Activation of T cells requires both TCR-specific ligation and costimulation through accessory molecules during T cell priming. IFNgamma is a key cytokine responsible for macrophage activation during Mycobacterium tuberculosis (Mtb) infection while IL-10 is associated with suppression of cell mediated immunity in intracellular infection. In this paper we evaluated the role of IFNgamma and IL-10 on the function of cytotoxic T cells (CTL) and on the modulation of costimulatory molecules in healthy controls and patients with active tuberculosis (TB). gamma-irradiated-Mtb (i-Mtb) induced IL-10 production from CD14(+) cells from TB patients. Moreover, CD3(+) T cells of patients with advanced disease also produced IL-10 after i-Mtb stimulation. In healthy donors, IL-10 decreased the lytic activity of CD4(+) and CD8(+) T cells whereas it increased gammadelta-mediated cytotoxicity. Furthermore, we found that the presence of IL-10 induced a loss of the alternative processing pathways of antigen presentation along with a down-regulation of the expression of costimulatory molecule expression on monocytes and macrophages from healthy individuals. Conversely, neutralization of endogenous IL-10 or addition of IFNgamma to either effector or target cells from TB patients induced a strong lytic activity mediated by CD8(+) CTL together with an up-regulation of CD54 and CD86 expression on target cells. Moreover, we observed that macrophages from TB patients could use alternative pathways for i-Mtb presentation. Taken together, our results demonstrate that the presence of IL-10 during Mtb infection might contribute to mycobacteria persistence inside host macrophages through a mechanism that involved inhibition of MHC-restricted cytotoxicity against infected macrophages.

Rats were fed for 6 weeks on a low fat (LF) diet or on high fat diets containing safflower oil [SO; rich in n-6 polyunsaturated fatty acids (PUFAs)] or fish oil (FO; rich in n-3 PUFAs). Lymph-borne dendritic cells (L-DC) were isolated after cannulation of the thoracic duct and were used as antigen [keyhole limpet hemocyanin (KLH)]-presenting cells in an ex vivo assay that used KLH-sensitized spleen lymphocytes as the responder cells. FO feeding significantly diminished the antigen presentation activity of L-DC compared with L-DC from rats fed each of the other diets. The antigen presentation activity of L-DC from rats fed the SO diet was greater than that of L-DC from rats fed the LF diet. Feeding the FO diet significantly reduced both the proportion of CD2-positive L-DC and the level of CD2 expression on L-DC compared with feeding each of the other diets; the proportions of L-DC staining positive for CD40, CD18, CD54, CD11a, and MHC II were unaffected by diet. However, FO feeding reduced the level of expression of CD18, CD11a, MHC II, and CD54 on L-DC compared with feeding the other two diets; the level of expression of CD40 was unaffected by diet. This is the first study to report effects of dietary fatty acids on dendritic cells. The suppressive effect of FO feeding may account for some of the beneficial effects of n-3 polyunsaturated fatty acids observed in clinical settings, such as prolonged survival of grafts and diminished chronic inflammatory responses. However, such an effect may also be detrimental because host defense toward bacterial and other antigens could be compromised.

Self-renewal and differentiation of B-cell precursors is dependent on interactions with bone marrow (BM) stromal cells and associated extracellular matrix. We have recently developed an interleukin (IL)-7-dependent, BM-derived stromal cell culture that supports the growth of normal human B-cell precursors. In the current study, we have characterized the constitutive expression, cytokine-regulated expression, and function of adhesion molecules on BM stromal cells that are critical for adhesion of B-cell precursors. Flow cytometric analysis showed that cultured adult BM stromal cells expressed higher constitutive levels of vascular cell adhesion molecule (VCAM)-1 than intercellular adhesion molecule (ICAM)-1 (CD54). IL-1 beta upregulated VCAM-1 and CD54 in a dose-dependent manner, whereas IL-4 upregulated VCAM-1, but had no effect on CD54. In contrast, transforming growth factor (TGF)-beta decreased the level of BM stromal cell VCAM-1. Using an assay to measure the adhesion of 51Cr-labeled B-cell precursors to BM stromal cells, we observed a direct correlation between cytokine-regulated levels of VCAM-1 and the capacity of stromal cells to support the adhesion of B-cell precursors. Blocking studies using a panel of monoclonal antibodies (MoAb) showed that adhesion of B-cell precursors to untreated and cytokine-treated (IL-1 beta, IL-4) BM stromal cells was mediated by very late antigen (VLA)-4 (CD49d/CD29) and VCAM-1. Adhesion of B-cell precursors could also be enhanced by direct stimulation with MoAb to the CD29 subunit. Our collective results indicate that B-cell precursor/BM stromal cell adhesion is mediated by a VLA-4-VCAM-1 interaction, which in turn can be regulated at the level of the BM stromal cell by cytokines that specifically increase or decrease cell surface VCAM-1.

The tyramide signal amplification (TSA) method has recently been introduced to improve the detection sensitivity of immunohistochemistry. We present three examples of applying this method to immunofluorescence confocal laser microscopy: (1) single labeling for CD54 in frozen mouse brain tissue; (2) double labeling with two unconjugated primary antibodies raised in the same host species (human immunodeficiency virus type 1 p24 and CD68) in paraffin-biopsied human lymphoid tissue; and (3) triple labeling for brain-derived neurotrophic factor, glial fibrillary acidic protein, and HLA-DR in paraffin-autopsied human brain tissue. The TSA method, when properly optimized to individual tissues and primary antibodies, is an important tool for immunofluorescence microscopy. Furthermore, the TSA method and enzyme pretreatment can be complementary to achieve a high detection sensitivity, particularly in formalin-fixed paraffin-embedded archival tissues. Using multiple-label immunofluorescence confocal microscopy to characterize the cellular localization of antigens, the TSA method can be critical for double labeling with unconjugated primary antibodies raised in the same host species.

OBJECTIVE

We investigated expression of toll-like receptor (TLR) in labial salivary glands of patients with Sjögren's syndrome (SS) and functional TLR expression in the cultured salivary gland cell line.

METHODS

Expression of TLR2, TLR3, TLR4, and myeloid differentiation factor 88 (MyD88) in labial salivary glands was examined by immunohistochemistry. Human salivary gland (HSG) cell-line cells were cultured with TLR ligands [peptidoglycan, poly (I:C) and lipopolysaccharide], and CD54 expression and interleukin 6 (IL-6) production was studied. Phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, and Akt was examined by Western blotting. Activation of nuclear factor-kappaB (NF-kappaB) p65 in HSG cells was studied by NF-kappaB p65 nuclear translocation by microscopic immunofluorescence or chemiluminescent electrophoretic mobility shift assay and detection of NF-kappaB p65 phosphorylation.

RESULTS

TLR2, TLR3, TLR4, and MyD88 were more strongly expressed in the labial salivary glands of SS patients (n =12) than in control subjects (n = 4), and were found in salivary-infiltrating mononuclear cells as well as acinar cells and ductal epithelial cells. In cultured HSG cells, a similar expression pattern was observed, and TLR ligands stimulated CD54 expression and IL-6 production. TLR ligands induced phosphorylation of ERK, JNK, and p38 in HSG cells, but not Akt phosphorylation or activation of NF-kappaB p65.

CONCLUSIONS

Although the putative ligands remain to be determined, our study indicated the activation of the TLR-mediated immune response in SS, and suggested that the TLR effect is mediated through the mitogen-activated protein kinase pathway.

Inflammation and infection of the gut can be followed by reactive arthritis at a distant joint. Leukocyte recruitment into synovium is essential for this process, but nothing is known about the endothelial adhesion molecules in synovial membrane which direct the homing of activated, gut-derived leukocytes to joints. Here we analyzed the expression of the known endothelial adhesion molecules in inflamed synovium and their function in binding of mucosal leukocytes. Intercellular adhesion molecule-1 (ICAM-1/CD54) and vascular adhesion protein-1 (VAP-1) were most prominently expressed in synovial vessels. All other adhesion molecules were found at lower levels in inflamed synovia, except mucosal addressin which was absent. Binding of macrophages isolated from lamina propria of the gut to synovial endothelium was almost entirely P-selectin-dependent. In contrast, small intestinal lymphocytes and immunoblasts both relied mainly on VAP-1 in recognition of synovial vessels. Thus, endothelial P-selectin and VAP-1 mediate binding of mucosal effector cells to synovium in a leukocyte subtype-selective manner. Antiadhesive therapy against these inducible molecules should ablate the pathogenetic cascade leading to inappropriate homing of leukocytes to joints in arthritis.

Satellite glial cells (SGC) in sensory ganglia tightly envelop the neuronal cell body to form discrete anatomical units. This type of glial cell is considered neuroectoderm-derived and provides physical support to neuron somata. There are scattered hints in the literature suggesting that SGC have an immune-related function within sensory ganglia. In this study, we addressed the hypothesis that SGC are tissue-resident APC. The immune phenotype and function of a large series (n = 40) of human trigeminal ganglia (TG) were assessed by detailed flow cytometry, in situ analyses, and functional in vitro assays. Human TG-resident SGC (TG-SGC) uniformly expressed the common leukocyte marker CD45, albeit at lower levels compared with infiltrating T cells, and the macrophage markers CD14, CD68, and CD11b. In addition, TG-SGC expressed the myeloid dendritic cell (DC) marker CD11c, the T cell costimulatory molecules CD40, CD54, CD80, and CD86 and MHC class II. However, the mature DC marker CD83 was absent on TG-SGC. Functionally, TG-SGC phagocytosed fluorescent bacteria, but were unable to induce an allogeneic MLR. Finally, TG-infiltrating T cells expressed the T cell inhibitory molecules CD94/NKG2A and PD-1, and the interacting TG-SGC expressed the cognate ligands HLA-E and PD-L1, respectively. In conclusion, the data demonstrate that human TG-SGC have a unique leukocyte phenotype, with features of both macrophages and immature myeloid DC, indicating that they have a role as TG-resident APC with potential T cell modulatory properties.

OBJECTIVE

Natural killer (NK)-92 cells are effective against a broad range of malignant targets both in vitro and in vivo. Interleukin-15 (IL-15) is an important cytokine for NK cell development and differentiation. IL-15 gene-modified NK-92 cells need to be characterized and their clinical implications investigated.

METHODS

IL-15 cDNA was inserted into a pcDNA3 eukaryotic expression vector and the recombinant vector (pcDNA3-IL15) was tranfected into NK-92 cells. The IL-15 gene-modified NK-92 cells (NK92-IL15) were cloned and characterized with regard to their cytokine production, proliferation, cytotoxicity and surface phenotype.

RESULTS

NK92-IL15 cells continuously produced a high level of IL-15 in culture supernatant, which made the cells proliferate significantly more rapidly in response to stimulation with low doses of IL-2 or IL-15; the cumulative number of cells in long-term culture was also significantly higher. NK92-IL15 cells became adherent to plastic and their expression of CD54 increased, which may explain their improved proliferating potential, like adherent NK cells. NK92-IL15 cells were more strongly cytotox against a broad range of target tumor cells than the parent NK-92 cells, and this increased cytotoxicity was correlated to the increased expression of cytotoxic effector molecules, such as perforin, Fas ligand and IFNgamma, and up- or down-regulated expression of activating or inhibitory NK cell receptors (NKG2D or NKG2A/CD94).

CONCLUSIONS

These results demonstrate that NK92-IL15 cells are promising for adoptive cellular immunotherapy.

Five zones in the secondary follicles of human tonsils are described, which are distinguished by the phenotype of their constituent cells. Moving from the apex to the base of the follicle the zones are termed: follicular mantle, outer zone, apical light zone, basal light zone and dark zone. The dark zone contains proliferating, CD77high, centroblasts and thin, widely spaced processes of follicular dendritic cells (FDC). The apical and basal light zones on the other hand contain a dense network of FDC which express CD21 and CD54 strongly. The FDC of the apical light zone differ from those of the basal light zone by their high expression of CD23. Centroblasts of the dark zone give rise to non-proliferating centrocytes which move apically through the light zone (Eur. J. Immunol. 1991. 21:2951). The centrocytes of the basal light zone are more pyroninophilic, more closely-packed and larger than those in the apical light zone. Consequently, by conventional histology the basal light zone appears to be part of the dark zone. The nomenclature adopted, however, adheres to the convention that the dark zone is filled with proliferating centroblasts. Cells undergoing apoptosis were identified both in the dark and light zones, but more than half of these cells were located in the basal light zone. This is consistent with the concept that the progeny of cells which undergo somatic mutation in their immunoglobulin variable region genes in the dark zone migrate to the light zone where they are selected on the basis of their capacity to bind to antigen held on FDC. Cells receiving an antigen-dependent signal survive while those that do not kill themselves by apoptosis. The outer zone does not contain CD23high cells and in this way is distinguished from the adjacent follicular mantle and apical light zone. It contains small lymphoid cells, blasts and plasmacytoid cells. Many cells of the outer zone express CDw75 strongly. The outer zone also extends as a narrow band around the dark zone. Possible roles of FDC and T cells of the light zone and outer zone in inducing centrocytes to differentiate to memory cells or plasmablasts are discussed.

Macrophages isolated from various tissues manifest differences in cell shape, the expression of surface markers, as well as metabolic and functional activities. However, the heterogeneity of macrophages expressing the same marker in different tissues has not been fully addressed. In the present study, mouse F4/80+ peritoneal exudate macrophages (PEMs) and splenic macrophages (SPMs) appeared similar in most respects. But the percentages of cells expressing CD80, CD40, MHC-II, TLR2, or TLR4, but not CD11c, CD54, or CD23, in freshly isolated F4/80+ SPMs were significantly higher than those in PEMs, whereas the levels of CD86+ cells in F4/80+ SPMs were markedly lower than those in PEMs. After lipopolysaccharide (LPS) stimulation, F4/80+ SPMs expressed significantly higher levels of CD86, CD40, or MHC-II than F4/80+ PEMs, but not CD11c, CD80, CD54, or CD23. F4/80+ SPMs had remarkably lower non-opsonic phagocytotic capacity against chicken RBCs or allo-T cells than PEMs as determined by two-photon microscopes and flow cytometry. SPMs produced markedly more NO than PEMs when cultured with LPS or allo-T cells. Furthermore, SPMs exhibited stronger immunogenicity than PEMs, as determined by the ability to stimulate T cell proliferation, delayed type hypersensitivity, and IFN-gamma production. The data showed the differences between SPMs and PEMs with regard to the phenotypes, phagocytosis, and immunogenicity, which may offer important information for us to better understand the distinguished immune responses of macrophages in spleens and the peritoneal cavity.

The contribution of Epstein-Barr virus (EBV) strain variation to the pathogenesis of virus-associated tumours remains unknown. Given the central role of LMP1 in EBV-induced transformation, much interest has focused on the influence of LMP1 sequence variation on the signaling pathways and multiple downstream phenotypic consequences of LMP1 expression. The identification of LMP1 variants with a common 10-amino-acid deletion and additional point mutations (typified by the CAO-LMP1 isolate) in EBV strains associated with nasopharyngeal carcinoma prompted us to examine the effect of stable prototype B95.8-LMP1 and CAO-LMP1 expression on the phenotype and differentiation of SCC12F human epithelial cells. Both forms of LMP1 were able to induce expression of the antiapoptotic A20 protein and provide protection from tumour necrosis factor-alpha-induced cytotoxicity. Although B95.8-LMP1 induced growth inhibition, expression of certain cell surface molecules (CD40, CD44, and CD54), and secretion of interleukin-6 and -8 in SCC12F cells, stable CAO-LMP1 expression failed to elicit these effects. Furthermore, B95. 8-LMP1, but not CAO-LMP1, induced alterations in cell morphology and blocked epithelial cell differentiation. Both B95.8-LMP1 and CAO-LMP1 induced similar levels of nuclear factor-kappaB activation, but the ability of CAO-LMP1 to activate the AP-1 pathway was relatively impaired. These data highlight significant functional differences between the prototype B95.8-LMP1 and the CAO-LMP1 variant when stably expressed in human epithelial cells and suggest that continued analysis of LMP1 variants will help to further dissect the signaling pathways activated by LMP1 as well as provide insights into the contribution of LMP1 sequence variation to the pathogenesis of EBV-associated tumours.

Human tonsil germinal center (GC) B cells rapidly undergo apoptosis in culture. Annexin-V binding shows an early event in this process. In the present study, this method has been used to label apoptotic GC B cells and to analyze additional surface molecules. The expression of all of the molecules studied was reduced in apoptotic (annexin-V(+)) GC B cells, and the reduction was more marked for CD11a, CD21, CD22, CD49d, and CD54, molecules that participate in survival interaction for GC B cells. The analysis of CD54, one of the molecules that was more drastically reduced, showed that GC, but not mantle zone, B cells actively secrete CD54 to the culture supernatant (SN). The secreted CD54 was partly released from the GC B cells in a particulate form as demonstrated by centrifugation. Further experiments using filtration, fluorescence microscopy, electron microscopy, and flow cytometry analysis showed that GC B cells released to the culture SN a population of spherical membranous vesicles of about 0.18 micrometers in size, similar to the blebs described in other apoptosis systems. Bleb formation depended on active metabolism, Ca(2+), and, in part, on microfilament integrity. GC B-cell-derived blebs were clearly associated with apoptosis, as antiapoptotic stimuli prevented their formation. In addition, GC B-cell-derived blebs contained the adhesion molecules previously studied. Consequently, bleb formation might contribute to the surface molecule loss occurring in apoptotic GC B cells. Finally, a chemotaxis assay showed that GC B-cell blebs were chemotactic for human monocytes, suggesting that this mechanism might operate in vivo.

The consequences of internalization of Staphylococcus aureus by HUVEC with respect to their adhesiveness for human monocytes and granulocytes were investigated. Viable and UV-killed, but not heat-killed, S. aureus were internalized by HUVEC, which required participation of the endothelial cytoskeleton. S. aureus-infected HUVEC displayed increased surface expression of CD106 (VCAM-1), CD54 (ICAM-1), and MHC I molecules. Expression of CD62P (P-selectin), CD62E (E-selectin), CD31 (PECAM-1), and CD102 (ICAM-2) was not affected. Concomitantly, these HUVEC expressed a time- and inoculum size-dependent hyperadhesiveness for monocytes and granulocytes. Monocyte adhesion reached maximal levels (approximately 60% adhesion) 23 h after the initial 1 h period of infection of HUVEC with about 50 bacteria per single HUVEC. To induce maximal (approximately 20%) adhesion of granulocytes, five times higher concentrations of HUVEC-infecting bacteria were required. Using the appropriate mAb, granulocyte adhesion to S. aureus-infected HUVEC was shown to be entirely mediated by the beta2 (CD11/CD18) integrins. Monocyte adhesion to these HUVEC was largely (approximately 70%) dependent on both CD11a/CD18 (LFA-1) and CD49d/CD29 (VLA-4). This demonstrates that infection of HUVEC with S. aureus potentiates CD11/CD18-mediated granulocyte adhesion and shifts the mechanism of monocyte adhesion from being completely CD11/CD18 dependent to one that also utilizes the VLA-4/VCAM-1 dependent pathway. Together, these findings indicate that in response to internalization of S. aureus, vascular endothelial cells may initiate recruitment of monocytes and granulocytes, which may be an important initial event in the pathogenesis of endovascular diseases.

Acute P. falciparum malaria is associated with loss of in vitro T cell responsiveness to antigenic stimulation, and with high plasma levels of soluble interleukin 2 receptor (IL 2R). In the present study peripheral T cells from acute P. falciparum malaria patients from a malaria-endemic area of Sudan were analyzed for expression of cell surface antigens associated with T lymphocyte adhesion, activation and maturation. The results were compared to results from T cells obtained from the same donors either before the attack, or during convalescence. Most donors showed a remarkable loss of T cells with high expression of the surface marker LFA-1 (CD11a/CD18) during the clinical episode, in addition to the functional changes described above. Two donors that did not show phenotypic changes were furthermore characterized by having an unabated proliferative response and normal plasma IL 2R levels. All peripheral CD3+ T lymphocytes expressed LFA-1, which had a clearly bimodal distribution on these cells. The T cell subpopulation having high LFA-1 expression (LFA-1++) was composed of both memory and unprimed T cells, according to their expression of CD45RA and CD45R0. Analysis of expression of membrane-bound IL 2R (CD25) and ICAM-1 (CD54) did not reveal in vivo activated T cells in the peripheral blood of the patients. Taken together, these data suggest that circulating T cells recognizing parasite antigens are temporarily withdrawn from peripheral circulation during P. falciparum malaria.

ICAM.1 (CD54) is a surface protein expressed on epithelial and other nonhematopoietic cells upon activation and is known to play an important role in the stimulation of T cells by the provision of cellular adhesion and costimulatory support. Sjogren's syndrome (SS) is an autoimmune exocrinopathy, which is characterized by chronic lymphocytic infiltration of exocrine glands and aberrant activation of epithelial tissues. To address the contribution of ICAM.1 in the pathogenesis of SS, the expression of this protein was studied by immunohistochemistry and flow cytometry in minor salivary gland (SG) biopsies as well as in cultured SG epithelial cell (SGEC) lines obtained from 18 SS patients and 16 controls. In biopsies from SS patients (but not controls), strong ICAM.1 was expressed by infiltrating mononuclear cells (52%) and by a significant proportion of periacinar myoepithelial cells (18%). In addition, a patchy pattern of moderate ICAM.1 expression was detected in 31% of ductal epithelia of SS patients. These ICAM.1-expressing epithelial and myoepithelial cells were observed throughout glandular tissues and were not confined in areas proximal to lymphoid infiltrates. In support to an intrinsic activation profile of SGEC in SS, long-term cultured non-neoplastic SGEC lines derived from SS patients displayed significantly upregulated spontaneous expression of ICAM.1, compared to controls (P < 0.05). The high expression of ICAM.1 protein by the salivary epithelium of SS patients is likely suggestive of its important role in the pathogenesis of the disorder. Further, our results support a model of intrinsic activation of salivary epithelial and myoepithelial cells in SS, whereby these cells actively participate in the induction and maintenance of lymphocytic infiltrates of patients.

Modulating signal transduction pathways represents a promising approach for altering the biological behaviour of haemopoietic malignancies. B-cell chronic lymphocytic leukaemia (B-CLL) cells were treated in vitro with CD40-ligand (CD40L) (CD154) or the protein kinase C modulator Bryostatin-1, exploring the effects on: (a) sensitivity to apoptosis induction by chemotherapeutic drugs (fludarabine, dexamethasone) or anti-Fas antibody; (b) expression of apoptosis-regulatory proteins (Bcl-2, Bcl-X, Mcl-1, Bax, Bak, BAG-1, Flip, XIAP); (c) expression of cell surface co-stimulatory antigens (CD80 [B7.1]; CD54 [ICAM-1]; CD70); and (d) expression of immune modulatory receptors (CD27, CD40, CD95 [Fas]). CD40L and Bryostatin decreased both spontaneous and drug-induced apoptosis in most B-CLL specimens tested. Apoptosis resistance was associated with CD40L- and Bryostatin-induced elevations in the anti-apoptotic Bcl-2 family protein Mcl-1. CD40L also induced striking increases in the levels of the anti-apoptotic protein Bcl-XL in B-CLLs. CD40L stimulated increases in the surface expression of CD40, CD54, CD69, CD70, CD80 and CD95, whereas Bryostatin induced expression of CD40, CD54, CD69 and CD95 but not the co-stimulatory molecules CD70 and CD80. Despite elevations in the expression of CD95 (Fas), anti-Fas antibodies failed to induce apoptosis of CD40L- and Bryostatin-treated B-CLL cells. This Fas-resistance was associated with increased expression of the Fas-antagonist Flip in CD40L-treated, and with elevations in the caspase inhibitor XIAP in Bryostatin-treated B-CLLs. The potential anti-apoptotic properties of CD40L and Bryostatin should be taken into consideration when employing these agents in clinical trials involving patients with B-CLL.

Increased expression of CD40 and CD40 ligand (CD40L or CD154) has been found in inflamed mucosa of human inflammatory bowel disease (IBD), and interactions between these molecules seem to be involved in local cytokine production by macrophages. However, the precise role of CD40 signaling in the pathogenesis of IBD is still poorly understood. The aim of the present study was to investigate the in vivo relevance of CD40 signaling in experimental colitis in SCID mice reconstituted with syngeneic CD45RBhighCD4+ T cells. The results demonstrated that CD40+ and CD40L+ cells as well as their mRNA levels were significantly increased in inflamed mucosa. Administration of anti-CD40L neutralizing mAb over an 8-wk period starting immediately after CD45RBhighCD4+ T cell reconstitution completely prevented symptoms of wasting disease. Intestinal mucosal inflammation was effectively prevented, as revealed by abrogated leukocyte infiltration and decreased CD54 expression and strongly diminished mRNA levels of the proinflammatory cytokines IFN-gamma, TNF, and IL-12. When colitic SCID mice were treated with anti-CD40L starting at 5 wk after T cell transfer up to 8 wk, this delayed treatment still led to significant clinical and histological improvement and down-regulated proinflammatory cytokine secretion. These data suggest that the CD40-CD40L interactions are essential for the Th1 inflammatory responses in the bowel in this experimental model of colitis. Blockade of CD40 signaling may be beneficial to human IBD.

We have isolated and characterized the human cardiac mast cell (CMC) and compared this novel mast cell (MC type with MC obtained from uterus, skin, and lung. Heart tissue was obtained from 14 patients with cardiomyopathy (CMP, heart transplantation). CMC were isolated by enzymatic digestion using collagenase, pronase-E, hyaluronidase, and DNAse. Substantial amounts of CMC (0.5% to 1.5% of isolated cells) were found in the atrial appendages but not in ventricular digests or other sites of the heart (< 0.1%). In situ staining of atrial tissue revealed the presence of CMC in the myocardium (2.16 +/- 0.7 MC/mm2), endocardium (2.24 +/- 0.9 MC/mm2), and epicardium. As assessed by combined toluidine blue/immunofluorescence staining with monoclonal antibodies (MoAbs), isolated CMC expressed surface IgE, the receptor for stem cell factor (c-kit receptor/CD117), the p24 antigen (CD9), the Pgp-1 homing receptor (CD44), the pan leukocyte antigen (CD45), and the ICAM-1 antigen (CD54). CMC were not recognized by MoAbs to lymphocyte function associated antigen 2 (LFA-2; CD2), T-cell receptor (TcR; CD3), T4 antigen (CD4), LFA-1 alpha-chain (CD11a), C3biR alpha-chain (CD11b), CR4 alpha-chain (CD11c), LPS-R related Ag (CD14), 3-FAL/x-hapten (CD15), Fc gamma RIII (CD16), lactosylceramid (CDw17), the B-cell antigen CD19, or CR1 (CD35). In situ expression of leukocyte antigens on CMC was demonstrable by indirect immunoperoxidase staining technique and double-labeling immunohistochemistry. Almost all CMC (90%) reacted with MoAbs against tryptase and chymase and thus were MCTC. Cardiac mast cells were also stained by the heparin-binding dye Berberine sulfate and expressed measurable amounts of histamine (4.6 +/- 1.4 pg per cell). Cross linking of either IgE receptor or SCF receptor (c-kit) on CMC resulted in histamine secretion (non-specific release: < 6% of total histamine, alpha IgE induced: 12% to 52%; SCF-induced release: 9% to 18%), whereas neither substance P (a skin MC agonist) nor the basophil agonist FMLP showed an effect on CMC. Together, the CMC is an MCTC primarily located in the appendage of the atrium. This novel type of MC exhibits surface membrane antigen and functional properties similar to those of lung and uterus MC.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S-1-P) are both low molecular weight lysophospholipid (LPL) ligands that are recognized by the Edg family of G protein-coupled receptors. In endothelial cells, these two ligands activate Edg receptors, resulting in cell proliferation and cell migration. The intercellular adhesion molecule-1 (ICAM-1, CD54) is one of many cell adhesion molecules belonging to the immunoglobulin superfamily. This study showed that LPA and S-1-P enhance ICAM-1 expression at both the mRNA and protein levels in human umbilical cord vein endothelial cells (HUVECs). This enhanced ICAM-1 expression in HUVECs was first observed at 2 h postligand treatment. Maximal expression appeared at 8 h postligand treatment, as detected by flow cytometry and Western blotting. Furthermore, the effects of S-1-P on ICAM-1 expression were shown to be concentration dependent. Prior treatment of HUVECs with pertussis toxin, a specific inhibitor of G(i), ammonium pyrrolidinedithiocarbamate and BAY 11-7082, inhibitors of the nuclear factor (NF)-kappaB pathway, or Clostridium difficile toxin B, an inhibitor of Rac, prevented the enhanced effect of LPL-induced ICAM-1 expression. However, pretreatment of HUVECs with exoC3, an inhibitor of Rho, had no effect on S-1-P-enhanced ICAM-1 expression. In a static cell-cell adhesion assay system, pretreatment of LPL enhanced the adhesion between HUVECs and U-937 cells, a human mononucleated cell line. The enhanced adhesion effect could be prevented by preincubation with a functional blocking antibody against human ICAM-1. These results suggest that LPLs released by activated platelets might enhance interactions of leukocytes with the endothelium through a G(i)-, NF-kappaB-, and possibly Rac-dependent mechanism, thus facilitating wound healing and inflammation processes.

Thymic nurse cells are known to interact with T cells and play a role in their functional maturation. However, the role of nurse cells in B cell maturation and differentiation is less well established, especially at extralymphoid sites. To address this issue, nurse-like cell clones from bone marrow and synovial tissue of patients with RA (RA-NLC) were established and characterized. RA-NLC constitutively expressed CD29, CD49c, CD54 (ICAM-1), CD106 (VCAM-1), CD157 (BST-1), and class I MHC molecules, and secreted IL-6, IL-7, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). Bone marrow-derived and synovial RA-NLC differed in that the former secreted IL-7 and expressed a greater density of CD157 constitutively and after stimulation with IFNgamma, whereas the latter secreted G-CSF and more IL-6. Stimulation of both bone marrow and synovial RA-NLC induced expression of CD40 and class II MHC, but not CD154 (CD40L) or CD35. RA-NLC rescued peripheral B cells from spontaneous apoptosis and promoted survival of B cells for>> 4 wk. B cell survival was blocked by antibodies to CD106 or CD157. RA-NLC also increased Ig production from B cells. After long-term culture (4-6 wk) with RA-NLC, but not alone or with fibroblasts, outgrowth of B cells was observed. All B cell lines derived from these cultures had been transformed by EBV, although the RA-NLC themselves were not infected with EBV. Precursor frequency analysis indicated that approximately 1 in 12,500 peripheral B cells could give rise to these EBV-transformed B cell lines upon coculture with RA-NLC. These results indicate that RA-NLC from bone marrow and synovium have the capacity to rescue B cells from spontaneous apoptosis, facilitate Ig production, and promote the outgrowth of EBV-transformed B lymphoblastoid cells. These findings suggest that RA-NLC may play a role in the local and systemic hyperreactivity of B cells characteristic of rheumatoid arthritis.

BACKGROUND

Dysfunctional antigen presentation may underlie the impaired antibody response to hepatitis B vaccination in hemodialysis patients. Dendritic cells are considered to be the most important antigen presenting cells, but their presence and function in hemodialysis patients is unclear. Granulocyte-monocyte-colony stimulating factor (GM-CSF) has been given successfully to hemodialysis patients to increase the proportion of responders to hepatitis B vaccination. Although GM-CSF acts on both monocytes and dendritic cells, the mechanisms underlying its adjuvant quality are largely unknown.

METHODS

In this study we analyzed monocytes and dendritic cells in the peripheral blood of hemodialysis patient that had responded to a standard hepatitis B vaccination procedure (responders), patients who had not responded (nonresponders), and healthy controls. The nonresponders were given two additional booster vaccines, both preceded by administration of GM-CSF the day before.

RESULTS

After two booster vaccinations with GM-CSF, six out of seven patients developed a protective antibody response to hepatitis B. The memory T-cell response to tetanus toxoid was significantly lower in nonresponders compared to controls. The monocytes of dialysis patients and healthy controls showed a similar expression of relevant cell surface molecules. However, the numbers of circulating dendritic cells were on average 50% reduced compared to healthy controls, with a further reduction after GM-CSF administration. This was accompanied by a decrease of T-cell proliferation in antigen presentation assays. Monocytes showed increased major histocompatibility complex (MHC) class II, CD54, and CD40 expression, while their antigen-presenting capacity remained unchanged.

CONCLUSIONS

GM-CSF is an effective adjuvant for hepatitis B vaccination in primary nonresponding hemodialysis patients, but paradoxically decreases the antigen presenting capacity of peripheral blood mononuclear cells and the number of circulating dendritic cells.

Blood eosinophil numbers may be elevated in allergy, inflammatory bowel disease and eosinophilic esophagitis. The aim of this study was to examine whether circulating eosinophils display distinct phenotypes in these disorders and if different patterns of eosinophilic chemoattractants exist. Blood eosinophils from patients with symptomatic eosinophilic esophagitis (EoE; n = 12), ulcerative colitis (n = 8), airway allergy (n = 10) and healthy controls (n = 10) were enumerated and their surface markers analyzed by flow cytometry. Plasma levels of pro-eosinophilic cytokines were quantified in parallel. Data were processed by multivariate pattern recognition methods to reveal disease-specific patterns of eosinophil phenotypes and cytokines. EoE patients had higher numbers of eosinophils with enhanced expression of CD23, CD54, CRTH2 and CD11c and diminished CCR3 and CD44 expression. Plasma CCL5 was also increased in EoE. Although allergic patients had increased interleukin (IL)-2, IL-3, IL-5 and granulocyte macrophage colony-stimulating factor plasma concentrations, their blood eosinophil phenotypes were indistinguishable from those of healthy controls. Decreased eosinophilic expression of CD11b, CD18, CD44 and CCR3, but no distinctive pattern of eosinophil chemoattractants, characterized ulcerative colitis. We propose that eosinophils acquire varying functional properties as a consequence of distinct patterns of activation signals released from the inflamed tissues in different diseases.

NK cells and certain CTL can recognize and lyse targets without restriction by the MHC. NK cells do not express CD3/TCR complexes and the membrane receptors participating in MHC-unrestricted cytotoxicity are largely unknown. We demonstrate that YT2C2, a human NK leukemia cell line, expresses the CD28 differentiation Ag and can spontaneously lyse both murine and human cell lines expressing B7, a B cell- activation Ag that is a ligand for CD28. The participation of CD28/B7 interactions in MHC-unrestricted cytotoxicity mediated by YT2C2 cells was demonstrated by correlation of target sensitivity with levels of B7 expression, inhibition of cytotoxicity by anti-CD28 or anti-B7 mAb, and by making both murine and human cell lines susceptible to YT2C2-mediated lysis by genetic transfection with expression vectors containing B7 cDNA. However, CD28/B7 interactions alone were insufficient to initiate cytotoxicity. mAb inhibition experiments and selection of CD54- (intercellular adhesion molecule-1) deficient B cell targets indicated that CD11a/18 (lymphocyte function-associated Ag-1) also cooperated in CD28/B7-dependent cytotoxicity. The requirement for both CD28/B7 and lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interactions in YT2C2-mediated MHC-unrestricted cytotoxicity was confirmed by demonstrating that efficient lysis of murine L cells required cotransfection with both B7 and intercellular adhesion molecule-1. These findings support the concept that MHC-unrestricted cytotoxicity may not be due to a unique receptor, but may result from interactions between an appropriate array of "adhesion" molecules with their ligands.

Osteoclast precursors (OCPs) circulate in the mononuclear fraction of peripheral blood (PB), but their abundance and surface characteristics are unknown. Previous studies suggest that the receptor activator for NF-kappaB (RANK) on cytokine-treated OCPs in mouse bone marrow interacts with osteoprotegerin ligand (OPGL/TRANCE/RANKL/ODF) to initiate osteoclast differentiation. Hence, we used a fluorescent form of human OPGL (Hu-OPGL-F) to identify possible RANK-expressing OCPs in untreated peripheral blood mononuclear cells (PBMCs) using fluorescence-activated cell sorting analysis. Monocytes [CD14-phycoerythrin (PE) antibody (Ab) positive (+) cells, 10-15% of PBMCs] all (98-100%) co-labelled with Hu-OPGL-F (n>> 18). T lymphocytes (CD3-PE Ab+ cells, 66% of PBMCs) did not bind Hu-OPGL-F; however, B cells (CD19-PE Ab+ cells, 9% of PBMCs) were also positive for Hu-OPGL-F. All Hu-OPGL-F+ monocytes also co-labelled with CD33, CD61, CD11b, CD38, CD45 and CD54 Abs, but not CD34 or CD56 Abs. Hu-OPGL-F binding was dose dependent and competed with excess Hu-OPGL. When Hu-OPGL-F+, CD14-PE Ab+, CD33-PE Ab+, Hu-OPGL-F+/CD14-PE Ab+ or Hu-OPGL-F+/CD33-PE Ab+ cells were cultured with OPGL (20 ng/ml) and colony-stimulating factor (CSF)-1 (25 ng/ml), OC-like cells readily developed. Thus, all freshly isolated monocytes demonstrate displaceable Hu-OPGL-F binding, suggesting the presence of RANK on OCPs in PB; also, OCPs within a purified PB monocyte population form osteoclast-like cells in the complete absence of other cell types in OPGL and CSF-1 containing medium.