Making a friend from a foe: expressing a GroEL gene from the whitefly Bemisia tabaci in the phloem of tomato plants confers resistance to tomato yellow leaf curl virus.

Some (perhaps all) plant viruses transmitted in a circulative manner by their insect vectors avoid destruction in the haemolymph by interacting with GroEL homologues, ensuring transmission. We have previously shown that the phloem-limited begomovirus tomato yellow leaf curl virus (TYLCV) interacts in vivo and in vitro with GroEL produced by the whitefly vector Bemisia tabaci. In this study, we have exploited this phenomenon to generate transgenic tomato plants expressing the whitefly GroEL in their phloem. We postulated that following inoculation, TYLCV particles will be trapped by GroEL in the plant phloem, thereby inhibiting virus replication and movement, thereby rendering the plants resistant. A whitefly GroEL gene was cloned in an Agrobacterium vector under the control of an Arabidopsis phloem-specific promoter, which was used to transform two tomato genotypes. During three consecutive generations, plants expressing GroEL exhibited mild or no disease symptoms upon whitefly-mediated inoculation of TYLCV. In vitro assays indicated that the sap of resistant plants contained GroEL-TYLCV complexes. Infected resistant plants served as virus source for whitefly-mediated transmission as effectively as infected non-transgenic tomato. Non-transgenic susceptible tomato plants grafted on resistant GroEL-transgenic scions remained susceptible, although GroEL translocated into the grafted plant and GroEL-TYLCV complexes were detected in the grafted tissues.

A role for NF-kappaB-dependent gene transactivation in sunburn.

Exposure of skin to ultraviolet (UV) radiation is known to induce NF-kappaB activation, but the functional role for this pathway in UV-induced cutaneous inflammation remains uncertain. In this study, we examined whether experimentally induced sunburn reactions in mice could be prevented by blocking UV-induced, NF-kappaB-dependent gene transactivation with oligodeoxynucleotides (ODNs) containing the NF-kappaB cis element (NF-kappaB decoy ODNs). UV-induced secretion of IL-1, IL-6, TNF-alpha, and VEGF by skin-derived cell lines was inhibited by the decoy ODNs, but not by the scrambled control ODNs. Systemic or local injection of NF-kappaB decoy ODNs also inhibited cutaneous swelling responses to UV irradiation. Moreover, local UV-induced inflammatory changes (swelling, leukocyte infiltration, epidermal hyperplasia, and accumulation of proinflammatory cytokines) were all inhibited specifically by topically applied decoy ODNs. Importantly, these ODNs had no effect on alternative types of cutaneous inflammation caused by irritant or allergic chemicals. These results indicate that sunburn reactions culminate from inflammatory events that are triggered by UV-activated transcription of NF-kappaB target genes, rather than from nonspecific changes associated with tissue damage.

Cloning of a second dendritic cell-associated C-type lectin (dectin-2) and its alternatively spliced isoforms.

Using a subtractive cDNA cloning strategy, we isolated previously five novel genes that were expressed abundantly by the murine dendritic cell (DC) line XS52, but not by the J774 macrophage line. One of these genes encoded a unique, DC-associated C-type lectin, termed "dectin-1." Here we report the characterization of a second novel gene that was also expressed in a DC-specific manner. Clone 1B12 encoded a type II membrane-integrated polypeptide of 209 amino acids containing a single carbohydrate recognition domain motif in the COOH terminus. The expression pattern of this molecule, termed "dectin-2," was almost indistinguishable from that for dectin-1; that is, both were expressed abundantly at mRNA and protein levels by the XS52 DC line, but not by non-DC lines, and both were detected in spleen and thymus, as well as in skin resident DC (i.e. Langerhans cells). Interestingly, reverse transcriptase-polymerase chain reaction and immunoblotting revealed multiple bands of dectin-2 transcripts and proteins suggesting molecular heterogeneity. In fact, we isolated additional cDNA clones encoding two distinct, truncated dectin-2 isoforms. Genomic analyses indicated that a full-length dectin-2 (alpha isoform) is encoded by 6 exons, whereas truncated isoforms (beta and gamma) are produced by alternative splicing. We propose that dectin-2 and its isoforms, together with dectin-1, represent a unique subfamily of DC-associated C-type lectins.

Identification of a novel, dendritic cell-associated molecule, dectin-1, by subtractive cDNA cloning.

Dendritic cells (DC) are special subsets of antigen presenting cells characterized by their potent capacity to activate immunologically naive T cells. By subtracting the mRNAs expressed by the mouse epidermus-derived DC line XS52 with the mRNAs expressed by the J774 macrophage line, we identified five novel genes that were expressed selectively by this DC line. One of these genes encoded a type II membrane-integrated polypeptide of 244 amino acids containing a putative carbohydrate recognition domain motif at the COOH-terminal end. This molecule, termed "dectin-1," was expressed abundantly at both mRNA and protein levels by the XS52 DC line, but not by non-DC lines (including the J774 macrophage line). Dectin-1 mRNA was detected predominantly in spleen and thymus (by Northern blotting) and in skin-resident DC, i.e. Langerhans cells (by reverse transcription-polymerase chain reaction). Affinity-purified antibody against dectin-1 identified a 43-kDa glycoprotein in membrane fractions isolated from the XS52 DC line and from the dectin-1 cDNA-transfected COS-1 cells. His-tagged recombinant proteins containing the extracellular domains of dectin-1 showed marked and specific binding to the surface of T cells and promoted their proliferation in the presence of anti-CD3 monoclonal antibody at suboptimal concentrations. These in vitro results suggest that dectin-1 on DC may bind to as yet undefined ligand(s) on T cells, thereby delivering T cell co-stimulatory signals. Not only do these results document the efficacy of subtractive cDNA cloning for the identification of unique genes expressed by DC, they also provide a framework for studying the physiological function of dectin-1.

Dendritic cells undergo rapid apoptosis in vitro during antigen-specific interaction with CD4+ T cells.

The terminal fate of dendritic cells (DC) remains relatively uncertain. In this study, we tested the hypothesis that DC undergo apoptosis after Ag-specific interaction with T cells. When splenic DC isolated from BALB/c mice were cocultured with HDK-1 T cells (a keyhole limpet hemocyanin (KLH)-specific CD4+ Th1 clone) in the presence of KLH, they showed conspicuous cell death as measured by propidium iodide (PI) uptake and chromatin condensation, whereas they remained relatively intact when incubated with either T cells or KLH alone. Likewise, the long term DC line XS52, which was established from BALB/c mouse epidermis, also died rapidly (within 2 h), and they exhibited characteristic DNA laddering when cocultured with HDK-1 T cells in the presence of KLH. RT-PCR and FACS analyses revealed the expression of CD95 (Fas) by XS52 DC and of CD95 ligand (CD95L) (Fas ligand) by activated HDK-1 T cells, suggesting a functional role for these molecules. In fact, anti-CD95L mAb inhibited partially (50%) T cell-mediated XS52 cell death, and coupling of surface CD95 with anti-CD95 mAb triggered significant XS52 cell death, but only in the presence of cycloheximide. Thus, ligation of CD95 (on DC) with CD95L (on T cells) is one, but not the only, mechanism by which T cells induce DC death. Finally, DC isolated from the CD95-deficient mice were found to be significantly more efficient than DC from control mice in their capacity to induce delayed type hypersensitivity responses in vivo. We propose that T cell-induced DC apoptosis serves as a unique down-regulatory mechanism that prevents the interminable activation of T cells by Ag-bearing DC.

UVB radiation interrupts cytokine-mediated support of an epidermal-derived dendritic cell line (XS52) by a dual mechanism.

We have established long-term dendritic cell lines from the epidermis of newborn mice. These cell lines (XS series) proliferate maximally in response to granulocyte/macrophage-colony stimulating factor, as well as to CSF-1, which is produced by skin-derived NS fibroblast lines and by keratinocytes (albeit in smaller amounts). The purpose of this study was to examine the impact of UVB radiation on CSF-1-mediated interaction of dendritic cells with fibroblasts and keratinocytes. Exposure of NS cells to UVB radiation (unfiltered FS20 sunlamp) decreased CSF-1 production at mRNA and protein levels. Both changes occurred in a dose-dependent fashion, with 50 J/m2 causing a significant reduction. UVB radiation also downregulated CSF-1 mRNA expression by Pam 212 keratinocytes. UVB exposure of XS cells diminished the surface expression of CSF-1 receptors, with 50 J/m2 causing a significant reduction. Thus, UVB radiation interrupts CSF-1-mediated cell-cell interaction by a dual mechanism: downregulating CSF-1 production and abrogating CSF-1 receptor expression. Importantly, granulocyte/macrophage-colony stimulating factor receptor expression by XS cells was also inhibited by UVB radiation, once again, with 50 J/m2 producing significant inhibition. We propose that the resulting CSF-1 deficiency in epidermal microenvironment and unresponsiveness by dendritic cells to relevant growth factors may contribute to UVB-mediated loss of resident epidermal dendritic cells (i.e., Langerhans cells) in skin.

Interleukin (IL)-15 promotes the growth of murine epidermal gamma delta T cells by a mechanism involving the beta- and gamma c-chains of the IL-2 receptor.

Dendritic epidermal T cells (DETC) are skin-specific members of the epithelial gamma delta T-cell family in mice. We have reported previously that the growth of DETC is promoted by interleukin (IL)-2 in an autocrine fashion, or by IL-7, which is secreted by neighboring keratinocytes. Here we report that DETC growth is promoted by IL-15, a newly discovered T-cell growth factor that is produced in lymphoid as well as nonlymphoid tissues. Recombinant IL-15 promoted the growth of the 7-17 DETC line in a time- and dose-dependent fashion. Using monoclonal antibodies against alpha-, beta-, or gamma c-chains of the IL-2 receptor complex, we observed that the combination of anti-beta chain and anti-gamma c chain antibodies blocked IL-15 responsiveness completely, whereas anti-alpha chain had no effect. These results indicate that this gamma delta T-cell line uses the beta/gamma c heterodimer for proliferative responses to IL-15. Antibodies against IL-2 or IL-7 did not block IL-15-driven proliferation of 7-17 DETC, indicating that IL-15 promotes their growth in an IL-2- and IL-7-independent manner. Both the surface expression of beta/gamma c heterodimers and the IL-15 responsiveness of 7-17 DETC were highest 1 to 8 days after concanavalin A stimulation, and both declined substantially 21 days after stimulation, illustrating regulation by the state of cell activation. Working with epidermal cells that were freshly procured from CBA mice, we noted that IL-15 promoted conavalin-A-triggered growth of Thy-1+ cells (i.e., DETC), but not of the Thy-1- cells. The gamma c-chain was not expressed by freshly procured DETC, becoming detectable within 48 h after concanavalin A stimulation. We propose that IL-15 facilitates the growth of epithelial gamma delta T cells by a beta/gamma c receptor-dependent mechanism.

T cell-dependent secretion of IL-1 beta by a dendritic cell line (XS52) derived from murine epidermis.

IL-1 beta has been reported to play an essential role in the induction of T cell-mediated immune responses in skin, and Langerhans cells are considered to be the primary source of IL-1 beta in epidermis. We have established recently a long-term dendritic cell line (XS52) from mouse epidermis. This line resembles resident epidermal Langerhans cells in many respects, including the potent capacity to present a protein Ag (KLH) to a CD4+ Th1 clone (HDK-1) and the expression of IL-1 beta mRNA. We sought to determine whether XS52 cells secrete IL-1 beta upon Ag-dependent interaction with T cells and, if so, to elucidate the mechanism. Despite constitutive expression of mRNA for both IL-1 beta and the IL-1 beta-converting enzyme (ICE), XS52 cells secreted no detectable IL-1 beta spontaneously. When they were cultured with HDK-1 T cells and KLH, relatively large amounts of IL-1 beta (17.5-kDa form) were detected in the culture supernatant. IL-1 beta was secreted by LPS-stimulated XS52 cells, but not by LPS- or Con A-stimulated HDK-1 cells, suggesting that IL-1 beta is secreted primarily by XS52 cells in the coculture system. Incubation with HDK-1 cells alone or with KLH alone caused no IL-1 beta secretion, indicating the requirement for both T cells and Ag. IL-1 beta secretion was associated with a striking up-regulation of IL-1 beta mRNA and a modest up-regulation of ICE mRNA and enzymatic activity. IL-1 beta secretion was blocked by Ac-YVAD-CHO (a peptide inhibitor of ICE), CTLA4-Ig fusion protein, or anti-Ia mAb. IL-1 beta secretion was triggered in a T cell-independent manner by either CTLA4-Ig or anti-Ia mAb in immobilized forms. Thus, the XS52 dendritic cell line secretes, by an ICE-dependent mechanism, biologically relevant amounts of IL-1 beta upon Ag-dependent interaction with T cells, with both Ia molecules and B7-related molecules playing essential roles.

Colony-stimulating factor-1 secreted by fibroblasts promotes the growth of dendritic cell lines (XS series) derived from murine epidermis.

We have established recently from mouse epidermis long-term dendritic cell lines (XS series) that resemble epidermal Langerhans cells (LC) by their surface phenotype, Ag-presenting profile and cytokine mRNA profile. The growth of XS lines was promoted maximally by granulocyte-macrophage-CSF or by a factor secreted by NS lines, which are fibroblastic cell lines established from dispase-separated specimens of mouse epidermis. The purpose of this study was to determine the identity of XS cell growth factor secreted by NS cells. We report the following: 1) NS cells express constitutively mRNA for CSF-1; 2) XS cells express the CSF-1R at mRNA and protein levels; 3) rCSF-1 mimics NS culture supernatant in its ability to promote XS cell growth; 4) NS supernatant-dependent XS cell growth is blocked completely by each of two Abs against the CSF-1R. We conclude that CSF-1 is responsible for the XS growth-promoting activity secreted by NS lines. We also report the following: 5) LC freshly isolated from skin express CSF-1R mRNA; and 6) fibroblasts derived from specimens of dermis also express mRNA and secrete large amounts (50-100 ng/ml) of CSF-1. These observations give rise to a new concept that dermal fibroblasts may support the survival and growth of LC (and their precursors) through the paracrine effect of elaborated CSF-1.

Cytokine-dependent regulation of growth and maturation in murine epidermal dendritic cell lines.

We have recently established dendritic cell (DC) lines (XS series) from the epidermis of newborn mice by repeated feeding with granulocyte/macrophage-colony-stimulating factor (GM-CSF) and culture supernatants from skin-derived stromal cell lines (NS series). XS lines resemble resident Langerhans cell (LC), which are immature DC that reside in epidermis, by their surface phenotype and antigen-presenting profile. XS lines further resemble resident LC in that they express mRNA for interleukin-1 beta and macrophage inflammatory protein (MIP)-1 alpha, and by the absence of mRNA for IL-6. Their growth is promoted by GM-CSF, colony-stimulating factor-1 (CSF-1), or NS culture supernatant, and inhibited by interferon-gamma or tumor necrosis factor-alpha. The expression by the XS lines of Ia molecules is up-regulated by GM-CSF, and down-regulated by NS supernatant. These results suggest the existence of negative regulatory mechanisms in which the growth and/or maturation of DC is suppressed by selected cytokines.

Successive generation of antigen-presenting, dendritic cell lines from murine epidermis.

Dendritic cells are specialized APCs that exhibit an extraordinary capacity to activate naive T cells. Langerhans cells (LC), as epithelial tissue-specific members of this family, play key roles in the induction of T cell-mediated immunity against environmental, infectious, and tumor-associated Ags in skin. A major limitation in studying the biology of dendritic cells or LC has been the absence of stable, long-term cell lines. To overcome this limitation, we have established a series of APC lines (XS series) from newborn BALB/c mouse epidermis. XS lines, which have grown for more than 12 mo in culture, exhibit high similarity to LC freshly procured from skin in terms of: a) tissue of derivation (epidermis), b) phenotype (lalow/CD45+/E-cadherin+/B7-1-), c) shape (elongated dendrites), and d) Ag-presenting profile (modest ability to activate naive, allogeneic T cells and remarkable ability to present a protein Ag to primed CD4+ T cells). The availability of XS lines as well as the methodologies used for their growth enhance our capability of studying the biology of LC at biochemical and molecular levels.

In vivo evidence that ultraviolet B-induced suppression of allergic contact sensitivity is associated with functional inactivation of Th1 cells.

Having previously shown in vitro that ultraviolet B (UVB)-treated Langerhans cells (LC) can induce antigen-specific proliferative unresponsiveness and tolerance in Th1 (but not Th2) cells, we wanted to determine whether cutaneous exposure to UVB radiation prior to hapten-painting would produce similar differential effects in hapten-reactive Th1 and Th2 T cells in vivo. C3H/HeN mice were exposed to UVB (200 J/m2/day) through abdominal skin on days -4 through -1, followed by painting dinitrofluorobenzene (DNFB) on the irradiated skin on days -1 and 0. Induction of allergic contact sensitivity (CS) was assayed by ear swelling responses to DNFB and by the proliferative responses of draining lymph node cells (LNC) to DNBS. UVB-irradiated and hapten-painted mice (in comparison to a control panel of unirradiated and DNFB-painted mice) displayed suppressed ear swelling responses to DNFB and suppressed LNC proliferation to DNBS. However, LNC from either panel of mice proliferated well in response to exogenous interleukin 2 (IL-2). To examine effects on Th1 and Th2 cells, lymphokines were assayed from supernatants of DNBS-stimulated LNC. The Th1-associated lymphokines, interferon-gamma and IL-2, were the predominant cytokines detected in samples taken from unirradiated and DNFB-painted mice. Both of these cytokines were reduced markedly in samples from UVB-treated and DNFB-painted mice. Except for miniscule amounts of IL-10, no Th2-associated lymphokines were detected in LNC supernatants from either panel of mice. These results suggest that UVB-induced suppression of CS in vivo is associated with functional inactivation of hapten-reactive Th1 cells.

ICAM-1 and LFA-1 on mouse epidermal Langerhans cells and spleen dendritic cells identify disparate requirements for activation of KLH-specific CD4+ Th1 and Th2 clones.

Expression of the adhesion molecules ICAM-1 and LFA-1 (CD11a/CD18) on mouse epidermal Langerhans cells (LC) and on spleen dendritic cells (DC) from BALB/c mice was examined by staining with specific mAb and was evaluated by flow cytometry. LC were shown to express both ICAM-1 and LFA-1, whereas spleen DC expressed only LFA-1. The contribution of these adhesion molecules to LC- or DC-induced activation of keyhole limpet hemocyanin (KLH)-specific, Iad-restricted, Th1 or Th2 clones was investigated in mAb blocking studies. At optimal doses, anti-CD11a or anti-CD18 mAb completely inhibited Th1 proliferation induced by either LC or DC. Anti-ICAM-1 also abrogated Th1 proliferation induced by LC, but only moderately reduced Th1 proliferation induced by DC. Inhibition in these experiments was specific, since isotype-matched control Ab against other Ag constitutively expressed on LC (NLDC 145) or DC (33D1) had no effect on Th1 proliferation. In marked contradistinction, the capacity of LC to present KLH to our Th2 clones was resistant to treatment with the same mAb against ICAM-1, CD11a or CD18. We conclude that interactions between ICAM-1 and LFA-1 on epidermal LC and LFA-1 on spleen DC with their respective ligands on our Th1 clones are required for optimal presentation of protein Ag to Th1. Our results also indicate that neither ICAM-1 nor LFA-1 is required for the analogous activation of our Th2 clones by LC.

Distorted antigen-presenting function of Langerhans cells induced by tumor necrosis factor alpha via a mechanism that appears different from that induced by ultraviolet B radiation.

Tumor necrosis factor alpha (TNF alpha) has been shown to mimic 2 effects of ultraviolet B (UVB) radiation in mice: morphologic damage to epidermal Langerhans cells (LC) and the inability to mount a normal contact hypersensitivity (CH) response. Our previous studies have shown LC to be the target of the immune tolerance evoked by UVB radiation, both during induction of CH in vivo and during presentation of protein antigen to CD4+ Th1 cells (Th1) in vitro. To determine whether these influences of TNF alpha and of UVB radiation on LC are related, 2 sets of experiments were performed. We first examined the effect of recombinant TNF alpha on the capacity of epidermal cells enriched for LC (IEC) to present keyhole limpet hemocyanin (KLH) to KLH-specific and Iad-restricted Th1. Addition of TNF alpha to co-cultures of IEC and Th1 significantly reduced proliferation in a dose-dependent manner. This inhibition was specific since it was reversed by neutralizing Ab against TNF alpha. That TNF alpha blocked Th1 proliferation by acting directly on LC is supported by 2 findings: 1) selective treatment of IEC prior to co-culturing also led to failure to present KLH; and 2) TNF alpha did not reduce Th1 proliferation stimulated by phorbol myristate acetate plus ionomycin, or by IL-2. We next examined the capacity of anti-TNF alpha Ab to protect LC from loss of antigen-presenting cell (APC) function induced by a single dose of 200 J/m2 UVB. Anti-TNF alpha Ab tested over a broad dose range did not prevent or restore the ability of UVB-irradiated IEC to present KLH to Th1.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of inflammatory cytokines and phorbol esters on the adhesion of U937 cells, a human monocyte-like cell line, to endothelial cell monolayers and extracellular matrix proteins.

The accumulation of mononuclear phagocytes at sites of chronic inflammation is dependent on an increase in the rate of extravasation of blood-borne monocytes through the vascular endothelium into the connective tissue. Once the monocytes have emigrated into the connective tissue, they may differentiate into tissue macrophages, presumably following interactions with extracellular matrix proteins. To study these processes, we tested the effects of cytokines and phorbol esters on the adhesion of U937 cells, a human monocyte-like cell line, to cultured endothelial cells (EC) and to matrix proteins. In the absence of cytokines, very few of the U937 cells adhered to EC (5% or less in most experiments). When EC were pretreated for optimal periods of time (4-8 hr) with recombinant interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor-alpha (TNF alpha), or lymphotoxin (LT; also known as TNF-beta), 35-85% of the U937 cells were able to bind. Interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) did not stimulate U937-EC binding, even though IFN-gamma was shown to increase EC adhesiveness for T lymphocytes. Phorbol esters also greatly stimulated U937-EC adhesion but, in this case, the increase was due to an action on the U937 cells. A monoclonal antibody (MAb), 60.3, against the CD11/CD18 family of leukocyte adhesion molecules partially inhibited the adhesion of untreated and phorbol ester-treated U937 cells to noncytokine-treated EC. However, that MAb had no effect on U937 cell binding to TNF-alpha-treated EC. Thus U937 cells use both CD11/CD18-dependent and -independent mechanisms to adhere to EC. In the absence of stimulating agents, only a small proportion of the U937 cells (2-20%) adhered to fibronectin (FN), and almost none bound to either laminin (LN) or gelatin (denatured type I collagen). In the presence of phorbol esters, a much larger proportion of the U937 cells adhered to FN, with only slight increases in the proportion of cells which bound to LN or gelatin. Additional adhesion assays performed in the presence of a pentapeptide containing the amino acid sequence arg-gly-asp (RGD), which is part of one of the cell-binding domains of FN, demonstrated that the RGD-containing peptide almost totally blocked the phorbol ester-induced adhesion of U937 cells to FN. In contrast, the peptide had no inhibitory effect on the phorbol ester-induced binding of U937 cells to EC.

Ultraviolet B radiation converts Langerhans cells from immunogenic to tolerogenic antigen-presenting cells. Induction of specific clonal anergy in CD4+ T helper 1 cells.

We have recently demonstrated that a single dose (200 J/m2) of UVB radiation abrogates the capacity of mouse epidermal Langerhans cells (LC) or splenic adherent cells (SAC) to present keyhole limpet hemocyanin (KLH) to Ag-specific, MHC-restricted CD4+ Th1 cells. In the present study we determined whether such Th1 unresponsiveness represented long-lasting immunologic tolerance. To address this question, Th1 were preincubated with KLH-pulsed UVB-LC or UVB-SAC, then isolated and restimulated with unirradiated APC (LC or SAC) plus KLH or with exogenous rIL-2 in the absence of APC. Preincubation with KLH and UVB-LC or UVB-SAC rendered Th1 unresponsive to subsequent restimulation with APC and KLH. In addition, such Th1 were defective in their autocrine IL-2 production, but could respond normally to exogenous rIL-2, indicating that unresponsiveness was due to functional inactivation and not to cell death. Th1 unresponsiveness was Ag-specific, MHC-restricted, and long lasting (greater than 16 days). In addition, it appears that Th1 unresponsiveness is not due to the release of soluble suppressor factors from UVB-LC or UVB-SAC because supernatants from such cells had no effect on Th1 proliferation. Addition of unirradiated allogeneic SAC during preincubation prevented the induction of unresponsiveness by UVB-LC or UVB-SAC, suggesting that UVB interferes with the capacity of LC or SAC to deliver a costimulatory signal(s) that can be provided by allogeneic SAC. We conclude that UVB can convert LC or SAC from immunogenic to tolerogenic APC.

Endothelial cell activation induced by tumor necrosis factor and lymphotoxin.

Alterations in the morphology and histochemistry of vascular endothelial cells (EC) have been repeatedly observed at sites of chronic inflammation and immune reactions. These changes, which are most prominent in the EC postcapillary venules present in areas with large lymphocytic infiltrates, include the acquisition of a columnar or cuboidal morphology, the development of ribonuclease-sensitive metachromasia, and an increase in intracellular organelles. Thus, EC at sites of inflammation appear to be activated and to demonstrate increased metabolic activity. This study reports that both tumor necrosis factor-alpha (TNF) and lymphotoxin (LT) can activate cultured human umbilical vein EC, as measured by: 1) increased adhesiveness for lymphocytes, 2) increased cell metabolism, as measured by RNA and protein synthesis, and 3) increased cell volume. Although gamma interferon (IFN-gamma) and interleukin-1 (IL-1) have been shown previously to stimulate EC adhesiveness for lymphocytes, these two cytokines had only marginal effects on EC RNA and protein synthesis, and both caused a decrease in EC volume. These findings suggest that TNF and LT play a role in the type of activation of EC in vivo that leads to the development of tall endothelium and increased lymphocyte emigration.

Inhibition by IL-1 of endothelial cell activation induced by tumor necrosis factor or lymphotoxin.

Previous studies in this and other laboratories have demonstrated that IL-1, lymphotoxin (LT), and TNF rapidly stimulate a number of proinflammatory properties in cultured endothelial cells (EC) including cell-surface procoagulant activity and increased adhesivity for lymphocytes, monocytes, and polymorphonuclear leukocytes. In addition, we have demonstrated that LT and TNF, but not IL-1, stimulate increases in EC RNA synthesis, protein synthesis, and cellular volumes, changes which may correspond to the hypertrophy of EC seen at sites of inflammation in vivo. It is reported here that both human rIL-1 alpha and rIL-1 beta totally inhibit the increases in EC RNA synthesis, protein synthesis, and cell volumes induced by either TNF or LT. As little as 0.1 ng/ml of either IL-1 was sufficient to totally block the activation of EC induced by 100-fold higher concentrations (10 ng/ml) of either LT or TNF. The relevance of these findings to the regulation of inflammatory responses is discussed.

IgG and IgM rheumatoid factor synthesis in rheumatoid synovial membrane cell cultures.

The detection of rheumatoid factors (RFs) in synovial membranes and fluids of patients with rheumatoid arthritis (RA) has suggested that local production of these antiimmunoglobulin autoantibodies may have a role in the pathogenesis of synovitis. To quantitate RF synthesis in the rheumatoid synovial membrane, 12 synovial specimens were obtained from patients with seropositive RA, 5 from patients with seronegative RA, and 6 from patients with other arthritides. Single cell suspensions were cultured, and supernatants were analyzed for IgG, IgM, IgG-RF, and IgM-RF by solid-phase radioimmunoassays. IgM-RF was detected in all of the 12 seropositive culture supernatants, and IgG-RF was detected in 8 of the 12. Addition of cycloheximide to the cultures resulted in a greater than or equal to 40% decreased in the amount of IgM-RF. A similar decrease in IgG-RF occurred in the 4 cultures in which the largest amounts of IgG-RF were detected. IgM-RF synthesis represented 7.3 +/- 0.7% (mean +/- SEM) of the total IgM produced, and IgG-RF represented 2.6 +/- 1.1% (mean +/- SEM) of the IgG synthesized in those cultures with detectable IgG-RF. Cultures of synovial membrane cells (SMC) from seronegative RA patients or patients with other arthritides did not contain detectable amounts of IgM-RF or IgG-RF. Selective synthesis of RF by seropositive synovium was suggested by the observation that the fractions of synthesized IgM with RF activity were greater in the SMC supernatants than in paired sera in all cases, and the fractions of IgG with RF activity were greater in the SMC supernatants of 3 of the 4 cases in which substantial amounts of IgG-RF were produced. Comparison of the percentages of newly synthesized IgM with RF activity in paired cultures of SMC and peripheral blood mononuclear cells similarly indicated selective synthesis of IgM-RF by the synovium. These results demonstrate active and selective synthesis of both IgG-RF and IgM-RF by seropositive SMC. However, RFs account for only a minor fraction of the total Ig produced.