Screening of a pig cDNA expression library with human serum in order to identify new xenoantigens.
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Animals↗
Biomedical subjects
I Anegon
Publications and source records attributed to I Anegon.
Protection of rat endothelial cells from primate complement-mediated lysis by expression of human CD59 and/or decay-accelerating factor.
The present study analyzed the ability of human decay-accelerating factor (DAF) and CD59 to protect rat endothelial cell (EC) clones from human and primate complement-mediated lysis. By flow cytometry and Scatchard analysis, we show that human DAF and/or CD59 cDNAs under the transcriptional control of elongation factor 1-alpha promoter were expressed at levels similar to or higher than that of a human EC line. Human DAF and CD59 were released from the surface of transfected rat cells by phosphatidylinositol phospholipase C, demonstrating that the two molecules were linked to the cell membrane by means of a glycolipid anchor. The functional activity of the two human C regulatory proteins expressed on rat EC lines was studied using an in vitro assay of C-dependent cytotoxic in which rat EC were incubated with human or nonhuman primate sera as sources of xenogeneic natural antibodies and C. We demonstrate that human and monkey xenogeneic natural antibodies bind to rat cells and induce lysis by a C-dependent mechanism involving mainly the C direct activation pathway. Our data indicate that human DAF and CD59, expressed either alone or in combination, abrogated all EC cytotoxicity, even in the presence of 50% human serum. This protective phenotype was correlated with decreased membrane attack complex (CD59 and/or DAF transfectants) and C3 deposition (DAF transfectants) on EC surface. Antibodies against the transfected molecules abolished the protection against C-mediated lysis.
Animals↗
Rat interleukin-2 immunoglobulin M fusion proteins are cytotoxic in vitro for cells expressing the IL-2 receptor and can abolish cell-mediated immunity in vivo.
A hybrid cDNA coding for a fusion protein between rat interleukin 2 (IL-2) and a truncated heavy chain from rat immunoglobulin M (IgM) was constructed. The rat IL-2 and rat IgM CH2-3-4 hybrid gene was subcloned into a vector (PKCR6) for expression of the fusion molecule in Chinese hamster ovary (CHO) cells. Cells transfected with the hybrid cDNA secrete multimeric forms of the fusion protein (IL-2-Mu). Size analysis of the construct revealed that the majority (95%) of the secreted proteins have a high mw (> 500 kDa). The IL-2-Mu construct bind specifically to cells bearing the IL-2 receptors (IL-2R) with a binding affinity around 5 nM. The specific binding to IL-2R leads to T cell proliferation or, if rabbit complement is added, to T cell lysis. Multimeric forms (> 500 kDa) of the fusion protein mediate complement-dependent lysis but trigger only weak proliferation when compared with the low-mw forms (< 500 kDa). In contrast, the latter only efficiently mediate T cell proliferation without inducing complement-dependent lysis. After intravenous administration of CHO supernatant containing IL-2-Mu, or purified IL-2-Mu proteins into rats, the fusion proteins disappeared from the circulation with a t1/2 of 1 hr. The circulating IL-2-Mu constructs in the rat serum retained their capacity to induce complement-dependent lysis of IL-2R-bearing T cells in vitro. Furthermore, the IL-2-Mu construct was able to suppress the delayed-type hypersensitivity (DTH) reaction (an IL-2R, T helper cell-dependent event) in mice. A weak immune response (antirat IL-2-Mu antibodies) was observed when rats received multiple daily injections of the construct.
Animals↗
Human natural antibodies to porcine platelets.
The specificity of human natural antibodies directed against blood cells from pigs was investigated by ELISA and immunoblotting. Both IgG and IgM were identified as xenoantibodies reacting with pig platelets adsorbed to microplates. The antibodies could be absorbed on platelets as well as on RBC, suggesting that the corresponding antigens are expressed on the surface of a variety of cells. Galactose (20 mM) and melibiose (10 mM) partially inhibited (approximately 50%) the binding of antibodies to platelets, whereas lactose and cellobiose (300 mM) did not. On immunoblots, platelet glycoproteins of 115, 125, 135, 180, and 210 kDa were specifically revealed with human sera diluted 1/20. In contrast with the results obtained by ELISA, xenoantibodies reactive with blotted glycoproteins were of the IgM class and the binding was not significantly inhibited by galactose or melibiose. "Anti-Gal" antibodies, purified from human sera by affinity chromatography on a melibiose-Sepharose immunoabsorbent, represented only a minor portion of the antibodies reactive with porcine platelets. Purified anti-Gal antibodies bound to the 115- and 135-kDa components, whereas the antibodies in the nonretained fraction revealed the 125-kDa molecule. As deduced from these data, human serum contains natural antibodies of both IgG and IgM classes directed to several porcine antigens. Gal-reactive structures were identified on the 115- and 135-kDa platelet glycoproteins, which might be homologous to their counterpart on endothelial cells. Also, the present work suggests that a majority of the natural antibodies reacted with other unidentified structures.
Animals↗
Decreased anti-donor major histocompatibility complex class I and increased class II alloantibody response in allograft tolerance in adult rats.
Permanent tolerance to allografts can be induced in adult rats by donor-specific transfusions (DST) prior to transplantation. We have previously reported, in a model of heart allograft, the presence of a heavy leukocyte infiltrate, in the allograft which displayed a strong allospecific cytotoxicity when tested in vitro against donor cells, and a strong accumulation of mRNA for granzyme A and perforin in vivo. In contrast, there was a major decrease in the accumulation of mRNA for interleukin-2 and interferon-gamma. These results suggested that the DST-induced tolerance was associated with a decrease in type-1 T helper (Th1) cell function. The major role of preformed antibodies in xeno and allorejection is clearly established. Nevertheless, the consequences of alloantibody production in acute rejection and tolerance induction remains to be elucidated. We here analyze the alloantibody response in rejecting and DST-treated recipients. We show that, after transplantation, tolerant recipients, in contrast to rejecting ones, mount a low IgM alloresponse that switches to low IgG production. Detailed analysis of IgG alloantibodies in DST-treated recipients revealed that their production decrease was not equally distributed. Whereas rejecting animals mounted a strong anti-class I and II IgG alloantibody response, DST-treated recipients produced anti-class II and low titers of anti-class I IgG alloantibodies. Furthermore, among IgG subclasses, tolerant recipients predominantly produced IgG2a, a profile which, in the rat, is compatible with a Th2-controlled response. Finally, the passive transfer of immune serum from rejecting animals to DST-treated recipients could abrogate the tolerance. We suggest that the absence of anti-class I alloantibodies combined with preserved and/or increased anti-class II production plays a major role in graft tolerance in this model. These results reinforced the role of alloantibodies in rejection and in induction of tolerance.
Animals↗
Presence of leukaemia inhibitory factor and interleukin 6 in porcine uterine secretions prior to conceptus attachment.
Leukaemia inhibitory factor (LIF) plays an important role in embryo development and implantation. We detected peak LIF activity in porcine uterine luminal fluids (ULF) at day 12 of gestation and during day 7 and 13 of the oestrous cycle. A radio-receptor competition assay showed the presence of a molecule in ULF specifically binding to human LIF receptor (LIF-R). LIF activity was partially neutralized by anti-human LIF antibody. Interleukin-6 (IL-6) activity was detected in ULF throughout the oestrous cycle and pre-implantation period. An anti-murine alpha chain (gp80) of IL-6 receptor (IL-6R) specifically neutralized this activity. LIF and IL-6 mRNA were only detected in day 11 endometrium. The presence of LIF or IL-6 in the uterine cavity has not been previously reported. Our results extend LIF production by endometrium during the oestrous cycle and pre-implantation period to another mammalian species other than mouse.
Animals↗
Specificity of natural antibodies directed against cells from pigs.
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ABO Blood-Group System↗
Similar levels of granzyme A and perforin mRNA expression in rejected and tolerated heart allografts in donor-specific tolerance in rats.
Congenic LEW.1W (RT1u) heart grafts in LEW.1A (RT1u) recipient rats are rejected within 15 +/- 6 days. Tolerance (> 100 days) can be induced by pretransplant donor-specific blood transfusions. In both cases, the graft is heavily infiltrated by recipient cells, and class I and class II molecules of the major histocompatibility complex are strongly expressed. Moreover, T lymphocytes extracted from both tolerated and rejected grafts are similarly cytotoxic in vitro against donor cells. However, it cannot be excluded that this cytotoxicity does not operate in vivo. To answer this important question, we have studied the expression of granzyme A and perforin mRNA expression, in situ, by Northern blotting. Our data show that the two corresponding mRNAs accumulate with the same kinetic and at the same level in rejected and tolerated grafts. These results strongly suggest that infiltrating cells are cytotoxic in vivo and that a "cellular rejection" does occur in the "tolerated" grafts. In addition, these findings show that cytotoxic T lymphocytes are not sufficient to impair graft survival and lead to a revaluation of the relevance, in general, terms of granzyme and perforin expression as a correlate of rejection.
Animals↗
High and low affinity receptors for human interleukin for DA cells/leukemia inhibitory factor on human cells. Molecular characterization and cellular distribution.
Radioiodinated recombinant human interleukin DA (HILDA)/leukemia inhibitory factor (LIF) purified from conditioned medium of Chinese hamster ovary transfected cells enabled the identification of specific receptor sites on a variety of human cell types. Using low concentrations (up to 500 pM) of the ligand iodinated at a high specific radioactivity, high affinity receptors (equilibrium dissociation constant Kd in the range of 30-100 pM) were first demonstrated. They were expressed at low levels by human peripheral blood monocytes but not by lymphocytes, NK cells, granulocytes, and platelets. The myelomonocytic cell line THP1 as well as the T lymphoma cell line HSB2 and the lymphoblastoid B cell line DAB were also receptor-negative. In contrast, most of the non-lymphoid tumoral cell lines tested, including melanomas, neuroblastomas, and carcinomas, expressed high affinity HILDA/LIF receptors at variable levels (Bmax from 20 to 600 sites/cell). The kinetics of HILDA/LIF high affinity binding to the choriocarcinoma JAR cell line were characterized at 4 degrees C with association and dissociation rate constants of k1 = 2.2 10(9) M-1 min-1 and k-1 = 0.0084 min-1, respectively, corresponding to a steady-state dissociation constant k1/k-1 = 3.8 pM. The subsequent use of higher concentrations of HILDA/LIF labeled at a lower specific radioactivity enabled the identification of a low affinity component on several cell lines (Kd in the range of 1-4 nM; Bmax from 1,000 to 5,000 sites/cell). On JAR cells, this low affinity component was characterized by association and dissociation rate constants at 4 degrees C of k1 = 7.3 10(7) M-1 min-1 and k-1 = 0.19 min-1, respectively (k-1/k1 = 2.6 nM). Affinity cross-linking of HILDA/LIF to JAR cells showed two cross-linked species under both reducing and nonreducing conditions corresponding to receptor species of 120 and 250 kDa, respectively. Whereas both bands had similar intensities under high affinity conditions, the higher band predominated under low affinity conditions. Our data suggest that the 250-kDa chain could constitute the low affinity binding component whereas the association of both 250- and 120-Da subunits would form the high affinity structure.
Animals↗
HILDA/LIF is present in the urine of rejecting kidney graft recipients.
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Graft Rejection↗
Regulation of HILDA/LIF gene expression in activated human monocytic cells.
We have previously shown that human monocytes express HILDA/LIF gene and its protein product upon stimulation. In the present study we have investigated some of the possible mechanisms involved in the regulation of HILDA/LIF gene expression in activated human monocytes and compared them to those of granulocyte colony-stimulating factor (G-CSF) and IL-1 beta gene expression. In the absence of added stimuli HILDA/LIF mRNA was barely detectable in isolated monocytes. HILDA/LIF mRNA accumulation was weakly induced by stimuli such as LPS or phorbol ester. However, a synergy was observed when each stimulus was used in combination with 1,25-dihydroxyvitamin D3. Nuclear run-on analysis did not detect an increase in HILDA/LIF gene transcription upon stimulation with LPS or phorbol ester in combination with 1,25-dihydroxyvitamin D3. HILDA/LIF mRNA half-life showed an increase when phorbol ester and 1,25-dihydroxyvitamin D3 were used in combination over that obtained for each stimuli alone. HILDA/LIF mRNA expression was largely inhibited when monocytes were stimulated in the presence of cycloheximide (CHX) added either at the onset or 4 h after the beginning of the stimulation period. When CHX was added at later time points, 2 h before cell harvesting, HILDA/LIF mRNA levels were superinduced when compared with those in cells stimulated in the absence of CHX. This superinduction can be at least partially explained by post-transcriptional mechanisms, since HILDA/LIF mRNA half-life in CHX-superinduced cells was significantly increased when compared with values on stimulated CHX-untreated cells. In contrast to HILDA/LIF, G-CSF and IL-1 beta mRNA accumulation showed a different response pattern to the same stimuli, and a augmentation of their gene transcription was detected by the run-on technique in activated monocytic cells when compared with controls. Furthermore, G-CSF and Il-1 beta mRNA levels were superinduced in monocytes stimulated in the presence of CHX from the onset of the stimulation period. These studies indicate that, when compared to G-CSF and IL-1 beta, the expression of HILDA/LIF gene is under the control of relatively specific regulatory mechanisms. HILDA/LIF gene expression is controlled by newly synthesized proteins acting at early and late time points of the stimulation period and with opposite effects on HILDA/LIF mRNA levels. Finally, HILDA/LIF mRNA levels are regulated by post-transcriptional mechanisms of mRNA stabilization probably controlled through labile newly synthesized proteins.
Blotting, Northern↗
Control of HILDA/LIF gene expression in activated human monocytes.
In the present study we have investigated some of the mechanisms involved in HILDA/LIF gene expression in activated human monocytes and compared them to those of G-CSF gene expression, another monocyte-derived cytokine. In the absence of added stimuli, HILDA/LIF mRNA was barely detectable in monocytes. HILDA/LIF mRNA accumulation was weakly induced by stimuli such as LPS or phorbol ester alone, and in a synergistic manner when they were used in combination with 1,25-dihydroxyvitamin D3. Nuclear run-on transcription analysis in U937 cells did not detect an increase of HILDA/LIF gene transcription upon phorbol ester and 1,25-dihydroxyvitamin D3 stimulation. Posttranscriptional control of HILDLA/LIF mRNA levels by an increase in mRNA half-life was demonstrated in the synergy between phorbol ester and 1,25-dihydroxyvitamin D3 and in the superinduction of HILDA/LIF transcript accumulation when CHX was added to stimulated cells shortly before cell harvesting. HILDA/LIF mRNA expression was largely inhibited when U937 cells were treated with CHX either at the onset or 4 h after the beginning of the stimulation period. When CHX was added 2 h before cell harvesting, a superinduction of mRNA accumulation was obtained. G-CSF mRNA accumulation showed a different pattern of response to the same stimuli, in particular a higher rate of response to LPS. In contrast to HILDA/LIF, an augmentation of G-CSF gene transcription was detected in activated monocytic cells when compared to controls. These studies indicate that HILDA/LIF gene expression by phorbol ester- and 1,25-dihydroxyvitamin D3-treated human monocytes has a relatively specific regulation, as compared to G-CSF gene expression, and that it is largely dependent on posttranscriptional mechanisms probably acting through labile, newly synthesized proteins.
Calcitriol↗
Production of human interleukin for DA cells (HILDA)/leukemia inhibitory factor (LIF) by activated monocytes.
We demonstrate mRNA accumulation and production of HILDA/LIF by human activated Mo, monocyte-derived macrophages and myelomonocytic cell lines. Among the various stimuli tested, the synergistic combination of phorbol diester and VD3 was the most potent inducer of HILDA/LIF gene expression. The kinetics of mRNA accumulation on activated Mo showed a stimulation peak at 24 hr which declined thereafter. HILDA/LIF activity in culture supernatants was detected at 24 hr and reached a plateau at 72 hr of culture. In contrast to Mo, PBL did not accumulate HILDA/LIF mRNA upon culture with PDBu and VD3, whereas PHA and the combination of PDBu and A23187 induced HILDA/LIF mRNA accumulation and secretion in the culture supernatant. To exclude the possibility that HILDA/LIF was produced by contaminating PBL, highly enriched Mo preparations were used, which were devoid of T cells as assessed by the absence of TCR-beta chain mRNA transcripts. HILDA/LIF production by monocytic cells was further documented by the capacity of stimulated U937 cell conditioned medium to compete with 125I-labeled nHILDA/LIF for binding to its receptor on murine M1 cells. Under the synergistic effect of PDBu and VD3 stimulation, Mo-derived macrophages as well as HL-60 and U937 cell lines accumulated HILDA/LIF mRNA and produced this cytokine with identical kinetics as for Mo. Finally, we show that HILDA/LIF mRNA accumulation in U937 cells upon stimulation with PDBu, or the combination of PDBu and VD3, was inhibited in the presence of the protein synthesis inhibitor CHX. These results document for the first time that human Mo, when stimulated appropriately in vitro, can express the HILDA/LIF gene and its product, and that intermediate proteins must be newly synthesized in this process.
Cell Differentiation↗
Interleukin 2 receptor in rat allograft rejection.
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Animals↗
Interleukin 2 receptor in rat heart allograft rejection.
Soluble interleukin 2 receptors (S-R-IL-2) of truncated Tac chain, produced in vitro during T lymphocyte activation, may represent an in vivo marker of an alloimmune reaction. We analyzed serum S-R-IL-2 production during acute heart allograft rejection and compared soluble and membranous Tac chain (blood lymphocytes and graft invading cells) regulation during rejection. Serum S-R-IL-2 was tested in an immunoradiometric assay, with a combination of two mouse IgG1 anti-IL2-R mAbs (ART18 and OX39). Membranous Tac chain was analyzed by immunochemistry in graft tissue, and by immunofluorescence on blood and spleen leukocytes. Four experimental groups were used: untreated allogeneic, untreated syngeneic, CsA-treated (10 mg/kg/day for 15 days) allogeneic and CsA-treated syngeneic graft recipients. In the untreated allogeneic group, S-R-IL-2, tested every day until rejection (9.14 +/- 1.6 days), increased as early as day 3 after transplantation, peaked at day 6, and plateaued thereafter. The allograft was infiltrated at day 5 by Tac chain-positive cells (10% of OX1 cells and 84% of OX19 cells). A small percentage of mononucleated cells was labeled in blood, but not in spleen, by ART18 and OX39 at day 7 only. In contrast, in untreated syngeneic and CsA-treated allogeneic combinations, there was no increase of baseline S-R-IL-2 level (P less than 0.001), and graft infiltrate did not contain IL-2-R positive cells. CsA treatment prolonged heart allograft survival (41.3 +/- 2.8 days). Baseline S-R-IL-2 levels during treatment were lower than those observed in untreated animals. In the CsA-treated allogeneic group, after CsA treatment interruption, S-R-IL-2 levels significantly increased, reaching a plateau at day 37. Results suggest that S-R-IL-2 measurement can be useful for clinical diagnosis of allograft rejection.
Animals↗
Combined assay of adenosine deaminase, purine nucleoside phosphorylase, and lactate dehydrogenase in the early clinical evaluation of B-chronic lymphocytic leukemia.
The levels of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), lactic dehydrogenase (LDH), and LDH isoenzyme patterns (LD1 to LD5) have been measured in lymphocyte extract from 28 patients with B-chronic lymphocytic leukemia (B-CLL). The activities of ADA, PNP, and LDH have been correlated with two morphological groups of B-CLL classified according to the percentage of large, nongranular, atypical lymphocytes (AL) in peripheral blood: "typical" B-CLL (less than 10% of AL, 21 cases) and "atypical" B-CLL (10-25% of AL, seven cases). Patients with atypical B-CLL had significantly (P less than 0.001) higher activities of ADA (0.46 +/- 0.17 U/10(9) cells), PNP (1.74 +/- 1.0 U/10(9) cells), and LDH (48.3 +/- 9.7 U/10(9) cells) than patients with typical B-CLL (ADA, 0.29 +/- 0.1 U/10(9) cells; PNP, 0.58 +/- 0.23 U/10(9) cells; and LDH, 29 +/- 10 U/10(9) cells). In addition, the "treatment-free period" was also significantly (P less than 0.025) shorter in the group of atypical B-CLL compared with the typical B-CLL group. No clear-cut statistical differences in lymphocyte surface markers or in several other prognostic factors between the two subgroups of B-CLL were found. The present study supports the idea that in B-CLL the simultaneous determination of ADA, PNP, and LDH might be helpful in better understanding the pathophysiology, prognosis, and natural history of the disease.
Adenosine Deaminase↗
Immune interferon enhances functional properties of human granulocytes: role of Fc receptors and effect of lymphotoxin, tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor.
We report here a comparative study of the effects of several cytokines known to affect myeloid cell differentiation on functional properties of human mature granulocytes. We show that recombinant interferon-gamma (rIFN-gamma), recombinant granulocyte/macrophage-colony stimulating factor (rGM-CSF), recombinant tumor necrosis factor (rTNF), and lymphotoxin (LT) purified to homogeneity are potent stimulators of polymorphonuclear cells (PMN) activity. All cytokines enhance antibody-dependent cell-mediated cytotoxicity (Ab-CMC) mediated by human PMN; however, rGM-CSF, rTNF, and LT have an immediate and short-lived effect on the PMN, whereas the activation by rIFN-gamma requires several hours of induction but can be observed up to 24 to 48 hr of culture. Only the effect of rIFN-gamma is in part dependent on induction of a high-affinity FcR for monomeric IgG on PMN, as suggested by two-color sorting analysis, and on mechanisms that result in prolonged survival of PMN in a functionally active state to mediate oxidative burst, phagocytosis, and bactericidal activity. Greater enhancement of Ab-CMC is obtained by using rIFN-gamma in combination with the other cytokines. Our data indicate that cytokines previously defined on the basis of their cytotoxic effects mediate a wide spectrum of activities on mature myeloid cells and provide evidence for their possible role in vivo, alone or in combination with rIFN-gamma, in modulating functional activities of cells responsible for non-adaptive systems of defense.
Antibody-Dependent Cell Cytotoxicity↗
Preferential proliferation of natural killer cells among peripheral blood mononuclear cells cocultured with B lymphoblastoid cell lines.
In this report, we show that in vitro stimulation of human peripheral blood mononuclear cells (PBMC) with B lymphoblastoid cell lines results in preferential proliferation of cells with the phenotypic, genotypic and functional characteristics of natural killer (NK) cells. This culture system offers a useful method for obtaining large numbers of pure NK cells on which biochemical, molecular and functional studies can be performed. Using this culture system, and average 25-fold increase in NK cell number is obtained, whereas the number of T cells is increased only 3-fold. At early times, activation of both T and NK cells occurs, as detected by the presence of activation antigens on both cell types, but actual proliferation of NK cells starts on day 6 of culture. Elimination of CD3(+)/CD5(+) T cells from the cultured cells gives homogeneous preparations of large numbers of CD16(+)/NKH-1(+) cells that have the morphology of large granular lymphocytes, are powerful effectors of both spontaneous and antibody-dependent cell-mediated cytotoxicity, and rapidly proliferate in the presence of recombinant interleukin 2 (rIL-2). As fresh NK cells, NK cell-enriched preparations from 10-day cultures of Daudi-stimulated PBMC do not show rearrangement of the gene for the beta-chain of the T cell antigen receptor; the 1.3-kb functional transcript of the beta-chain gene was not expressed in NK cells, but the 1.0-kb truncated transcript was present in all preparations. Our data indicate that proliferation of both T and NK cells is dependent upon IL-2 production in the culture, because an anti-IL-2 antiserum completely suppresses proliferation. Because T cells, and in particular CD4(+) T cells, are required for preferential proliferation of NK cells, the NK cell stimulation induced by the B cell line is probably in part indirect, and due to induction of IL-2 production by allogeneic stimulation of CD4(+) cells. However, the B cell lines also need to interact directly with NK cells because neither allogeneic PBMC nor high doses of rIL-2 are sufficient to induce preferential proliferation of NK cells.
Antibodies, Monoclonal↗