IP-10-induced recruitment of CXCR3 host T cells is required for small bowel allograft rejection

BACKGROUND & AIMS: Chemokines mediate cell trafficking in inflammatory states such as allograft rejection. However, their role in small-bowel allograft rejection has not been defined. The aim of this study was to examine the roles of type 1 helper T-cell chemokines in small-bowel allograft rejection. METHODS: Mucosal histology, chemokine messenger RNA (real-time polymerase chain reaction), and cell isolates were examined in small-bowel allografts and isografts. Interferon-gamma-inducible protein-10/CXC chemokine receptor (CXCR) 3 interactions were specifically evaluated by using allografts from interferon-gamma-inducible protein-10(-/-) donors and adoptive transfer of CXCR3(-/-) T cells into recombination activating gene (RAG)-1(-/-) recipients of small-bowel allografts. RESULTS: Type 1 helper T-cell cytokine (interferon-gamma) and chemokine (interferon-gamma-inducible protein-10, monokine induced by interferon-gamma, macrophage-inflammatory protein-1 alpha, and regulated on activation, normal T cells expressed and secreted) messenger RNA up-regulation was detected (real-time polymerase chain reaction) by postoperative day 3 in small-bowel allografts. Interferon-gamma-inducible protein-10(+/+) small-bowel allograft rejection was associated with a dramatic (>7-fold) increase in CXCR3(+) host T cells in the graft lamina propria. With interferon-gamma-inducible protein-10(-/-) small-bowel allografts, CXCR3(+) host T-cell infiltration of the graft lamina propria was markedly decreased and rejection was significantly delayed. Whereas adoptive transfer of wild-type B6 (CXCR3(+/+)) T cells into B6 (RAG-1(-/-)) recipients induced rapid rejection of CB6F1 small-bowel allografts, rejection was significantly delayed (29.2 +/- 8.7 days vs. 16.5 +/- 3.1 days; P < 0.01) in B6 (RAG-1(-/-)) mice reconstituted with T cells from B6 (CXCR3(-/-)) mice. CONCLUSIONS: Recruitment of CXCR3(+) host T cells by donor derived interferon-gamma-inducible protein-10 may precipitate small-bowel allograft rejection. These data highlight the importance of type 1 helper T cell-related chemokines in promoting cell-mediated rejection responses in small-bowel allografts and suggest that interferon-gamma-inducible protein-10 is an attractive therapeutic target for humanized monoclonal antibody strategies.     

The dietary antioxidants resveratrol and quercetin protect cells from exogenous pro-oxidative damage.

In the colorectal epithelium oxidative stress is observed endogenously in premalignant adenoma cells or induced by nutritional factors like fatty acid hydroperoxides (LOOH). Bioactive phenols like resveratrol and quercetin can quench reactive oxygen species and protect from pro-oxidative damage. Our study used colorectal adenoma and carcinoma cell lines to assess antioxidant protective effects of resveratrol and quercetin. It demonstrated that both compounds efficiently protect from oxidative stress induced by LOOH. Effective concentrations (10 microM resveratrol and 1 microM quercetin) can easily be reached in the intestinal lumen after consumption of plant foods or food supplements. Both compounds prevent LOOH-induced formation of intracellular H2O2, stimulation of cyclooxygenase-2 and vascular endothelial growth factor. For reduction of endogenous H2O2 levels in colorectal tumor cells higher antioxidant-concentrations are needed in all cell lines. Quercetin (10 microM) alone even increased H2O2 in LT97 adenoma cells and stimulated VEGF production. Resveratrol and quercetin also induced 10-30% and 40-60% cell loss respectively by apoptosis. In summary, this indicates that resveratrol and quercetin have little protective capacity in absence of exogenous stress. They are however highly efficient in protecting against nutrition induced oxidative stress damage suggesting that this constitutes the major part of their tumor protective activity.     

Study of the effects of cardiovascular drugs in heart cell cultures

Compounds that produce myocardial pathology in vivo can be separated into two main classes—those that are directly toxic to the myocardium and those that are considered to act by way of an indirect vascular or neurologically based mechanism. An in vitro model of myocardium without nervous or systemic influences can be used to differentiate between direct myocardial cytotoxic effects and indirect cardiac pathology arising in vivo from exaggerated vascular or neural pharmacological effects of a number of drugs. In this study direct-acting cardiotoxic compounds are distinguished from those causing cardiac pathology by indirect mechanisms by their different pattern of effects in chick embryonic myocardial myocyte reaggregates (MMRs) cultures. The toxicity of the direct-acting cardiotoxic drugs allylamine (positive control, 50 μ ) and doxorubicin were compared with digoxin and isoprenaline, which show both direct and indirect mechanisms in vivo, and the indirectly acting hydralazine and pinacidil. Changes in spontaneous beating activity (SBA), leakage of lactate dehydrogenase (LDH) and cell morphology by light and transmission electron microscopy were used to assess toxicity. The MMRs were cultured for up to 24 hr in a series of concentrations of the five compounds in the range 0.1 to 10,000 μ . Allylamine, doxorubicin, digoxin and, to a lesser extent, isoprenaline were highly toxic to the MMRs, as shown by alterations in SBA, LDH leakage and cellular morphology. In contrast, hydralazine showed a very mild degree of toxicity at the highest concentrations in the absence of LDH leakage; treatment with pinacidil did not show any evidence of morphological degeneration but did cause a dose-related inhibition of SBA. These results are consistent with the view that doxorubicin and digoxin are directly toxic to myocardial cells and also suggests that this is an important mechanism in vivo for isoprenaline. The absence of a significant degree of toxicity with hydralazine and pinacidil is consistent with an indirect toxic mechanism.     

Immuno-suppressive lymphocyte factors—III. Complete purification and partial characterization of human inhibitor of DNA synthesis

Inhibitor of DNA synthesis (IDS) is an immunoregulatory T lymphocyte factor first characterized in the rat. It appears to cause the non-specific immune suppression seen in certain tolerant disease states.We have produced IDS from human peripheral blood lymphocytes, purified it to homogeneity and partiallycharacterized it. Human IDS has an isoelectric point of 3.40 and a molecular weight of 20,000. However, in the supernatants containing IDS activity, it usually exists in an aggregated tetrameric form which is also biologically active. IDS is not cytotoxic and is glycoprotein in nature. The activity depends on an intact carbohydrate moiety. Human IDS is identical to rat IDS except for a slightly higher isoelectric point. The biologic role is undetermined at present, but might be similar to that of rat IDS.     

Human fetal brain antigen expression common to tumors of neuroectodermal tissue origin

The antigenic relationship between human tumors of neuroectodermal origin and fetal brain were further investigated by characterization of two hybridoma antibodies derived from a fusion of P3-NS1/1-Ag 4-1 (NSI) myeloma cells and splenocytes hyperimmunized to second trimester human fetal brain homogenate. Monoclonal antibodies (MAs) 1H8cl 2 and 1H8cl 3 were analyzed by cell surface radioimmunoassay (CS-RIA), quantitative absorption, indirect immunofluorescence, and peroxidase-antiperoxidase (PAP) immunohistology. MA 1H8cl 3 is the more broadly reactive, binding to 9/14 glioblastoma (GBM), 2/3 neuroblastoma, 1/2 melanoma, and 1 medulloblastoma cell line(s) by CS-RIA analysis, and to 12/15 GBM, fetal brain, spleen, and liver, and adult spleen by PAP analysis. MA 1H8cl 2 is more restricted, binding to 7/14 GBM, 2/3 neuroblastoma, 1 medulloblastoma, and 2/3 fetal skin fibroblast cell line(s) by CS-RIA, and to 9/15 GBM and fetal brain and spleen by PAP analysis. Control non-central nervous system tumors and normal adult tissue including brain, thymus, lymph node, liver, kidney, lung, skin, and pancreas, were unreactive with both 1H8cl 2 and 1H8cl 3 by CS-RIA, PAP, and absorption analysis. The data presented here establish the unique nature of the detected antigenic specificities as compared to previously described oncofetal and onconeural antigens, and define two immune reagents which are operationally specific for tumors of neuroectodermal origin within the adult central nervous system.     

Loss of receptor activity for mouse erythrocytes precedes tumour promoter-induced maturation of chronic lymphocytic leukaemia cells

The tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation in lymphocytes from patients with chronic lymphocytic leukaemia (CLL). The differentiation was detected by the appearance of cytoplasmic immunoglobulin (CIg) and a plasmacytoid morphology, and was accompanied by the loss of the ability of the CLL cells to rosette with mouse erythrocytes (M). Loss of M rosetting occurred rapidly (within 10 min), was not prevented by cycloheximide or actinomycin D and was elicited by low concentrations of TPA (ED₅₀ 8 × 10^?10 M). Inhibition of rosette formation was induced by other phorbol diester promoters, but not non-promoting derivatives.Loss of the capacity to form M rosettes occurs before events which are evidently related to genome activation.     

Screening for cytotoxicity in neuroblastoma cells. I. Dependence of growth inhibition on the presence of serum

The growth-inhibitory effects of a variety of potentially toxic compounds on neuroblastoma cells in defined, serum-free medium were compared with those in serum-containing medium. For 13 of 21 compounds tested, concentrations between 2 and 10(5) times higher were required for 50% inhibition of growth in serum-containing medium. The ranking of substances for their potency in inhibiting growth was thereby different in the two different culture conditions. The presence of bovine serum albumin (BSA) in the medium had similar effects as serum on the dose-response curves.     

Suppression of murine allogeneic cell interactions by sex hormones

Investigations have been carried out on the action of several steroid hormones on lymphocyte functions in inbred strains of mice. The recognitive, proliferative and effector phases of allogeneic cell interactions in vitro were assessed using a mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML). In MLR containing Balb/c (responder) and C57BL/6 (stimulator) splenocytes DNA synthesis was markedly reduced in the presence of progesterone, cortisol or estradiol. In CML, progesterone and estradiol (1–5 μg/ml) blocked in vitro generation of cytotoxic lymphocytes, while cultures with cortisol were partially inhibited. None of these hormones suppressed the cytotoxic activity of previously sensitized effector cells generated in vitro. Cultures containing testosterone expressed both normal DNA synthesis in MLR and cytotoxic activity in the CML test. These findings suggest a selective pattern of immunosuppression by sex hormones which may be important in preventing graft rejection or graft-versus-host interactions which may arise as a consequence of fetal engraftment during pregnancy.     

Diverse signalling by different chemokines through the chemokine receptor CCR5

We have investigated the signalling properties of the chemokine receptor, CCR5, using several assays for agonism: stimulation of changes in intracellular Ca(2+) or CCR5 internalisation in CHO cells expressing CCR5 or stimulation of [(35)S]GTPgammaS binding in membranes of CHO cells expressing CCR5. Four isoforms of the chemokine CCL3 with different amino termini (CCL3, CCL3(2-70), CCL3(5-70), CCL3L1) were tested in these assays in order to probe structure/activity relationships. Each isoform exhibited agonism. The pattern of agonism (potency, maximal effect) was different in the three assays, although the rank order was the same with CCL3L1 being the most potent and efficacious. The data show that the amino terminus of the chemokine is important for signalling. A proline at position 2 (CCL3L1) provides for high potency and efficacy but the isoform with a serine at position 2 (CCL3(2-70)) is as efficacious in some assays showing that the proline is not the only determinant of high efficacy. We also increased the sensitivity of CCR5 signalling by treating cells with sodium butyrate, thus increasing the receptor/G protein ratio. This allowed the detection of a change in intracellular Ca(2+) after treatment with CCL7 and Met-RANTES showing that these ligands possess measurable but low efficacy. This study therefore shows that sodium butyrate treatment increases the sensitivity of signalling assays and enables the detection of efficacy in ligands previously considered as antagonists. The use of different assay systems, therefore, provides different estimates of efficacy for some ligands at this receptor.