Pregnancy is characterized by the presence of generalized leukocyte activation. We used flow cytometry to investigate changes in phenotype and intracellular cytokines of circulating granulocytes, monocytes, and T lymphocytes of pregnant women during gestation. We report that peripheral circulation of pregnancy is characterized by an increased percentage of granulocytes and a decrease in lymphocytes. The proportion of monocytes remains stable throughout gestation; however, a progressive up-regulation of surface markers CD11a, CD54, and CD64 was detected. Monocytes also showed higher production of interleukin (IL)-12 and IL-1beta compared with the nonpregnant state, and granulocytes had greater potential to synthesize IL-8. All these changes were particularly marked in late gestation. T lymphocytes did not have any characteristics of the activated state and showed a decreased IL-6 production. These findings demonstrate that activation of maternal monocytes and granulocytes increases during pregnancy and support the idea that pregnancy results in an elevation of the innate immune system and suppression of the adaptive immune system.

Extracellular ATP mediates numerous biological activities by interacting with plasma membrane P2 purinergic receptors. Recently, P2 receptors have been described on dendritic cells (DC), but their functional role remains unclear. Proposed functions include improved Ag presentation, cytokine production, chemotaxis, and induction of apoptosis. We investigated the effects of ATP and of other P2 receptor agonists on endocytosis, phenotype, IL-12 secretion, and T cell stimulatory capacity of human monocyte-derived DC. We found that in the presence of extracellular ATP, DC transiently increase their endocytotic activity. Subsequently, DC up-regulate CD86, CD54, and MHC-II; secrete IL-12; and exhibit an improved stimulatory capacity for allogeneic T cells. These effects were more pronounced when chemically modified ATP derivatives with agonistic activity on P2 receptors, which are resistent to degradation by ectonucleotidases, were applied. Furthermore, ATP and TNF-alpha synergized in the activation of DC. Stimulated with a combination of ATP and TNF-alpha, DC expressed the maturation marker CD83, secreted large amounts of IL-12, and were potent stimulators of T cells. In the presence of the P2 receptor antagonist suramin, the effects of ATP were completely abolished. Our results suggest that extracellular ATP may play an important immunomodulatory role by activating DC and by skewing the immune reaction toward a Th1 response through the induction of IL-12 secretion.

We studied the in vitro effects of butyric acid on differentiation, maturation and function of dendritic cells (DC) and macrophages (M(Phi)) generated from human monocytes. A non-toxic dose of butyrate was shown to alter the phenotypic differentiation process of DC as assessed by a persistence of CD14, and a decreased CD54, CD86 and HLA class II expression. The more immature differentiation stage of treated cells was confirmed further by their increased phagocytic capability, their altered capacity to produce IL-10 and IL-12, and their weak allostimulatory abilities. Butyrate also altered DC terminal maturation, regardless of the maturation inducer, as demonstrated by a strong down-regulation of CD83, a decreased expression of CD40, CD86 and HLA class II. Similarly, butyrate altered M(Phi) differentiation, down-regulating the expression of the restricted membrane antigens and reducing the phagocytic capacity of treated cells. To investigate further the mechanism by which butyrate hampers the monocyte dual differentiation pathway, we studied the effects of 1,25(OH)2D3 alone or in combination with butyrate on the phenotypic features of DC. Unlike 1,25(OH)2D3, butyrate inhibited DC -differentiation without redirecting it towards M(Phi). Combined treatment gave rise to a new cell subset (CD14(high), CD86 and HLA-DR(low)) phenotypically distinct from monocytes. These results reveal an alternative mechanism of inhibition of DC and M(Phi) differentiation. Altogether, our data demonstrate a novel immune suppression property of butyrate that may modulate both inflammatory and immune responses and support further the interest for butyrate and its derivatives as new immunotherapeutic agents.

Resting CD45RO+, mature/memory, T cells are phenotypically distinct from intermediate CD45RO+/CD45RA+ and CD45RA+, immature/virgin, T cells, and are characterized by high levels of expression of a number of adhesion molecules, such as CD2, CD18, CD58 and CD29. The kinetics of up-regulation of molecules, like CD25 and CD54 associated with activation, were similar in both subsets and suggested that their high level expression was associated with later events rather than initial recognition and signal transduction. CD45RA+ T cells, unlike CD45RO+ T cells, were unable to proliferate in response to mitogenic combinations of CD2 monoclonal antibodies (mAb), although in combination with submitogenic doses of PMA both up-regulation of cell-surface molecules and proliferation occurred. In addition, recruitment of CD45RA+ T cells by CD2 mAb-activated CD45RO+ T cells can occur.

We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM-1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombin resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-kappa B site (-533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-kappa B site (-223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-kappa B site is critical for thrombin inducibility. NF-kappa B-directed luciferase activity increased approximately 3-fold when cells transfected with the plasmid pNF-kappa BLUC containing five copies of consensus NF-kappa B site linked to a minimal adenovirus E1B promoter-luciferase gene were exposed to thrombin, indicating that activation of NF-kappa B was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-kappa Bp65 (Rel A) to downstream NF-kappa B site of the ICAM-1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH2 terminus of proteolytically activated receptor-1, mimicked thrombin's action in inducing ICAM-1 expression. These data indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-kappa Bp65 binding to the downstream NF-kappa B site of ICAM-1 promoter after proteolytically activated receptor-1 activation.

Cell-cell adhesion events play critical roles in the sequential migrations and multiple specific cell-cell interactions which B cells undergo during normal development and function. We have observed that mAb to several B cell-associated molecules, including mAb to CD19, CD37, and CD40, induce homotypic aggregation of freshly isolated human B cells. The aggregation of B cells induced by CD40 mAb was due to activation of a cell-cell adhesion system, and not due to agglutination by mAb, because 1) in addition to being energy dependent and cation dependent, the aggregation was blocked by inhibitors of messenger RNA and protein synthesis; and 2) a mouse B cell line transformed with intact human CD40 aggregated in response to CD40 mAb, whereas a line expressing surface CD40, but lacking the cytoplasmic tail and previously shown incapable of transmitting a signal from the cell surface, did not aggregate. The aggregation, although of slow onset, was persistent and of high avidity. In addition, CD40 mAb induced increased surface expression of intercellular adhesion molecule-1 (CD54), a ligand for CD11a/CD18 (LFA-1), and CD18 mAb blocked aggregation. CD40 mAb also augmented the ability of dense B cells to stimulate the proliferation of allogeneic T cells via a CD18-dependent process. We conclude that signaling through CD40, elicited by cross-linking the CD40 protein on the cell surface, activates the CD18/intercellular adhesion molecule adhesion system; in addition, CD40 cross-linking may activate a second adhesion system since CD40 mAb induced aggregation of the B cell line Ramos, which does not express surface CD18. B cell adhesion may be triggered by signaling through multiple surface proteins, thereby lending specificity of activation to adhesion systems which are broadly expressed.

To characterize the molecules responsible for synovial fibroblast-T lymphocyte (TL) cross-talk in rheumatoid arthritis (RA), synovial fibroblasts from patients with established RA (RASFibs) were cocultured with TLs from peripheral blood of early RA patients (RAPBTL). TLs from peripheral blood of healthy controls and from synovial fluid of RA served as controls. Adhesion molecules and cytokines were determined by flow cytometry, ELISA, and real-time PCR. RAPBTL (n = 20) induced an up-regulation of ICAM-1, intracellular IL-8, IL-6, IL-15, and surface IL-15 in cocultured RASFibs. In turn, RAPBTL showed an up-regulation of TNF-alpha, IFN-gamma, IL-17, CD25, and CD69 expression. Responses seen with TLs from peripheral blood of healthy controls (n = 20) were significantly lower, whereas responses with TLs from synovial fluid of RA (n = 20) were maximal. Blocking Abs to IL-15 and CD54, but not an isotype-control Ab, down-regulated the increased TL cytokine and activation marker expression. Abs to CD69, CD11a, IL-17, TNF-alpha, and IFN-gamma significantly decreased the up-regulation of RASFib cytokine and CD54 expression. Cocultures using 0.4- micro m inserts did not result in up-regulation of surface molecules or cytokines. Methotrexate significantly inhibited RASFib/TL cross-talk signals and decreased adhesion of TL to RASFibs. In summary, RASFib production of IL-15 induces the proinflammatory cytokines TNF-alpha, IFN-gamma, and IL-17 in cocultured TLs through a cell contact-dependent mechanism. In turn, these cytokines stimulate the expression of IL-15, IL-8, and IL-6 in RASFibs, thereby creating a feedback loop that favors persistent synovial inflammation. Methotrexate seems to disrupt this loop by decreasing cell adhesion.

OBJECTIVE

To assess the safety and efficacy of a monoclonal antibody (MAb) to intercellular adhesion molecule 1 (ICAM-1; CD54) in rheumatoid arthritis (RA).

METHODS

A phase I/II, open-label, dose-escalation study of 32 patients.

RESULTS

During treatment, a peripheral CD3+/CD4+ lymphocytosis was noted, and several patients demonstrated transient cutaneous anergy, which suggests that therapy modified T cell recirculation. Thirteen of the 23 patients who received 5 days of treatment demonstrated clinical improvement through day 29, and 9 of 23 through day 60. Adverse effects were minor and transient.

CONCLUSIONS

Anti-ICAM-1 MAb therapy was well tolerated, resulted in a transient alteration in T lymphocyte recirculation, and effected clinical improvement in some RA patients.

The antigenic phenotype of human fetal osteoclasts was compared with that of human tissue macrophages and macrophage polykaryons in foreign body lesions using a large number of monoclonal antibodies directed against myeloid (granulocyte/mononuclear phagocyte) antigens. Osteoclasts expressed a restricted range of macrophage-associated antigens including CD13, CD15A, CD44, CD45, CD54, (ICAM-1), CD71 (transferrin receptor), and CD68. These antigens were also present on macrophages and macrophage polykaryons both of which also strongly expressed CD11a,b,c, CD18, (LFA family), CD14, CD31, CD36, CD37, CD39 and CD43 antigens. There was also weak and occasional expression of CD16 (FcRIII), CD25 (interleukin 2 receptor), CD32 (FcRII), CD35 (C3b receptor) and HLA-DR by macrophage polykaryons. The presence of some macrophage associated antigens on osteoclasts is consistent with their originating from cells of the mononuclear phagocyte system. The numerous differences in antigenic phenotype between osteoclasts and macrophage polykaryons, however, suggest that their pathways of development and differentiation are not identical. The differences discerned in antigenic phenotype should also permit distinction between these polykaryons (and possibly their mononuclear precursors) in normal and diseased tissues.

Under selective pressure from host cytotoxic T lymphocytes, many viruses have evolved to downregulate major histocompatibility complex (MHC) class I and/or T-cell costimulatory molecules from the surface of infected cells. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two proteins, MIR-1 and MIR-2, that serve this function during lytic replication. In vivo, however, KSHV exists in a predominantly latent state, with less than 5% of infected cells expressing discernible lytic gene products. Thus, mechanisms of immune evasion that depend on genes expressed only during lytic replication are unlikely to be active in most KSHV-infected cells. As a result, we searched for evidence of similar defensive strategies extant during latency, employing culture systems that strongly favor latent KSHV infection. We measured cell surface levels of immunomodulatory proteins on both primary dermal microvascular endothelial cells (pDMVEC) infected through coculture with induced primary effusion lymphoma cells and telomerase-immortalized DMVEC infected directly with cell-free virus. Employing a panel of antibodies against several endothelial cell surface proteins, we show that de novo infection with KSHV leads to the downregulation of MHC class I, CD31 (PE-CAM), and CD54 (ICAM-I) but not CD58 (LFA-3) or CD95 (Fas). Furthermore, flow cytometry with a fluorescently labeled monoclonal antibody to the latency-associated nuclear antigen (LANA) revealed that downregulation occurred predominantly on KSHV-infected (LANA-positive) cells. Although the vast majority of infected cells displayed this downregulation, less than 1% expressed either immediate-early or late lytic proteins detectable by immunofluorescence. Together, these results suggest that downregulation of immunomodulatory proteins on the surface of target cells may represent a constitutive mode of immune evasion employed by KSHV following de novo infection.

BACKGROUND

Epidemiologic studies show that exposure to ambient particulate matter leads to asthma exacerbation. Diesel exhaust particles (DEPs), a model pollutant, act as an adjuvant for allergic sensitization. Increasing evidence shows that this effect could be mediated by an effect on dendritic cells (DCs).

OBJECTIVE

Our aim was to elucidate the mechanism by which pro-oxidative DEP chemicals change DC function so that these antigen-presenting cells strengthen the immune response to an experimental allergen.

METHODS

We exposed murine bone marrow-derived DCs and a homogeneous myeloid DC line, BC1, to DEPs and organic extracts made from these particles to determine how the induction of oxidative stress affects cellular maturation, cytokine production, and activation of antigen-specific T cells.

RESULTS

DEP extracts induced oxidative stress in DCs. This change in redox equilibrium interfered in the ability of Toll-like receptor agonists to induce the expression of maturation receptors (eg, CD86, CD54, and I-A(d)) and IL-12 production. This perturbation of DC function was accompanied by decreased IFN-gamma and increased IL-10 induction in antigen-specific T cells. The molecular basis for the perturbation of DC function is the activation of a nuclear factor-erythroid 2 (NF-E2)-related factor 2-mediated signaling pathway that suppresses IL-12 production. NF-E2-related factor 2 deficiency abrogates the perturbation of DC function by DEPs.

CONCLUSIONS

These data provide the first report that pro-oxidative DEP chemicals can interfere in T(H)1-promoting response pathways in a homogeneous DC population and provide a novel explanation for the adjuvant effect of DEPs on allergic inflammation.

CONCLUSIONS

These data clarify the adjuvant effect of particulate air pollutants in allergic inflammatory disease.

Type II alveolar epithelial cells (AEC) can produce various antimicrobial and proinflammatory effector molecules. This, together with their abundance and strategic location, suggests a role in host defense against pulmonary pathogens. We report that murine type II AEC, like their human counterparts, express class II major histocompatibility complex (MHC). Using a murine model of pulmonary tuberculosis, we find that type II AEC become activated and have increased cell surface expression of class II MHC, CD54, and CD95 following infection. Type II AEC use the class II MHC pathway to process and present mycobacterial antigens to immune CD4+ T cells isolated from mice infected with Mycobacterium tuberculosis. Therefore, not only can type II AEC contribute to the pulmonary immunity by secreting chemokines that recruit inflammatory cells to the lung, but they can also serve as antigen-presenting cells. Although type II AEC are unlikely to prime naïve T cells, their ability to present antigens to T cells demonstrates that they can participate in the effector phase of the immune response. This represents a novel role for type II AEC in the immunological response to pulmonary pathogens.

The murine melanoma cell line B16.F10 (H-2b) was used to study specific T cell responses that reject tumors. Stable B16 transfectants were established that express viral Ags, either the hepatitis B surface Ag (HBsAg) or the large tumor Ag (T-Ag) of SV40. B16 cells and their transfected sublines were CD40+ CD44+ but expressed no (or low levels of the) costimulator molecules CD154 (CD40L), CD48, CD54, CD80, and CD86. Surface expression of MHC class I (Kb, Db) and class II (I-Ab) molecules by B16 cells was low, but strikingly up-regulated by IFN-gamma. CD95 (Fas) and CD95 ligand (CD95L (FasL)) were "spontaneously" expressed by B16 cells growing in vitro in serum-free medium; these markers were strikingly up-regulated by IFN-gamma. B16 cells coexpressing CD95 and CD95L were irreversibly programmed for apoptosis. In vitro, noninduced B16 transfectants stimulated a specific IFN-gamma release response, but no cytolytic response (in a 4-h assay) in MHC class I-restricted CTL; in contrast, IFN-gamma-induced B16 targets were efficiently and specifically lysed by CTL. In vivo, B16 transfectants were specifically rejected by DNA-vaccinated syngeneic hosts through a T-dependent immune effector mechanism. The tumors showed evidence of massive apoptosis in vivo during the rejection process. The data suggest that CTL-derived IFN-gamma enhances an intrinsic suicide mechanism of these tumor cells in addition to facilitating lytic interactions of effectors with tumor targets.

Differentiation and maturation of monocytes are accompanied by the expression of specific surface glycoproteins, the secretion of cytokines, and the capacity to respond to ligands. These changes may be influenced by interactions with hormones, soluble lymphocytic products, or direct contact with lymphocytes. We have studied two distinct pathways in the differentiation of a human monocytic cell line, THP-1: one being induced by IFN-gamma and the other by 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). In THP-1 cells, IFN-gamma induces cell surface expression of HLA-DR and CD54 and production of IL-1 beta, TNF-alpha, and IL-6. In contrast, 1,25(OH)2D3 increases cell surface expression of CD11b and CD14, but fails to stimulate cytokine production. Direct contact of THP-1 with stimulated fixed T cells markedly induces IL-1 beta, TNF-alpha, and IL-6 production by THP-1. Production is higher when THP-1 have been previously exposed to 1,25(OH)2D3 as compared to prior exposure to IFN-gamma. mAb raised against certain relevant cell surface glycoproteins on THP-1 were tested for their ability to block the response of THP-1 to T cells. Antibodies to CD11a, CD11b, and CD11c, alone or in combination, only partially blocked IL-1 beta production by THP-1, whereas antibodies to CD54 and CD14 did not. Thus other unknown structures on the THP-1 cells may be involved in the induction of THP-1 cytokine production by T cell contact.

BACKGROUND

Cloned glomerular endothelial cells (GENC) have many potential uses and applications in immunologic and physiologic studies. Propagation of GENC has been difficult and available homogeneous GENC, particularly from mice, are limited. Herein we report isolation, cloning, propagation, and characterization of GENC from mice.

METHODS

tsA58 immorto mice were used to isolate glomerular cells. Glomeruli were isolated by differential sieving, and decapsulated explants were cultured in permissive and optimal conditions for endothelial cells. The primary cells from glomerular outgrowths were expanded, taking advantage of the temperature-sensitive tsA58 gene, and then the cells were allowed to undergo spontaneous transformation. The cells were then sorted using anti-CD31 antibodies and their capacity to uptake acetylated-low-density lipoprotein (LDL). Individual subclones isolated by patch cloning were characterized using multiple markers.

RESULTS

One of the homogeneous clones was morphologically endothelial-like, positive for CD31, CD106, CD62E, CD54, and acetylated-LDL uptake, formed tubes, and was negative for epithelial and mesangial cell markers. The functional properties of this GENC clone appeared to be intact, and signaling pathway was not altered. Two of the clones displayed the characteristics of either visceral epithelial or mesangial cells.

CONCLUSIONS

The identified clones should have utility in multiple areas of investigation.

The attachment of erythrocytes infected with P. falciparum to human venular endothelium is the primary step leading to complications from severe and cerebral malaria. Intercellular adhesion molecule-1 (ICAM-1, CD54) has been implicated as a cytoadhesion receptor for P. falciparum-infected erythrocytes. Characterization of domain deletion, human/murine chimeric ICAM-1 molecules, and amino acid substitution mutants localized the primary binding site for parasitized erythrocytes to the first amino-terminal immunoglobulin-like domain of ICAM-1. The ICAM-1 binding site is distinct from those recognized by LFA-1, Mac-1, and the human major-type rhinoviruses. Synthetic peptides encompassing the binding site on ICAM-1 inhibited malaria-infected erythrocyte adhesion to ICAM-1-coated surfaces with a Ki of 0.1-0.3 mM, whereas the Ki for soluble ICAM-1 is 0.15 microM. These findings have implications for the therapeutic reversal of malaria-infected erythrocyte sequestration in the host microvasculature.

The current studies examined whether cytokine patterns indicative of an imbalance in Th1 and Th2 cells could be identified in PBMC of patients with active rheumatoid arthritis (RA). To investigate this possibility, a reproducible PCR technique to assess cytokine mRNA levels in PBMC was employed that minimized in vitro manipulation of the cells. Seven of 14 RA patients had increased mRNA levels for IL-2, 5/14 for IFN-gamma, 3/14 for IL-4, and 4/14 for the IL-2R alpha-chain, compared with normal donors. Whereas 4 patients had elevated mRNA for IL-2 and IFN-gamma, indicative of an increase in activated Th1 or Th0 cells, 1 of 14 patients expressed low levels of IL-2 and IFN-gamma and high levels of IL-4 mRNA. Seven RA patients were treated with a mAb to ICAM-1 (CD54). To determine whether changes in cytokine mRNA levels might be associated with and/or account for the anti-inflammatory effect of anti-ICAM-1 mAb therapy, changes in cytokine mRNA levels were assessed and correlated with clinical improvement. Anti-ICAM-1 mAb administration was followed by a prompt and transient increase of IFN-gamma mRNA. Elevation of IFN-gamma mRNA expression throughout the treatment period reflected a temporary increase in the number of circulating CD3+CD4+ T cells, suggestive of altered circulatory patterns of activated Th1-like cells and was related to clinical efficacy. The results indicate that elevated cytokine mRNA levels characteristic for Th1 cells can be detected in the PBMC in active RA and, furthermore, that anti-ICAM-1 mAb may be beneficial in RA by altering the recruitment of activated Th1-like cells into the synovium. This assumption further strengthens the hypothesis of a significant contribution of Th1-like cells to the pathogenesis of RA.

In their mammalian hosts, Leishmania are obligate intracellular parasites that mainly reside in macrophages. They are also phagocytosed by dendritic cells (DCs), which play decisive roles in the induction and shaping of T cell-dependent immune responses. Little is known about the role of DCs in the Leishmania life cycle. Here, we examined the ability of mouse bone marrow-derived DCs to serve as hosts for L. amazonensis. Both infective stages of Leishmania (metacyclic promastigotes and amastigotes) could be phagocytosed by DCs, regardless of whether they had previously been experimentally opsonized with either the complement C3 component or specific antibodies. Parasites could survive and even multiply in these cells for at least 72 hours, within parasitophorous vacuoles displaying phagolysosomal characteristics and MHC class II and H-2M molecules. We then studied the degree of maturation reached by infected DCs according to the parasite stage internalised and the type of opsonin used. The cell surface expression of CD24, CD40, CD54, CD80, CD86, OX40L and MHC class II molecules was barely altered following infection with unopsonized promastigotes or amastigotes from nude mice or with C3-coated promastigotes. Even 69 hours post-phagocytosis, a large proportion of infected DCs remained phenotypically immature. In contrast, internalisation of antibody-opsonized promastigotes or amastigotes induced DCs to mature rapidly, as shown by the over-expression of costimulatory, adhesion and MHC class II molecules. Thus, in the absence of specific antibodies (e.g. shortly after infecting naive mammals), infected DCs may remain immature or semi-mature, meaning that they are unable to elicit an efficient anti-Leishmania T cell response. Absence of DC maturation or delayed/incomplete DC maturation could thus be beneficial for the parasites, allowing their establishment and amplification before the onset of immune responses.

As part of the blood-retina barrier, the neuroectodermally-derived retinal pigment epithelial (RPE) monolayer is strategically positioned to interact with circulating leukocytes and regulate their access to the retina. We, therefore, studied whether human RPE cells express intercellular adhesion molecule-1 (ICAM-1), a specialized cell surface glycoprotein that binds the leukocyte function antigen-1 receptor present on all leukocytes. Using specific monoclonal antibody to ICAM-1, immunohistochemical staining of freshly-isolated primary and fourth passaged human RPE cells resulted in delicate reaction product that increased dramatically upon exposure to human recombinant (r) interferon-gamma (rIFN-gamma), interleukin-1-beta (rIL-1 beta), or tumor necrosis factor-alpha (rTNF-alpha). Fluorescence-activated cell sorting analysis demonstrated 2-fold increases in constitutive RPE ICAM-1 expression within 6 hours of exposure to physiologic concentrations of rIFN-gamma, rIL-1 beta, or rTNF-alpha. In standardized leukocyte adherence assays, cultured RPE cells showed avid binding of neutrophils that increased significantly after stimulation with rIFN-gamma, rIL-1 beta, or rTNF-alpha (p less than 0.001). In parallel assays, monoclonal antibody to either ICAM-1 on RPE cells, or subunits of leukocyte function antigen-1 receptors on leukocytes significantly blocked leukocyte binding to unstimulated (p less than 0.001) or rIFN-gamma-stimulated RPE cells (p less than 0.001). To demonstrate RPE ICAM-1 expression in intact human tissue, fresh uveoretinal explants were exposed to rIFN-gamma, rIL-1 beta, or rTNF-alpha and stained using mAb to ICAM-1. Tissue sections of cytokine-stimulated explants revealed dramatic increases in RPE ICAM-1 immunoreactivity over the low levels observed in unstimulated uveoretinal tissue. Our results indicate that: (a) ICAM-1 is expressed at low levels on unstimulated RPE cells, (b) RPE ICAM-1 may be augmented by inflammatory cytokines, and (c) RPE ICAM-1 is a functional receptor mediating leukocyte binding. ICAM-1 on RPE cells at the blood-retina barrier may regulate leukocytic infiltration in ocular diseases in which leukocytes are important pathogenetically and may be important to the generation of ocular immune responses.

BACKGROUND

Adipose tissue is an attractive and abundant source of multipotent stem cells. Human adipose stem cells (ASCs) have shown to have therapeutic relevancy in diverse clinical applications. Nevertheless, expansion of ASCs is often necessary before performing clinical studies. Standard in vitro cell-culture techniques use animal-derived reagents that should be avoided in clinical use because of safety issues. Therefore, xeno- and serum-free (XF/SF) reagents are highly desirable for enhancing the safety and quality of the transplanted ASCs.

METHODS

In the current study, animal component-free isolation and cell-expansion protocols were developed for ASCs. StemPro MSC SFM XF medium with either CELLstart™ CTS™ coating or Coating Matrix Kit were tested for their ability to support XF/SF growth. Basic stem-cell characteristics such as immunophenotype (CD3, CD11a, CD14, CD19, CD34, CD45RO, CD54, CD73, CD80, CD86, CD90, CD105, HLA-DR), proliferation, and differentiation potential were assessed in XF/SF conditions and compared with human serum (HS) or traditionally used fetal bovine serum (FBS) cultures.

RESULTS

ASCs cultured in XF/SF conditions had significantly higher proliferation rates compared with HS/FBS cultures. Characteristic immunophenotypes of ASCs were maintained in every condition; however, cells expanded in XF/SF conditions showed significantly lower expression of CD54 (intercellular adhesion molecule 1, ICAM-1) at low passage number. Further, multilineage differentiation potential of ASCs was maintained in every culture condition.

CONCLUSIONS

Our findings demonstrated that the novel XF/SF conditions maintained the basic stem cell features of ASCs and the animal-free workflow followed in this study has great potential in clinical cell therapies.

Comparative study of cultured human bone marrow and adipose tissue (lipoaspirate) mesenchymal stem cells was carried out. The main morphological parameters, proliferative activity, expression of surface and intracellular markers of these cells were characterized. Flow cytofluorometry and histological staining showed that both cell types exhibited similar expression of CD105, CD54, CD106, HLA-I markers, were positively stained for vimentin, ASMA, collagen-1, and fibronectin, but not HLA-DR, CD117, and hemopoietic cell markers. The cells underwent differentiation into adipocytes and osteoblasts under appropriate conditions of culturing. Incubation under neuroinductive conditions led to the appearance of a cell population positively stained for type III beta-tubulin (neuronal differentiation marker).

Intravascular Lymphomatosis (IL) is a rare and usually aggressive form of non-Hodgkin's lymphoma characterized by the growth of neoplastic cells within vascular lumina that usually presents with skin or central nervous system (CNS) involvement. The mechanism(s) for the selective intravascular growth of this neoplasm remain(s) unexplained. We now report clinical and immunohistologic data on surgical material from 6 cases of IL; in 4 of 6 cases, autopsies were performed. Our IL cases shared the following features: (1) B-cell lineage; (2) lack of skin involvement at presentation; (3) aggressive behavior; and (4) lack of extravascular lymphomatous masses; in addition, 1 case had an associated gastric low-grade MALT lymphoma. We studied by immunohistochemistry formalin-fixed, paraffin-embedded sections with monoclonal antibodies to molecules known to be involved in lymphocyte and endothelial adhesion phenomena, that is, CD29 (beta1 integrin subunit), CD43 (leukosialin), CD44 (H-CAM), CD54 (ICAM-1), embryonal N-CAM (e-NCAM), and EMA (episialin). In all cases, the surfaces of IL aggregates reacted for CD44 but were consistently negative for CD29; also absent was CD54. Conversely, the integrity of the endothelial cells was underscored by their even reactivity for CD29, CD44, and CD54. Given that CD29 is currently regarded as critical for lymphocyte trafficking in general and for transvascular migration in particular, and CD54 is also involved in transvascular lymphocyte migration, we conclude that their consistent absence in IL may contribute to its intravascular and disseminated distribution pattern. The rather frequent association of IL with various conventional lymphomas is known; yet, one of our cases appears to be the first report of IL associated with a low-grade MALT lymphoma.

A precise knowledge of the early events inducing maturation of resting microglia into a competent APC may help to understand the involvement of this cell type in the development of CNS immunopathology. To elucidate whether signals from preactivated T cells are sufficient to induce APC features in resting microglia, microglia from the adult BALB/c mouse CNS were cocultured with Th1 and Th2 lines from DO11.10 TCR transgenic mice to examine modulation of APC-related molecules and Ag-presenting capacity. Upon Ag-specific interaction with Th1, but not Th2, cells, microglia strongly up-regulated the surface expression of MHC class II, CD40, and CD54 molecules. Induction of CD86 on mouse microglia did not require T cell-derived signals. Acutely isolated adult microglia stimulated Th1 cells to secrete IFN-gamma and, to a lesser extent, IL-2, but were inefficient stimulators of IL-4 secretion by Th2 cells. Microglia exposed in vitro to IFN-gamma showed enhanced expression of MHC class II, CD40, and CD54 molecules and became able to restimulate Th2 cells. In addition to IFN-gamma, GM-CSF increased the ability of microglia to activate Th1, but not Th2, cells without up-regulating MHC class II, CD40, or CD54 molecules. These results suggest that interaction with Th1 cells and/or Th1-secreted soluble factors induces the functional maturation of adult mouse microglia into an APC able to sustain CD4+ T cell activation. Moreover, GM-CSF, a cytokine secreted by T cells as well as reactive astrocytes, could prime microglia for Th1-stimulating capacity, possibly by enhancing their responsiveness to Th1-derived signals.

Parasite Ag-specific T cell unresponsiveness and diminished IFN-gamma production are immunologic hallmarks of patent infection with lymph-dwelling filarial nematodes. Although this diminished responsiveness is directed primarily against the intravascular microfilarial (MF) parasite stage and mediated in part by reduced APC function, the mechanisms involved are not fully understood. In this report, we demonstrate that human dendritic cells (DC) exposed to live MF up-regulate both the cell surface and gene expression of CD54 (ICAM-1). Moreover, live MF result in a 3-fold increase in DC death compared with MF-unexposed DC, primarily due to apoptosis. Notably, microarray and real-time RT-PCR data indicate that live MF concurrently up-regulate mRNA expression of proinflammatory molecules such as IL-8, RANTES, IL-1alpha, TNF-alpha, and IL-beta in DC, the presence of which is also detected at the protein level, while inhibiting the production of IL-12 (p40 and p70) and IL-10. Soluble excretory-secretory products from live MF diminished IL-12 and IL-10 production and induced DC death, although to a lesser degree. Moreover, exposure of DC to live MF resulted in a decrease in the ability of DC to promote CD4(+) T cell production of IFN-gamma and IL-5. Our findings clearly suggest that the interaction between live MF and DC is complex but contributes to the hyporesponsiveness and parasite persistence associated with the MF(+) state in the infected human. These data further suggest that MF induce an orchestrated response in APC that leads to a diminished capacity to function appropriately, which in turn has significant consequences for CD4(+) T cells.