA1c < 8% (p = 0.04 vs. patients with HbA1c < 8%). In T1DM children analyzed 2 months after diagnosis, we found that those who were overweight (BMI>>/= 85th percentile) had higher levels of monocyte activation than those who were not (BMI </= 85th percentile) (p = 0.03). CD11b and HbA1c were significantly correlated (correlation coefficient 0.329, p = 0.02). Finally, monocytes from T1DM patients showed higher adhesion to HUVEC compared to controls.

CONCLUSIONS

Circulating immune cells from T1DM patients display many aspects of a proinflammatory state, as indicated by primed or activated monocytes. Obesity is an important factor in monocyte activation during diabetes.

Indirect immunofluorescence staining with monoclonal antibody (MoAb) CL203.4 of malignant cells from 269 patients with hematologic malignancies showed a heterogeneous expression of CD54/intercellular adhesion molecule-1 (ICAM-1). This marker was expressed by malignant cells of 57 out of 118 patients with myeloid malignancies and 69 out of 135 with B-lymphoid malignancies. On the other hand, CD54 was not detected on malignant cells of 16 patients with T-lymphoid malignancies. In myeloid malignancies, CD54 is preferentially expressed by "stem cell-derived" malignancies, being detectable on blast cells from almost all patients affected by chronic myelogenous leukemia in blast phase or myelodysplastic syndromes and by only 34% of patients with de novo acute myeloid leukemia (AML). The expression of CD54 did not correlate with any specific myeloid FAB subtype, although three cases of highly undifferentiated AML (FAB MO) displayed maximal levels of the antigen. The expression of CD54 in AML was significantly associated with that of CD34 and HLA-DR antigens. In B-lymphoid malignancies, CD54 expression appears to correlate with the differentiation stage of malignant cells, since B-origin acute lymphoblastic leukemias and conventional B-chronic lymphocytic leukemias (B-CLL; ie, "dim SIg" CLL) expressed lower levels of CD54 than more mature lymphoproliferative disorders ("bright SIg" CLL, prolymphocytic leukemias, and lymphoplasmacytic tumors). "High-grade" B-cell non-Hodgkin's lymphomas (B-NHL) express in general a higher level of CD54 than "low-grade" ones. This finding in conjunction with the expression of CD54 in all 17 patients with "bright SIg" CLL investigated (characterized by marked organomegaly and poor prognosis) suggest that the differential expression of CD54 in lymphoproliferative disorders may also relate to their degree of malignancy.

Antigen-presenting cells (APC) play a pivotal role in the initiation of the T cell-mediated and antigen-specific immune response. The suggested role of endogenous inhibitor cystatin C (CyC) is to modulate cysteine proteases (cathepsins) present in human APC. To test this hypothesis, dendritic cells (DC) were generated in vitro from isolated monocytes, and changes in content, localization, and secretion of CyC and cathepsins S, L, and H (CatS, -L, and -H, repsectively) were followed in response to interleukin-4, enabling monocyte differentiation, and to tumor necrosis factor alpha (TNF-alpha), enabling DC maturation. A large increase in intracellular CyC accompanied the differentiation of monocytes to immature DC, also shown by strong immunolabeling of Golgi in immature DC. On DC maturation, intracellular CyC levels decreased, and CyC was mostly absent from the Golgi. On prolonged incubation of mature DC with TNF-alpha, CyC was found located in the proximity of the plasma membrane, indicating that the transport of CyC from Golgi was not blocked as the result of the arrested exocytosis in mature DC. The secretion of CyC ceased, consistent with the peak of the surface expression of phenotypic markers (CD40, CD54, CD80, CD83, CD86, and major histocompatibility complex class II), characteristic for the mature DC stage, whereas the secretion of cathepsins did not correlate with the maturation stage. The difference in localization of CyC and of CatS, -L, and -H in immature and mature DC shows that the regulatory potential of CyC toward CatS, -L, and -H inside DC is limited. However, these interactions may occur extracellularly in lymph, as suggested by the large excess of CyC over secreted CatS, -L, and -H, and they may facilitate DC migration to lymph nodes.