Lung dendritic cells were identified by immunohistochemistry in lung tissue sections from C57BL/6 mice. Following isolation from the lungs using CD11c magnetic beads, the flow cytometric analysis of I-A(b+) and CD11c(+) cells indicated a mixed population of dendritic cells at different stages of maturation, with most expressing an immature phenotype. When cultured for 7 days with recombinant murine granulocyte-macrophage colony-stimulating factor, 99% of cells were CD11c(+) and had a morphology typical of immature dendritic cells. These cells were negative for CD34, CD14, and CD8 alpha antigens but expressed low levels of the myeloid marker F4/80 and moderate levels of MAC3. All expressed high levels of CD11a (LFA-1), CD11b (Mac1), and CD54 antigens, with low levels of class II major histocompatibility complex. Most cells expressed CD80 but only a small percentage of cells were positive for CD40 and CD86. Both overnight and 7-day cultures of lung dendritic cells were able to phagocytose Mycobacterium tuberculosis, and this was associated with the production of interleukin-12 and stimulation of both naïve and immune T cells to produce gamma interferon.

Sin Nombre virus (SNV) and Hantaan virus (HTN) infect endothelial cells and are associated with different patterns of increased vascular permeability during human disease. It is thought that such patterns of increased vascular permeability are a consequence of endothelial activation and subsequent dysfunction mediated by differential immune responses to hantavirus infection. In this study, the ability of hantavirus to directly induce activation of human lung microvascular endothelial cells (HMVEC-Ls) was examined. No virus-specific modulation in the constitutive or cytokine-induced expression of cellular adhesion molecules (CD40, CD54, CD61, CD62E, CD62P, CD106, and major histocompatibility complex classes I and II) or in cytokines and chemokines (eotaxin, tumor necrosis factor alpha, interleukin 1beta [IL-1beta], IL-6, IL-8, MCP-1, MIP-1alpha, and MIP-1beta) was detected at either the protein or message level in hantavirus-infected HMVEC-Ls. Furthermore, no virus-specific enhancement of paracellular or transcellular permeability or changes in the organization and distribution of endothelial intercellular junctional proteins was observed. However, infection with either HTN or SNV resulted in detectable levels of the chemokines RANTES and IP-10 (the 10-kDa interferon-inducible protein) in HMVEC-Ls within 72 h and was associated with nuclear translocation of interferon regulatory factor 3 (IRF-3) and IRF-7. Gamma interferon (IFN-gamma)-induced expression of RANTES and IP-10 could also be detected in uninfected HMVEC-Ls and was associated with nuclear translocation of IRF-1 and IRF-3. Treatment of hantavirus-infected HMVEC-Ls with IFN-gamma for 24 h resulted in a synergistic enhancement in the expression of both RANTES and IP-10 and was associated with nuclear translocation of IRF-1, IRF-3, IRF-7, and NF-kappaB p65. These results reveal a possible mechanism by which hantavirus infection and a TH1 immune response can cooperate to synergistically enhance chemokine expression by HMVEC-Ls and trigger immune-mediated increases in vascular permeability.

OBJECTIVE

Mesenchymal stem cells (MSC) are self-renewing, multipotent cells that are present in many adult tissues, including bone marrow, trabecular bone, adipose, and muscle. The presence of such cells of mesenchymal origin and their role during the wound healing of ocular injuries are currently being explored by many studies worldwide. In this study, we aimed to report the presence of mesenchymal cells resembling bone marrow-derived cells (MSC-BM) in the limbus of the human eye.

METHODS

Fresh limbal tissues obtained from human subjects undergoing limbal biopsy for ocular surface reconstruction were used to establish limbal mesenchymal cell (MC-L) cultures. The spindle cell outgrowths observed in extended limbal epithelial cultures (LECs) and from deepithelialized limbal tissues were serially passaged using a human corneal epithelial (HCE) medium, which contained epidermal growth factor (EGF) and insulin, and supplemented with fetal bovine serum (FBS). MSC cultures were established from human bone marrow samples using Dulbecco's Modified Eagles Medium (DMEM) supplemented with FBS. The mesenchymal cells from both extended limbal cultures (MC-L) and bone marrow (MSC-BM) were characterized by morphology and immunophenotyping using epithelial, mesenchymal, hematopoietic, and endothelial markers using fluorescent-activated cell sorting (FACS). Selective markers were further confirmed by immunostaining and reverse transcription polymerase chain reaction (RT-PCR). Stromal cells of both origins (limbal and bone marrow-derived) were also evaluated for colony forming ability and population doubling. Attempts were made to differentiate these into adipocytes and osteocytes using conditioned medium.

RESULTS

Spindle cells from extended limbal epithelial cultures as well as de-epithelialized human limbal tissues appeared elongated and fibroblast-like with oval vesicular nuclei. Both MC-L and MSC-BM showed colony forming ability in 14 days of plating. MC-L showed a population doubling of 22.95 while in MSC-BM, it was 30.98. Immunophenotyping of these cells by FACS and immunocytochemistry showed that the MC-L were positive for mesenchymal markers and negative for epithelial and hematopoietic markers similar to MSC-BM. The MC-L phenotype has thus been defined as MC-L(CD105, CD106, CD54, CD166, CD90, CD29, CD71, pax -6 +/ p75, SSEA1, Tra-1-61, Tra-1-81, CD31, CD34, CD45, CD11a, CD11c, CD14, CD138, Flk1, Flt1, VE-Cadherin -). The profile was further confirmed by RT-PCR. These cells also showed differentiation into adipocytes and osteocytes.

CONCLUSIONS

We demonstrated the presence of mesenchymal cells in the human limbus, similar to the bone marrow-derived MSC-BM. This presence suggests that these cells are unique to the adult stem cell niche.

Multiple myeloma (MM) is incurable, mainly because of cell adhesion-mediated drug resistance (CAM-DR). In this study, we performed functional screening using short hairpin RNA (shRNA) to define the molecule(s) responsible for CAM-DR of MM. Using four bona fide myeloma cell lines (KHM-1B, KMS12-BM, RPMI8226 and U266) and primary myeloma cells, we identified CD29 (beta1-integrin), CD44, CD49d (alpha4-integrin, a subunit of VLA-4), CD54 (intercellular adhesion molecule-1 (ICAM-1)), CD138 (syndecan-1) and CD184 (CXC chemokine receptor-4 (CXCR4)) as major adhesion molecules expressed on MM. shRNA-mediated knockdown of CD49d but not CD44, CD54, CD138 and CD184 significantly reversed CAM-DR of myeloma cells to bortezomib, vincristine, doxorubicin and dexamethasone. Experiments using blocking antibodies yielded almost identical results. Bortezomib was relatively resistant to CAM-DR because of its ability to specifically downregulate CD49d expression. This property was unique to bortezomib and was not observed in other anti-myeloma drugs. Pretreatment with bortezomib was able to ameliorate CAM-DR of myeloma cells to vincristine and dexamethasone. These results suggest that VLA-4 plays a critical role in CAM-DR of MM cells. The combination of bortezomib with conventional anti-myeloma drugs may be effective in overcoming CAM-DR of MM.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells exerting immunomodulatory effects on cells of the innate and adaptive immune system. It has been shown that an inflammatory milieu is required for the activation of MSC-mediated immunomodulation, and interferon-γ (IFN-γ) plays an important role in this process. We determined the influence of IFN-γ on human adipose-derived stem cells (ASCs) and human amniotic mesenchymal stromal cells (hAMSCs). We further evaluated the effect of MSCs on stimulated T-cells and peripheral blood mononuclear cells (PBMCs) in a cell-contact independent setting. On IFN-γ treatment, ASCs and hAMSCs possessed significantly higher antiproliferative properties and showed surface characteristics of nonprofessional antigen presenting cells (HLA-DR(+)CD40(med+)CD54(high)) with a possible regulatory phenotype (PD-L1(+)PD-L2(+)). The effect of ASCs and hAMSCs on cytokine secretion and T-cell activation was dependent on stimulation method and cellular context. Although ASCs and hAMSCs highly inhibited cytokine secretion of stimulated PBMCs, this was not observed in the case of purified T-cells. The presence of ASCs even favored the secretion of pro-inflammatory cytokines including IFN-γ by T-cells, although T-cell proliferation was efficiently inhibited. Further, ASCs enhanced the number of CD69(+) T-cells independent of the stimuli and cellular context. Interestingly, ASCs significantly suppressed CD25 expression on phytohemagglutinin stimulated PBMCs but had no effect on αCD3/αCD28 stimulated cells. Depending on the stimulation method and cellular context, immune cells create a specific cytokine milieu in vitro, thus differently influencing MSCs and, in turn, affecting their action on immune cells.

Presentation of antigen in the form of immune complexes to B lymphocytes by follicular dendritic cells (FDC) is considered to be a central step in the generation of memory B cells. During this process, which takes place in the microenvironment of the germinal center, B cells and FDC are in close physical contact. In the present study, we have explored the molecular basis of FDC-B cell interaction by using FDC and B cells derived from human tonsils. We found that FDC express high levels of the adhesion receptors intercellular adhesion molecule 1 (ICAM-1 [CD54]) and vascular cell adhesion molecule 1 (VCAM-1), while the B lymphocytes express lymphocyte function-associated antigen 1 (LFA-1 [CD11a/18]), very late antigen 4 (VLA-4 [CD49d], and CD44. Furthermore, we established that both the LFA-1/ICAM-1 and VLA-4/VCAM-1 adhesion pathways are involved in FDC-B lymphocyte binding, and therefore, these pathways might be essential in affinity selection of B cells and in the formation of B memory cells.

In Plasmodium falciparum malaria, large proportions of resident macrophages and circulating monocytes and leukocytes contain massive amounts of the malarial pigment, hemozoin. Previous studies have shown that important functions (e.g., the generation of the oxidative burst, the ability to repeat phagocytosis, and protein kinase C activity) were severely impaired in hemozoin-loaded monocytes. Expression of membrane antigens directly involved in the immune response and in the phagocytic process, and/or under protein kinase C control, in hemozoin-loaded human monocytes was studied. Expression of major histocompatibility complex (MHC) class II after gamma interferon stimulation was blocked in hemozoin-loaded monocytes at the protein expression and gene transcription levels but was preserved in control monocytes loaded with opsonized latex beads or anti-D(Rho)-immunoglobulin G (IgG)-opsonized human erythrocytes. Expression of CD54 (intracellular adhesion molecule 1) and CD11c (p150,95 integrin) was also decreased in hemozoin-loaded monocytes. Expression of MHC class I, CD16 (low-affinity Fc receptor for aggregated IgG), CD32 (low-affinity Fc receptor for aggregated IgG), CD64 (high-affinity receptor for IgG), CD11b (receptor for complement component iC3b [CR3]), CD35 (receptor for complement components C3b and C4b [CR1]), and CD36 (non-class-A scavenger receptor) was not specifically affected by hemozoin loading. These results suggest that hemozoin loading may contribute to the impairment of the immune response and the derangement of antigen presentation reported in previous studies of P. falciparum malaria.

Thrombin-induced expression of endothelial adhesivity toward neutrophils (PMN) was studied using human umbilical vein endothelial cells (HUVEC). HUVEC were challenged with human alpha-thrombin for varying durations up to 120 min, after which the cells were fixed with 1% paraformaldehyde and 51Cr-labeled human PMN were added to determine PMN adhesion. Endothelial adhesivity increased within 15 min after alpha-thrombin exposure, and the response persisted up to 120 min. Expression of endothelial adhesion proteins, P-selectin (GMP-140, PADGEM, CD62), and intercellular adhesion molecule-1 (ICAM-1; CD54) on the endothelial surface was quantitated by increase in the specific binding of anti-P-selectin mAb G1 and anti-ICAM-1 mAb RR1/1 labeled with 125I. P-selectin expression was maximal at 5-15 min alpha-thrombin exposure and decayed to basal levels within 90 min. In contrast, ICAM-1 activity increased at 30 min and remained elevated for 120 min after alpha-thrombin challenge. The initial endothelial adhesivity was dependent on P-selectin expression since PMN adhesion occurring within the first 30 min after alpha-thrombin challenge was inhibited by mAb G1. The later prolonged PMN adhesion was ICAM-1 dependent since this response was inhibited by mAb RR1/1 and to the same degree by the anti-CD18 mAb IB4. Anti-ELAM-1 mAb BB11 had no effect on adhesion of PMN to the alpha-thrombin-challenged cells. The initial P-selectin expression and PMN adhesion responses were reproduced by the 14-amino peptide (SFLLRNPNDKYEPF) (thrombin-receptor activity peptide; TRP-14) which comprised the NH2 terminus created by thrombin's proteolytic action on its receptors. However, TRP-14-induced PMN adhesion was transient, and TRP-14 did not cause ICAM-1 expression. The ICAM-1-dependent PMN adhesion mediated by alpha-thrombin was protein synthesis independent since ICAM-1 expression and PMN adhesion were not inhibited by cycloheximide pretreatment of HUVEC. Moreover, Northern blot analysis indicated absence of ICAM-1 mRNA signal up to 180 min after alpha-thrombin challenge. In conclusion, thrombin-induced endothelial adhesivity involves early- and late-phase responses. The initial reversible PMN adhesion is mediated by rapid P-selectin expression via TRP-14 generation. Thrombin-induced PMN adhesion is stabilized by a protein synthesis-independent upregulation of the constitutive ICAM-1 activity which enables the interaction of ICAM-1 with the CD18 beta 2 integrin on PMN.

THE protein CD43 (also known as sialophorin, leukosialin, large sialoglycoprotein or gp115) is expressed on the surface of T lymphocytes, monocytes, neutrophils, platelets and some B lymphocytes. Expression of CD43 is deficient and/or defective in the X-chromosome-linked immunodeficiency disorder Wiscott-Aldrich syndrome, suggesting that CD43 might have a role in T-cell activation. We have shown that expression of human CD43 in an HLA-DR-specific murine T-cell hybridoma enhances the antigen-specific response to stimulation by the human lymphoblastoid cell line Daudi, and that Daudi cells bind specifically to purified immobilized CD43. These data indicate that the specific interaction of CD43 with a ligand on the surface of Daudi cells might contribute to T-cell activation. Here we report evidence that intercellular adhesion molecule-1 (ICAM-1, or CD54), is a ligand for CD43.

Human polymorphonuclear leukocytes (PMNs or neutrophils) kill invading microorganisms with reactive oxygen species (ROS) and cytotoxic granule components. PMNs from individuals with X-linked chronic granulomatous disease (XCGD) do not produce ROS, thereby rendering these individuals more susceptible to infection. In addition, XCGD patients develop tissue granulomas that obstruct vital organs, the mechanism(s) for which are unknown. To gain insight into the molecular processes that contribute to the pathophysiology of XCGD, including formation of granulomas, we compared global gene expression in PMNs from XCGD patients and healthy control individuals. Genes encoding mediators of inflammation and host defense, including CD11c, CD14, CD54, FcgammaR1, FcalphaR, CD120b, TLR5, IL-4R, CCR1, p47(phox), p40(phox), IL-8, CXCL1, Nramp1, and calgranulins A and B, were up-regulated constitutively in unstimulated XCGD patient PMNs. By comparing transcript levels in normal and XCGD PMNs after phagocytosis, we discovered 206 genes whose expression changed in the presence and the absence of ROS, respectively. Notably, altered Bcl2-associated X protein synthesis accompanied defective neutrophil apoptosis in XCGD patients. We hypothesize that granuloma formation in XCGD patients reflects both increased proinflammatory activity and defective PMN apoptosis, and we conclude that ROS contribute directly or indirectly to the resolution of the inflammatory response by influencing PMN gene transcription.

Most of the immunosuppressive effects of interleukin-10 (IL-10) are related to functional inhibition of antigen-presenting cells (APC). Herein, we investigate the influence of recombinant (r)IL-10 on human dendritic cells (DC) purified from peripheral blood of healthy volunteers. First, we found that rIL-10 inhibited in a dose-dependent manner the proliferative responses as well as the production of IL-2 and interferon-gamma (IFN-gamma) in mixed lymphocyte reaction (MLR) between purified T cells and DC. This rIL-10 effect could be attributed to a direct effect on DC, as DC preincubated with rIL-10 were found to be deficient in the induction of alloreactive T cells even when anti-IL-10 neutralizing mAb was added at the time of MLR. Flow cytometric analysis indicated that rIL-10 did not modify the expression of ICAM-1 (CD54) and B7-1 (CD80), but decreased HLA-DR and B7-2 (CD86) expression at the DC surface. We conclude that the inhibitory effect of rIL-10 on primary alloreactive T cell responses involves down-regulation of class II MHC and B7-2 expression at the DC surface.

Ischemic acute kidney injury (AKI) triggers an inflammatory response which exacerbates injury that requires increased expression of endothelial adhesion molecules. To study this further, we used in situ hybridization, immunohistology, and isolated endothelial cells, and found increased Toll-like receptor 4 (TLR4) expression on endothelial cells of the vasa rectae of the inner stripe of the outer medulla of the kidney 4 h after reperfusion. This increase was probably due to reactive oxygen species, known to be generated early during ischemic AKI, because the addition of hydrogen peroxide increased TLR4 expression in MS1 microvascular endothelial cells in vitro. Endothelial TLR4 may regulate adhesion molecule (CD54 and CD62E) expression as they were increased on endothelia of wild-type but not TLR4 knockout mice in vivo. Further, the addition of high-mobility group protein B1, a TLR4 ligand released by injured cells, increased adhesion molecule expression on endothelia isolated from wild-type but not TLR4 knockout mice. TLR4 was localized to proximal tubules in the cortex and outer medulla after 24 h of reperfusion. Thus, at least two different cell types express TLR4, each of which contributes to renal injury by temporally different mechanisms during ischemic AKI.

In susceptible mouse strains, infection of mice with Plasmodium berghei ANKA (PbA) results in a lethal complication, cerebral malaria. Cerebral malaria is due to the immune response induced by the parasite, which results in an increased production of TNF, known to increase the expression of adhesion molecules on the endothelia. To investigate the role of the adhesion molecule ICAM-1 (CD54), we infected wild-type (+/+) and ICAM-1-deficient (-/-) mice with PbA. While +/+ mice died 6-8 days after infection, -/- mice survived>> 15 days. Parasitaemia was similar in +/+ and -/- mice. Serum TNF concentration was increased by the infection and was significantly higher in infected +/+ than in -/- mice. However, TNF mRNA levels in spleen, lungs, and brain were elevated in both infected +/+ and -/- mice. For IFN-gamma, serum levels were similar in both groups. A breakdown of the blood-brain barrier was evident in infected +/+ mice only. Interestingly, thrombocytopenia was profound in infected +/+, but practically absent in -/- mice. Moreover, macrophage sequestration was evident in brain venules and lung capillaries of +/+ mice and was significantly less important in the alveolar capillaries of infected -/- mice. In contrast, neutrophil sequestration in the lung was similar in both +/+ and -/- mice. Sequestration of parasitized red blood cells was significantly greater in the alveolar capillaries from +/+ than -/- mice. These results indicate that while the immune response is similar in both +/+ and ICAM-1(-/-) mice, the absence of mortality in ICAM(-/-) mice correlates with a decrease of macrophage and parasitized RBC trapping and a less severe thrombocytopenia.

Effective stimulation of CD4+ T cells in an immune response depends on activation signals transduced via not only the CD3-T-cell receptor (TCR) complex but also those generated by accessory cell-surface proteins, including some that mediate adhesion between T cells and antigen-presenting cells (APC). Three members of the Ig superfamily, CD54 [intercellular cell adhesion molecule 1 (ICAM-1)], CD58 [lymphocyte function-associated antigen 3 (LFA-3)], and B7, expressed on the surface of APC, have been shown to mediate both adhesion and signaling during T cell-APC interactions. Recently another member of the Ig superfamily, [vascular cell adhesion molecule 1 (VCAM-1; INCAM110)], has been identified. VCAM-1 mediates adhesion between endothelial cells and activated lymphocytes and certain tumor cells. Here, using a soluble VCAM-1 fusion protein with receptor globulin (Rg), we examined the role of VCAM-1 in T-cell activation. We observed that CD4+ T cells, which are inefficiently stimulated by immobilized anti-TCR-1 or anti-CD3 monoclonal antibody (mAb) alone, can be induced to proliferate when exposed to immobilized VCAM-1-Rg in conjunction with either immobilized anti-TCR-1 or immobilized anti-CD3 mAb. The costimulatory effects of VCAM-1-Rg on CD4+T cells is inhibited by mAb to either the CD29 (integrin beta 1)-CD49d [very late activation antigen 4 alpha (VLA-4 alpha)] complex on the surface of CD4+ T cells or to VCAM-1. Stimulation of CD4+ T cells with immobilized VCAM-1-Rg and anti-TCR or -CD3 mAb results in the synthesis of both interleukin 2 (IL-2) receptors and IL-2. In addition, anti-CD25 (anti-IL-2 receptor a) mAb significantly inhibited the VCAM-1-Rg/anti-TCR or -CD3 mAb-driven activation of CD4+ T cells, indicating that endogenously produced IL-2 is in part responsible for the observed T-cell proliferation. Collectively, these results suggest that VCAM-1 can play an important costimulatory role during the activation of CD4+ T cells.

We first identified and isolated cellular subpopulations with characteristics of mesenchymal progenitor cells (MPCs) in osteoarthritic cartilage using fluorescence-activated cell sorting (FACS). Cells from osteoarthritic cartilage were enzymatically isolated and analyzed directly or after culture expansion over several passages by FACS using various combinations of surface markers that have been identified on human MPCs (CD9, CD44, CD54, CD90, CD166). Culture expanded cells combined and the subpopulation derived from initially sorted CD9+, CD90+, CD166+ cells were tested for their osteogenic, adipogenic and chondrogenic potential using established differentiation protocols. The differentiation was analyzed by immunohistochemistry and by RT-PCR for the expression of lineage related marker genes. Using FACS analysis we found that various triple combinations of CD9, CD44, CD54, CD90 and CD166 positive cells within osteoarthritic cartilage account for 2-12% of the total population. After adhesion and cultivation their relative amount was markedly higher, with levels between 24% and 48%. Culture expanded cells combined and the initially sorted CD9/CD90/CD166 triple positive subpopulation had multipotency for chondrogenic, osteogenic and adipogenic differentiation. In conclusion, human osteoarthritic cartilage contains cells with characteristics of MPCs. Their relative enrichment during in vitro cultivation and the ability of cell sorting to obtain more homogeneous populations offer interesting perspectives for future studies on the activation of regenerative processes within osteoarthritic joints.

Gangliosides are ubiquitous membrane-associated glycosphingolipids, which are involved in cell growth and differentiation. Most tumor cells synthesize and shed large amounts of gangliosides into their microenvironment, and many studies have unraveled their immunosuppressive properties. In the present study we analyzed the effects of GM3 and GD3 gangliosides, purified from human melanoma tumors, on the differentiation of monocyte-derived dendritic cells (MoDC). At concentrations close to those detected in the sera from melanoma patients, both gangliosides dose-dependently inhibit the phenotypic and functional differentiation of MoDC, as assessed by a strong down-regulation of CD1a, CD54, CD80, and CD40 Ags and impaired allostimulatory function on day 6 of culture. Furthermore, GM3 and GD3 gangliosides decreased the viable cell yield and induced significant DC apoptosis. Finally, addition of GD3 to differentiating DC impaired their subsequent maturation induced by CD154. The resulting DC produced low amounts of IL-12 and large amounts of IL-10, a cytokine pattern that might hamper an efficient antitumor immune response. In conclusion, the results demonstrate that gangliosides impair the phenotypic and functional differentiation of MoDC and induce their apoptosis, which may be an additional mechanism of human melanoma escape.

Traumatic brain injury (TBI) is often accompanied by an acute inflammatory reaction mediated initially by neutrophils. Adhesion molecules expressed on vascular endothelium are requisite elements during recruitment of leukocytes at sites of inflammation. In a rat model of TBI the induction and persistent expression of E-selectin (CD62E) on cerebrovascular endothelium ipsilateral, but not contralateral, to the site of contusion was demonstrated (P < 0.05 at 4 and 48 h posttrauma). In addition, these studies confirmed up-regulation and prolonged expression of ICAM-1 (CD54) on endothelium in the traumatized hemisphere (P < 0.05 at 4, 24, 48, and 72 h posttrauma). It is of interest that increased expression of CD54 was noted on blood vessels in the contralateral, non-traumatized hemisphere 48 h posttrauma. Expression of a third endothelial adhesion molecule, PECAM-1 (CD31), was unchanged following trauma. Administration of a murine monoclonal antibody (TM-8) that inhibits the adhesive function of CD54 blocked a significant portion (37.9%) of neutrophil recruitment 24 h posttrauma (P = 0.04). Employing immunocytochemistry and a monoclonal antibody specific for rat neutrophils (RP-3), peak infiltration of neutrophils was shown to occur 48 h after trauma. In contrast to emigration of neutrophils from blood vessels within the contusion, however, entry of neutrophils occurred from the surrounding leptomeninges and choroidal vessels. These studies demonstrate the relevance of CD54 (ICAM-1) in recruitment of neutrophils following TBI. However, the majority of neutrophil influx relies on endothelial adhesion molecules other than CD54. Because emigration of neutrophils was shown to occur predominantly from vessels within the leptomeninges and choroid plexus, intrathecal delivery of agents that inhibit the adhesive interactions between neutrophils, endothelial CD54, and other endothelial adhesion molecules to be defined may offer a novel form of therapy to prevent the acute inflammatory response that follows TBI.

Recombinant adenovirus (rAd) infection is one of the most effective and frequently employed methods to transduce dendritic cells (DC). Contradictory results have been reported recently concerning the influence of rAd on the differentiation and activation of DC. In this report, we show that, as a result of rAd infection, mouse bone marrow-derived immature DC upregulate expression of major histocompatibility complex class I and II antigens, costimulatory molecules (CD40, CD80, and CD86), and the adhesion molecule CD54 (ICAM-1). rAd-transduced DC exhibited increased allostimulatory capacity and levels of interleukin-6 (IL-6), IL-12p40, IL-15, gamma interferon, and tumor necrosis factor alpha mRNAs, without effects on other immunoregulatory cytokine transcripts such as IL-10 or IL-12p35. These effects were not related to specific transgenic sequences or to rAd genome transcription. The rAd effect correlated with a rapid increase (1 h) in the NF-kappaB-DNA binding activity detected by electrophoretic mobility shift assays. rAd-induced DC maturation was blocked by the proteasome inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) or by infection with rAd-IkappaB, an rAd-encoding the dominant-negative form of IkappaB. In vivo studies showed that after intravenous administration, rAds were rapidly entrapped in the spleen by marginal zone DC that mobilized to T-cell areas, a phenomenon suggesting that rAd also induced DC differentiation in vivo. These findings may explain the immunogenicity of rAd and the difficulties in inducing long-term antigen-specific T-cell hyporesponsiveness with rAd-transduced DC.

TRAF5 [tumor necrosis factor (TNF) receptor-associated factor 5] is implicated in NF-kappaB and c-Jun NH(2)-terminal kinase/stress-activated protein kinase activation by members of the TNF receptor superfamily, including CD27, CD30, CD40, and lymphotoxin-beta receptor. To investigate the functional role of TRAF5 in vivo, we generated TRAF5-deficient mice by gene targeting. Activation of either NF-kappaB or c-Jun NH(2)-terminal kinase/stress-activated protein kinase by tumor necrosis factor, CD27, and CD40 was not abrogated in traf5(-/-) mice. However, traf5(-/-) B cells showed defects in proliferation and up-regulation of various surface molecules, including CD23, CD54, CD80, CD86, and Fas in response to CD40 stimulation. Moreover, in vitro Ig production of traf5(-/-) B cells stimulated with anti-CD40 plus IL-4 was reduced substantially. CD27-mediated costimulatory signal also was impaired in traf5(-/-) T cells. Collectively, these results demonstrate that TRAF5 is involved in CD40- and CD27-mediated signaling.

Fibroblast-like cells emerging from cultured human pancreatic endocrine and exocrine tissue have been reported. Although a thorough phenotypic characterization of these cells has not yet been carried out, these cells have been hypothesized to be contaminating fibroblasts, mesenchyme and/or possibly beta-cell progenitors. In this study, we expanded fibroblast-like cells from adult human exocrine pancreas following islet isolation and characterized these cells as mesenchymal stem cells (MSCs) based on their cell surface antigen expression and ability to differentiate into mesoderm. Analysis by flow cytometry demonstrated that pancreatic MSCs express cell surface antigens used to define MSCs isolated from bone marrow such as CD13, CD29, CD44, CD49b, CD54, CD90 and CD105. In addition, utilizing protocols used to differentiate MSCs isolated from other somatic tissues, we successfully differentiated pancreatic MSCs into: (1) osteocytes that stained positive for alkaline phosphatase, collagen, mineralization (calcification) and expressed osteocalcin, (2) adipocytes that contained lipid inclusions and expressed fatty acid binding protein 4 and (3) chondrocytes that expressed aggrecan. We also demonstrated that pancreatic MSCs are multipotent and capable of deriving cells of endodermal origin. Pancreatic MSCs were differentiated into hepatocytes that stained positive for human serum albumin and expressed endoderm and liver-specific genes such as GATA 4 and tyrosine aminotransferase. In addition, preliminary protocols used to differentiate these cells into insulin-producing cells resulted in the expression of genes necessary for islet and beta-cell development such as Pax4 and neurogenin 3. Therefore, multipotent MSCs residing within the adult exocrine pancreas could represent a progenitor cell, which when further manipulated could result in the production of functional islet beta-cells.

The malarial parasite Plasmodium falciparum has acted as a potent selective force on the human genome. The particular virulence of this organism is thought to be due to the adherence of parasitised red blood cells to small vessel endothelium through several receptors, including CD36, thrombospondin and intercellular adhesion molecule 1 (ICAM-1, CD54), and parasite isolates differ in their ability to bind to each. Immunohistochemical studies have implicated ICAM-1 as of potential importance in the pathogenesis of cerebral malaria, leading us to reason that if any single receptor were involved in the development of cerebral malaria, then in view of the high mortality of that complication, natural selection should have produced variants with reduced binding capacity. We therefore sequenced the N-terminal domain of ICAM-1 from a number of Africans and discovered a single mutation present at high frequency. Genotypes at this locus from samples from a case-control study indicated an association of the polymorphism with the severity of clinical malaria such that individuals homozygous for the mutation have increased susceptibility to cerebral malaria with a relative risk of two. These counterintuitive results have implications for the mechanism of malaria pathogenesis, resistance to other infectious agents and transplantation immunology.

The VLA-4 (CD49d/CD29) integrin is the only member of the VLA family expressed by resting lymphoid cells that has been involved in cell-cell adhesive interactions. We here describe the triggering of homotypic cell aggregation of peripheral blood T lymphocytes and myelomonocytic cells by mAbs specific for certain epitopes of the human VLA alpha 4 subunit. This anti-VLA-4-induced cell adhesion is isotype and Fc independent. Similar to phorbol ester-induced homotypic adhesion, cell aggregation triggered through VLA-4 requires the presence of divalent cations, integrity of cytoskeleton and active metabolism. However, both adhesion phenomena differed at their kinetics and temperature requirements. Moreover, cell adhesion triggered through VLA-4 cannot be inhibited by cell preincubation with anti-LFA-1 alpha (CD11a), LFA-1 beta (CD18), or ICAM-1 (CD54) mAb as opposed to that mediated by phorbol esters, indicating that it is a LFA-1/ICAM-1 independent process. Antibodies specific for CD2 or LFA-3 (CD58) did not affect the VLA-4-mediated cell adhesion. The ability to inhibit this aggregation by other anti-VLA-4-specific antibodies recognizing epitopes on either the VLA alpha 4 (CD49d) or beta (CD29) chains suggests that VLA-4 is directly involved in the adhesion process. Furthermore, the simultaneous binding of a pair of aggregation-inducing mAbs specific for distinct antigenic sites on the alpha 4 chain resulted in the abrogation of cell aggregation. These results indicate that VLA-4-mediated aggregation may constitute a novel leukocyte adhesion pathway.

Cell adhesion molecules (CAM) participate in interactions between lymphocytes, accessory cells, and target cells that are critical in the generation of effective immune responses. To characterize the involvement of CAM in NK and lymphokine activated killer (LAK) activities, we examined the expression of several CAM by freshly isolated human NK cells and by NK cells activated in vitro with IL-2, and compared this to CAM expression by T lymphocytes under similar conditions. Freshly isolated human NK cells were uniformly LFA-3 (CD58)+ and expressed two to three-fold higher surface levels of LFA-1 (CD11a/CD18) than resting T lymphocytes. More NK cells than T cells also expressed phenotypically detectable levels of intercellular adhesion molecule-1 (CD54). After in vitro incubation with IL-2, human NK cells demonstrated four- to sixfold increases in surface levels of CD11a/CD18, CD2, CD54, CD58, and the NK cell-associated Ag NKH-1 (CD56). Furthermore, essentially all NK cells became CD54+ within 3 days of exposure to IL-2. T cells did not demonstrate comparable up-regulation of CAM after incubation with IL-2. Increases in NK cell CAM expression were associated with enhanced formation of E:T cell conjugates, enhanced killing of NK-sensitive targets, and the induction of cytotoxicity for previously NK-resistant targets (LAK activity). The LAK activity induced by exogenous IL-2 could be partially inhibited by anti-CD2, anti-CD11a, or anti-CD54 antibodies and almost completely abrogated by anti-CD2 and anti-CD11a in combination. These studies suggest that CAM play a central role in the regulation of NK cytolysis, and that changes in CAM expression may alter the target cell specificity of activated NK effectors.

Human adult mesenchymal stem cells (MSCs) were first identified by Friedenstein et al. when observing a group of cells that developed into fibroblastic colony forming cells (CFU-F). Ever since, the therapeutic uses and clinical applications of these cells have increased research and interest in this field. MSCs have the potential to be used in tissue engineering, gene therapy, transplants and tissue injuries. However, identifying these cells can be a challenge. Moreover, there are no articles bringing together and summarizing the cell surface markers of MSCs in adults. The purpose of this study is to summarize all the available information about the cell surface characterization of adult human MSCs by identifying and evaluating all the published literature in this field. We have found that the most commonly reported positive markers are CD105, CD90, CD44, CD73, CD29, CD13, CD34, CD146, CD106, CD54 and CD166. The most frequently reported negative markers are CD34, CD14, CD45, CD11b, CD49d, CD106, CD10 and CD31. A number of other cell surface markers including STRO-1, SH2, SH3, SH4, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-I, DP, EMA, DQ (MHC Class II), CDIO5, Oct 4, Oct 4A, Nanog, Sox-2, TERT, Stat-3, fibroblast surface antigen, smooth muscle alpha-actin, vimentin, integrin subunits alpha4, alpha5, beta1, integrins alphavbeta3 and alphavbeta5 and ICAM-1 have also been reported. Nevertheless, there is great discrepancy and inconsistency concerning the information available on the cell surface profile of adult MSCs and we suggest that further research is needed in this field to overcome the problem.