Transforming growth factor-beta1 mediates coexpression of the integrin subunit alphaE and the chymase mouse mast cell protease-1 during the early differentiation of bone marrow-derived mucosal mast cell homologues.

BACKGROUND: Mucosal mast cells (MMC) play a central role in gut hypersensitivities and inflammation. They are morphologically, biochemically and functionally distinct from their connective tissue counterparts. Massive hyperplasia of MMC occurs 7-10 days after intestinal infection with nematodes but it has never been possible to replicate this phenomenon in vitro. OBJECTIVE: (1) To determine whether mouse bone marrow-derived mast cells (mBMMC) grown in the presence of transforming growth factor (TGF)-beta1 could develop over the same time frame (7-10 days) as MMC in parasitized mice. (2) To compare the early expression of surface receptors (integrins alphaE and beta7, c-kit and FcepsilonR) with that of the MMC-specific granule chymase mouse mast cell protease-1 (mMCP-1). METHODS: Mouse bone marrow cells were cultured in the presence of IL-9, IL-3 and Stem Cell Factor (SCF) with or without TGF-beta1. mBMMC were quantified after toluidine blue or Leishmans' staining. Expression of MMC-specific mouse mast cell proteases was analysed by ELISA, immunohistochemistry and RT-PCR. Surface antigen expression was characterized by flow cytometry and confocal microscopy. RESULTS: TGF-beta1 promotes the development of abundant MMC-like mBMMC from bone marrow progenitor cells with kinetics, which closely parallel that seen in vivo. mRNA transcripts encoding mMCP-1 and -2 are readily detectable by day 4 ex vivo in cultures grown in the presence of TGF-beta1. Between 30 and 40% and 75-90% of the cells in these cultures on days 4 and 7, respectively, have typical mast cell morphology, are c-kit+, FcepsilonR+, integrin alphaEbeta7+, and express and secrete abundant mMCP-1. The integrin alphaE subunit is coexpressed with mMCP-1. CONCLUSION: The kinetics of mMCP-1+/alphaE+ mBMMC development, regulated by TGF-beta1, are consistent with that seen in vivo in the parasitized intestine. The normally down-regulatory functions of TGF-beta1 in haematopoiesis are superseded in this culture system by its ability to promote the early expression of alphaE and mMCP-1.

Isolation and characterization of two novel A20-like proteins.

The transcription factor nuclear factor kappa B (NF-kappa B) plays a pivotal role in inflammatory processes through induction of adhesion molecules and chemokines. The zinc finger molecule A20 is an important negative regulator of NF-kappa B. The mechanism utilized by A20 is not fully understood, but A20 has been shown to bind to tumour-necrosis-factor-receptor-associated factor (TRAF) molecules, which are necessary for pro-inflammatory cytokine signalling. We report two novel genes, Cezanne (cellular zinc finger anti-NF-kappa B) and TRABID (TRAF-binding domain), with sequence similarity to A20. Co-immunoprecipitation studies indicated that TRAF6 was able to interact with both Cezanne and TRABID. In contrast, reporter gene experiments revealed a specific ability of Cezanne to down-regulate NF-kappa B. It is likely, therefore, that Cezanne participates in the regulation of inflammatory processes.

Studies on transcriptional regulation of the mucosal T-cell integrin alphaEbeta7 (CD103).

Integrin alphaEbeta7 is expressed almost exclusively by mucosal T cells and mucosal dendritic antigen-presenting cells (APCs) and is thought to be induced locally by transforming growth factor-beta (TGF-beta). In mice, mRNA for the alphaE subunit was found to be abundant in mucosal T cells but absent from other tissues. Exposure of a T-cell line to TGF-beta strongly up-regulated alphaE mRNA levels within 30 min, and nuclear run-on experiments established that regulation occurred at the level of transcription. The organization of the human alphaE gene and a very closely linked novel gene, ELG, was determined. The alphaE promoter was tested in T cells and fibroblasts and functioned equally well in both cell types and did not confer TGF-beta responsiveness. Regions of the promoter providing enhancer activity and phorbol 12-myristate 13-acetate (PMA) responsiveness were identified by deletion studies. DNAse 1 hypersensitivity analysis of 36 kb of the alphaE gene revealed one hypersensitive site, found only in alphaE+ cells, located near the transcription start points. These results show that, unlike the situation with other integrins, lineage specificity and cytokine responsiveness of alphaE transcription are not conferred by the proximal promoter. Specificity may depend on distant control elements that have not yet been identified.

Interleukin-13 protects endothelial cells from apoptosis and activation: association with the protective genes A20 and A1.

BACKGROUND: Chronic rejection is the major obstacle to long-term survival of allografts and is associated with graft endothelial cell activation and apoptosis. Recent reports have found an association between graft survival, presence of Th2 cytokines, and expression by endothelial cells of cytoplasmic "protective" molecules that prevent apoptosis and down-regulate the inflammatory process. METHODS: Cultured human umbilical vein endothelial cells (HUVEC) were used. Apoptotic cells were detected by staining with FITC-annexinV followed by flow cytometry. Expression of vascular cell adhesion molecule-1, E-selectin, and intercellular adhesion molecule-1 were also measured by flow cytometry. Transcripts were detected by reverse transcription-PCR and quantitation was achieved by co-amplification of competing, internal standard RNA. RESULTS: We demonstrate that exposure of HUVEC to interleukin (IL)-13 for 72 hr afforded partial protection from apoptosis induced by tumor necrosis factor-alpha/cycloheximide or serum starvation. Pretreatment with IL-13 also modulated induction of E-selectin after acute exposure to tumor necrosis factor-alpha or IL-1alpha. Protection was associated with transcription of the genes A1 and A20. Prolonged treatment with IL-13 had minimal proinflammatory effects and did not induce expression of E-selectin or vascular cell adhesion molecule-1 or increase intercellular adhesion molecule-1 above basal levels. CONCLUSIONS: Our data provide a possible explanation for the observed association between Th2 cytokines and expression of protective genes in the endothelium of long-surviving allografts and xenografts.

Diverse patterns of expression of the 67-kD laminin receptor in human small intestinal mucosa: potential binding sites for prion proteins?

It has been shown that the 67-kD laminin receptor (LR) may function as a receptor for Sindbis and tick-born encephalitis viruses. Recent data indicate that the 37-kD precursor (LRP) for this molecule acts as a receptor for prion proteins (PrP), self-proteins implicated in the pathogenesis of transmissible spongiform encephalopathies including new variant Creutzfeldt-Jakob disease (nvCJD). Laminin and PrP share the same binding site on LRP, which is incorporated into the mature LR as a functional binding domain. To localize PrP binding sites potentially relevant to oral infection, the expression of the LR in human small intestinal mucosa was studied. Expression of the LR was determined by immunohistochemistry in duodenal and jejunal biopsies using a monoclonal antibody (MLuC5) which specifically recognizes the 67-kD LR. Biopsy material was obtained from 39 control patients, 15 patients with ulcerative colitis, 15 patients with Crohn's disease and uninvolved small bowel, and 28 patients with active coeliac disease. Two distinctive patterns of LR expression were found within each group of patients. One pattern was characterized by LR expression in the brush border and Golgi apparatus region of villus and crypt enterocytes. Paneth cell secretory granules were positive for LR in these samples. Brush border expression of LR was found in approximately 40% of samples, with the exception of Crohn's disease (6.7% of samples were positive). Another pattern of LR expression was characterized by positively stained endothelium, while the epithelium was generally negative (45 of 97). The use of two polyclonal antibodies which recognize both the LRP and the LR confirmed brush border and paranuclear expression of the LR, but also showed varying cytoplasmic and apical surface immunoreactivity in MLuC5-negative epithelium, reflecting the distribution of LRP as opposed to the mature receptor. In conclusion, expression of the LR in the brush border and in Paneth cell secretory granules suggests that this molecule might be involved in both secretory and endocytotic functions. The major implication of intestinal epithelial/brush border expression of the LR may be an increased susceptibility to oral infection with prion proteins.

Alpha-galactosyl-mediated activation of porcine endothelial cells: studies on CD31 and VE-cadherin in adhesion and signaling.

BACKGROUND: Ligation of alpha-galactosyl epitopes on endothelial cells by naturally occurring human antibodies causes hyperacute rejection in porcine-to-human xenotransplantation. The alpha-galactosyl-specific lectin Bandeiraea simplicifolia isolectin B4 (IB4) has been reported to trigger endothelial "gap" formation and tyrosine phosphorylation of an unidentified 130-kDa protein. We have studied two 130-kDa junctional adhesion molecules, CD31 and VE-cadherin, in porcine aortic endothelial cells (PAECs) during IB4-mediated activation. The cellular distribution of these molecules, their susceptibility to tyrosine phosphorylation, and their capacity to bind IB4 or natural human antibodies have been determined. METHODS: Porcine CD31 and VE-cadherin were cloned. Recombinant proteins and monoclonal antibodies were prepared. The distribution and phosphorylation of CD31 and VE-cadherin in confluent PAECs activated with IB4 or human serum were studied by confocal microscopy and Western blotting, respectively. RESULTS: IB4 caused rapid redistribution of CD31 and VE-cadherin away from cell junctions and tyrosine-phosphorylation of CD31 but not VE-cadherin. A monoclonal antibody to CD31 also triggered tyrosine phosphorylation of this molecule, but brief exposure of PAECs to normal human serum did not. Tyrosine-phosphorylated CD31 complexed with SHP2 and other unidentified phosphoproteins. Both IB4 and natural human antibodies bound to porcine CD31 but not to VE-cadherin. Cell adhesion tests showed that porcine and human CD31 are functionally incompatible. CONCLUSIONS: Endothelial cell retraction during IB4-mediated activation of PAECs is associated with rapid loss of CD31 and VE-cadherin from cell junctions. CD31 becomes strongly tyrosine-phosphorylated and forms a cell signaling complex, which may have a significant role in the response of the xenograft vascular endothelium.

Structure-function analysis of the integrin beta 7 subunit: identification of domains involved in adhesion to MAdCAM-1.

Beta 7 integrins serve special roles in mucosal immunity. Alpha 4 beta 7-mediated adhesion to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) directs lymphocyte homing to the gut, and alpha E beta 7 mediates binding of lymphocytes to E-cadherin on epithelial cells. Since alpha 4 beta 7 mediates adhesion to MAdCAM-1 but alpha 4 beta 1 does not, we used beta 7/beta 1 chimeras to directly assess the importance of specific regions of beta 7 in MAdCAM-1 binding. We found a region of beta 7 (residues 46-386) that accounts for specificity of alpha 4 beta 7 binding to MAdCAM-1. We also used human/mouse and human/rat chimeric beta 7 subunits to map epitopes recognized by fifteen anti-beta 7 mAbs. Six of seven Abs that block adhesion to MAdCAM-1 and E-cadherin (Fib 21, 22, 27, 30, 504; Act-1) mapped to amino acid residues 176-250. Residues 176-250 lie within the region of beta 7 that specifies MAdCAM-1 binding and also within a region that has a predicted structure homologous to the metal ion-dependent adhesion site (MIDAS) domains of the integrin subunits alpha L and alpha M. Three new Abs that recognize beta 7 in the presence of Mn2+, but not Ca2+, and promote adhesion to MAdCAM-1, mapped to amino acids 46-149. One blocking and five other Abs mapped to other regions (amino acids 387-725). We conclude that a MIDAS-like domain serves a critical role in beta 7 integrin-mediated adhesion.

Distribution of beta 7 integrins in human intestinal mucosa and organized gut-associated lymphoid tissue.

Two alternative integrins involved in mucosal homing (alpha 4 beta 7) or epithelial retention (alpha E beta 7) of lymphocytes were examined in the human gut. The distribution of the beta 7 subunit [monoclonal antibody (mAb) M301] was bimodal in that it was strongly expressed by alpha E beta 7 + cells but weakly by alpha 4 beta 7 + cells. More than 90% of intraepithelial lymphocytes (IEL), including the minor subsets of CD4+, T-cell receptor (TCR) gamma/delta +, and CD3- cells, expressed alpha E beta 7 as did most lamina propria CD8+ (88%) and a fraction (36%) of CD4+ lymphocytes. Conversely, B-lineage cells (CD19+) and macrophages (CD68+) were negative. In gut-associated lymphoid tissue (GALT: Peyer's patches and appendix) only a few (< 5%) cells were positive for alpha E beta 7 (confined to CD8+ lymphocytes and CD11c+ putative dendritic cells). A relatively small fraction of IEL (30-50%) expressed alpha 4 beta 7 (mAb Act-1), while most (70%) lamina propria T and B lymphocytes, blasts, plasma cells and macrophages were positive. In GALT, T lymphocytes expressed similar levels of alpha 4 beta 7 as in the lamina propria whereas relatively few B lymphocytes (< 50%) were positive. Isolated lamina propria CD8+, CD4+, CD19+, and CD38+ cells contained mRNA for alpha 4 and the former three subsets as well as appendix CD8+ cells also for beta 7 while only lamina propria CD8+ cells had mRNA for alpha E. Together, the results suggested that alpha E beta 7 and alpha 4 beta 7 are differentially regulated in inductive sites and effector sites of the human gut. Because lymphoid cells at both sites expressed mainly alpha 4 beta 7, this integrin may be a homing receptor on memory and effector cells bound for lamina propria as well as on naive lymphocytes extravasating in GALT. Conversely, because alpha E beta 7 was mainly expressed by CD8+ cells in epithelium and lamina propria, it was probably induced after extravasation, in agreement with the observation that IEL and a fraction of lamina propria T lymphocytes (mainly CD8+ cells) generally expressed higher levels of beta 7 than most CD4+ and B cells. Also a subset of putative dendritic cells located near the follicle-associated epithelium of GALT expressed alpha E beta 7, perhaps reflecting epithelial interaction during primary immune responses.

Recognition of E-cadherin on epithelial cells by the mucosal T cell integrin alpha M290 beta 7 (alpha E beta 7).

The integrin alpha M290 beta 7 on the surface of a T cell hybridoma, MTC-1, mediated adhesion of these cells to the mouse epithelial cell line CMT93. This interaction was critically dependent on the presence of divalent cations; Mn2+ strongly promoted adhesion, Ca2+ was ineffective and Mg2+ gave intermediate results. Antibodies to molecules on the surface of CMT93 cells were tested for inhibition of adhesion. One monoclonal antibody (mAb) against E-cadherin, ECCD-2, was found to have significant inhibitory activity. Other mAb to E-cadherin and antibodies to other molecules had no effect. To show that inhibition by ECCD-2 was specific for adhesion mediated by alpha M290 beta 7, MTC-1 cells were induced to adhere to CMT93 via the LFA-1/ICAM-1 pathway. For this purpose, the epithelial cells were treated with interferon-gamma and tumor necrosis factor-alpha to induce ICAM-1 expression and, in addition, alpha M290 beta 7 on MTC-1 cells was down-regulated by culturing the cells in the absence of transforming growth factor beta. Under these circumstances adhesion of MTC-1 cells to CMT93 was inhibited by an antibody to LFA-1 but not by ECCD-2. Transfection of mouse L cells with cDNA for mouse E-cadherin enabled MTC-1 cells to adhere to them through the alpha M290 beta 7 integrin; this interaction was inhibited both by ECCD-2 and by blocking antibody against the integrin. These data strongly suggest that E-cadherin is a principal ligand for alpha M290 beta 7.

Distinct but overlapping epitopes are involved in alpha 4 beta 7-mediated adhesion to vascular cell adhesion molecule-1, mucosal addressin-1, fibronectin, and lymphocyte aggregation.

The mouse CD8+ T cell lymphoma TK1 expresses high levels of alpha 4 beta 7 integrin, which it can use to interact with multiple ligands including mucosal addressin-1 (MAdCAM-1), VCAM-1, and fibronectin. In addition, alpha 4 beta 7 can support TK1 cell aggregation. Here we have produced and characterized a panel of mAbs against alpha 4 beta 7 to define antigenic and functional epitopes associated with its distinct functions. One mAb, DATK32, is unique in recognizing an epitope specific to the alpha 4 beta 7 heterodimer. Furthermore, DATK32 induces TK1 cell aggregation yet inhibits TK1 cell adhesion to MAdCAM-1, VCAM-1, and fibronectin. Considered as a whole, the panel of anti-alpha 4 beta 7 mAbs studied define unique patterns of inhibition for alpha 4 beta 7 binding to each of its defined molecular ligands. We conclude that alpha 4 beta 7 interactions with MAdCAM-1, VCAM-1, and fibronectin can be modulated by Ab binding to distinct epitopes and thus probably involve functionally separable, although physically overlapping binding sites on this multifunctional integrin. These findings are consistent with the general observation that integrins use distinct, potentially differentially regulated interaction sites for adhesion to multiple ligands. Extension of these concepts to alpha 4 beta 7 has important considerations for understanding the roles of this integrin in lymphocyte homing to mucosal sites and in cell-cell interactions during the immune response.

Distinct binding specificities of integrins alpha 4 beta 7 (LPAM-1), alpha 4 beta 1 (VLA-4), and alpha IEL beta 7.

Integrin receptors are important for regulating lymphocyte recirculation and recruitment to sites of inflammation. Transfectants of the B cell lymphoma 38C13 were generated that differ exclusively in the expression of integrin beta 1 or beta 7 subunits allowing for a functional comparison of lymphocyte Peyer's patch HEV adhesion molecule 1 (LPAM-1) (alpha 4 beta 7) and very late antigen 4 (VLA-4) (alpha 4 beta 1) in an identical cellular environment. Whereas 38-beta 7 transfectants bound to purified and cellular mucosal addressin cell adhesion molecule (MAdCAM-1), unstimulated 38-beta 1 cells failed to bind MAdCAM-1. Treatment of 38-beta 1 cells with Mn2+ but not with PMA induced low level binding to MAdCAM-1. MAdCAM-1 adhesion of 38-beta 7 cells was constitutive and not enhanced by Mn2+ treatment. Similarly, MAdCAM-1-dependent adhesion to mucosal high endothelial venules was shown for 38-beta 7 but not for 38-beta 1 cells. The results therefore establish the LPAM-1-MAdCAM-1 interaction as the functionally dominant adhesion pathway for regulating lymphocyte homing to mucosal sites. Nonetheless, the activated VLA-4 on some lymphocytes may be involved in MAdCAM-1 recognition or promote binding to MAdCAM-1 in other tissues. By contrast, 38-beta 7 and 38-beta 1 transfectants did not differ in their binding capacity for vascular cell adhesion molecule 1 (VCAM-1) or fibronectin and LPAM-1 did not display any preference for interacting with either MAdCAM-1 or VCAM-1. LPAM-1 may therefore contribute significantly to cellular functions previously attributed to VLA-4. Interestingly, functional analysis of the intraepithelial lymphocyte integrin alpha IEL beta 7 which is structurally related to LPAM-1 did not reveal detectable binding activity for MAdCAM-1, VCAM-1, or fibronectin.

Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1.

The mucosal vascular addressin, MAdCAM-1, is an immunoglobulin superfamily adhesion molecule for lymphocytes that is expressed by mucosal venules and helps direct lymphocyte traffic into Peyer's patches (PP) and the intestinal lamina propria. We demonstrate that the lymphocyte integrin alpha 4 beta 7, also implicated in homing to PP, is a receptor for MAdCAM-1. Certain antibodies to alpha 4 and beta 7 integrin chains but not to the beta 2 integrin LFA-1 inhibit lymphocyte binding to purified MAdCAM-1 and to MAdCAM-1 transfectants. Lymph node lymphocytes, alpha 4 beta 7+ TK1 lymphoma cells, and a beta 7-transfected variant of an alpha 4+ B cell line, 38C13, bind constitutively to MAdCAM-1. Binding is enhanced by Mn(++)-induced integrin activation. The related integrin alpha 4 beta 1 supports efficient binding to VCAM-1 but not to MAdCAM-1, even after integrin activation, indicating that MAdCAM-1 is a preferential ligand for alpha 4 beta 7. Alpha 4 beta 7 can also bind VCAM-1, but this requires greater integrin activation than binding to MAdCAM-1. The findings imply a selective role for the interaction of alpha 4 beta 7 and MAdCAM-1 lymphocyte in homing to mucosal sites.

The mucosal T cell integrin alpha M290 beta 7 recognizes a ligand on mucosal epithelial cell lines.

The integrin alpha M290 beta 7 is expressed at high levels on mucosal T cells, particularly on those within the epithelium of the gut. We now report that a mouse T cell hybridoma, MTC-1, with similar surface expression of this molecule, adhered strongly to cells of the mouse rectal carcinoma line CMT93 and that adhesion was blocked completely by the monoclonal antibody (mAb) M290. Other mAb to the alpha M290 or beta 7 subunits had little or no inhibitory effect. M290 also inhibited adhesion of the hybridoma to cells of the mouse lung carcinomas CTM64/61 and KLN205 but had little or no effect on adhesion to seven other mouse epithelial cell lines or to the human colon carcinoma line, HT29. Intraepithelial lymphocytes (IEL) isolated from the small intestine of BALB/c mice displayed potent T cell receptor-dependent cytotoxic effector function against CMT93 in the presence of low concentrations of Phytolacca americana lectin. This cytotoxic activity also was inhibited by the M290 mAb. Treatment of CMT93 cells with tumor necrosis factor-alpha and interferon-gamma induced expression de novo of ICAM-1 and reduced the inhibitory effect of M290 in tests both for adhesion and cytotoxicity. In further experiments cytotoxic activity of IEL against the mastocytoma P815 was investigated. This target cell was considered not to possess a ligand for the integrin. In this case cytotoxic effector function was triggered by anti-CD3 mAb and, in contrast to results with CMT93, target cell lysis was increased in the presence of M290 and other antibodies to the integrin, suggesting a co-stimulatory effect. These results show that alpha M290 beta 7 recognizes a ligand on the surface of certain epithelial cell lines. Further, they provide the first clear indication that this integrin may play an important role in functional interactions between T cells and the mucosal epithelium.

A new antigenic determinant on intra-epithelial lymphocytes and its association with CD45.

Rat monoclonal antibodies were prepared against intra-epithelial lymphocytes (IEL) isolated from the gut of Balb/c mice and screened for selective reactivity with mucosal lymphocytes. One antibody, M371, identified a new surface antigen on 30-40% of IEL. It bound to very few, if any, lymphocytes within the lamina propria and to none in other lymphoid tissues; neither did it stain lymph node lymphocytes that had been stimulated in culture with mitogens or alloantigens. The data suggest that M371 identifies a sessile population of IEL and that expression of the antigen is induced locally in the epithelium. In addition to IEL, M371 bound to some goblet cells in the mid and distal small gut but not in the proximal region. Double-staining experiments showed that M371 was highly specific for IEL with the phenotype Lyt-2+, Lyt-3-, Thy-1-, CD3+ and stained a majority of cells in this subpopulation. M371 precipitated a surface molecule approximately 275,000 MW in size, which was also precipitated by antibodies to CD45. Treatment of fixed IEL with sodium periodate prevented staining by M371, suggesting involvement of carbohydrate in the epitope. The specificity of M371 was shown to differ from that of the antibodies CT1 and CT2, which identify a carbohydrate determinant of CD45 expressed on cytotoxic lymphocytes and IEL. The possibility that the gut epithelium provides an environment for the functional differentiation of thymus-independent mucosal T cells is discussed.

Studies on the specificity of antibodies to ovalbumin in normal human serum: technical considerations in the use of ELISA methods.

As part of a study designed to reveal information about molecular features of allergenic food proteins after absorption from the gut the specificity of antibodies in normal human serum to hen's egg ovalbumin was investigated using ELISA techniques. Preliminary investigations with monoclonal antibodies and hyperimmune rabbit antiserum specific for ovalbumin in its native and denatured form established that the molecule underwent an extensive conformational change on adsorption to polyvinyl chloride microtitre plates. The native conformation could be retained by using antibodies to couple the protein to the surface. Serum from 90% of healthy adult human donors contained IgG antibodies to ovalbumin. In nearly all cases the antibodies were specific predominantly for the native molecule and could not be absorbed with denatured ovalbumin or peptides prepared from it by cleavage with cyanogen bromide or trypsin. Antibodies to denatured ovalbumin were detected in most sera but at very low levels and were preferentially absorbed by the homologous antigen; peptides and native ovalbumin showing variable absorptive activity. Thus, although ovalbumin is ingested largely in a denatured form, the serum antibody response is stimulated mainly by topographic epitopes of the native molecule.

Monoclonal antibodies to the two most basic papaya proteinases.

The proteinases from Carica papaya include papain, isoenzymes of chymopapain and two proteinases A and B distinguished by their unusually high pI. The identity of one of the most basic proteinases has been questioned. The present report describes the preparation and characterisation of two monoclonal antibodies that react specifically with papaya proteinases A and B respectively and a third that identifies a common structural feature found in papain and proteinase A.

Induction of class II MHC antigens in cultured epithelial cells from rat gut.

A long-lived culture line of epithelial cells producing low levels of secretory component was derived from rat gut. Treatment of the cells with culture supernatants from rat spleen cells that had been stimulated with concanavalin A induced expression of class II antigens of the major histocompatibility complex and inhibited DNA synthesis.

Specific unresponsiveness to skin allografts in mice. IV. Immunological reactivity of mice treated with liver extracts, Bordetella pertussis, and antilymphocyte serum.

The strain-specific unresponsiveness to H-2 incompatible skin allografts induced by treatment of adult mice with single inoculations of donor strain liver extract and Bordetella pertussis vaccine, as well as three doses of antilymphocyte serum, has been investigated by several in vivo and in vitro methods, with a view to elucidating it mechanism. Lymphoid cells from mice with long surviving skin grafts were found to be reactive in graft-versus-host assays (as measured by splenomegaly or popliteal lymph node enlargement), and mixed lymphocyte culture tests gave positive results. Attempts to cause lethal runting of F1 hybrid mice injected at birth with spleen cells from unresponsive mice gave variable results. However, the injection of F1 hybrid cells into the footpads of unresponsive animals failed to elicit a significant host-versus-graft response. Although lymphoid cells from unresponsive animals did not include detectable numbers of cytotoxic cells, such cells could be generated by previous in vitro mixed lymphocyte culture stimulation or, to some degree, by the injection of the animals with F1 hybrid cells. Attempts to prevent mixed lymphocyte culture stimulation or cytotoxicity with serum from unresponsive mice failed at the serum concentrations used. The data indicate that long-term unresponsiveness in this system is maintained by the production in the hosts of factors that interfere with the cell-mediated response.