bluuebunny/crossref_metadata_2025 huggingface dataset:
Chimeric Fc receptors identify functional domains of the murine high affinity receptor for IgG.
Authors: Hulett M D , Osman N , McKenzie I F , Hogarth P M
Published: December 2022

Chimeric Fc gamma R have been generated between the mouse high affinity receptor for IgG (Fc gamma RI) and the low affinity receptor for IgG (Fc gamma RII) by exchanging the first two domains of the three-domain extracellular structure of Fc gamma RI with the homologous two-domain extracellular structure of Fc gamma RII. Studies of the affinity and specificity of binding of mouse Ig classes to these receptors defined functional regions of Fc gamma RI and showed some surprising results. After removal of the third extracellular domain of Fc gamma RI, the remaining two domains (domains 1 and 2) retained the capacity to bind Ig in the form of immune complexes, however, they bound monomeric IgG2a with a reduced affinity. Surprisingly, these two domains in the absence of the third domain bound not only IgG2a but also IgG1 and IgG2b, i.e., the third domain of Fc gamma RI suppresses the intrinsic capacity of the first two domains to act as a low affinity Fc gamma RII-like molecule. Linking the third extracellular domain of Fc gamma RI to the two extracellular domains of Fc gamma RII resulted in a receptor that retained the specificity and affinity of Fc gamma RII. Thus, the removal of domain 3 from Fc gamma RI resulted in the conversion of Fc gamma RI to an "Fc gamma RII-like" receptor. These findings indicate that domains 1 and 2 of Fc gamma RI form an Ig-binding motif, and although domain 3 is not essential for Fc binding by Fc gamma RI, it plays a crucial role in determining the specific high affinity interaction of Fc gamma RI with IgG2a.
Spontaneous activation of serum C1 in vitro. Role of C1 inhibitor.
Authors: Tseng Y , Poon P H , Zavodszky P , Schumaker V N
Published: December 2022

The temperature and ionic strength dependence of the spontaneous activation of C1 were determined for normal human serum, and the free energy, enthalpy, and entropy of spontaneous activation were calculated. The half-life of C1 in human serum was approximately 15 h at 37 degrees C. This half-life was markedly extended by dilution with C1-depleted serum, and an extrapolated upper limit of 40 to 50 h was reached at infinite dilution. Thus, the spontaneous activation of C1 in serum appeared to involve a dilution-sensitive reaction as well as a dilution-insensitive, first order reaction. A reaction mechanism was developed combining: 1) first order spontaneous activation of C1; 2) second order, C1-catalyzed activation of C1; and 3) second order inactivation of C1 by C1-inhibitor. A steady state equation was derived from this reaction mechanism, which provided a reasonable fit to the experimental data. The equation predicts that when the C1-inhibitor concentration decreases so that the steady state condition is lost, the concentration of C1 builds up quickly, and activation of most of the C1 occurs rapidly.
Differential T cell hyporesponsiveness induced by in vivo administration of intact or F(ab')2 fragments of anti-CD3 monoclonal antibody. F(ab')2 fragments induce a selective T helper dysfunction.
Authors: Hirsch R , Archibald J , Gress R E
Published: December 2022

Induction of peripheral T cell anergy associated with stimulation through the TCR complex in vivo has been described in mice using chemically modified APC, staphylococcal enterotoxin B, and intact anti-CD3 mAb. In the latter two models, T cell proliferation, IL-2R expression, and lymphokine production have been demonstrated before subsequent induction of hyporesponsiveness, whereas in the former model, these events have not been observed. To further investigate the relationship between mitogenicity and induction of peripheral hyporesponsiveness, mice were treated with either mitogenic intact anti-CD3 mAb or nonmitogenic F(ab')2 fragments of anti-CD3 mAb. T cells from F(ab')2-treated mice demonstrated a selective decrease in helper functions, with minimal effect on CTL function. Specifically, a marked reduction in ability of Th cells to secrete IL-2 when challenged in vitro with mitogen or alloantigen was observed, which persisted for at least 2 mo after mAb administration and which was independent of T cell depletion. Proliferative function was decreased in CD4+ T cells and could not be fully restored with addition of exogenous IL-2. A helper defect was also evident in vivo, in that F(ab')2-treated mice were deficient in their ability to reject MHC-disparate skin grafts, and in vivo administration of IL-2 reconstituted their ability to reject skin grafts normally. In contrast, T cells from mice treated with intact mAb demonstrated a significant decrease in both CTL and helper functions. A long term reduction in TCR expression on CD4+ cells from F(ab')2-treated mice, and on both CD4+ and CD8+ cells from intact mAb-treated mice was observed. These findings demonstrate that peripheral T cell hyporesponsiveness can be induced in vivo by binding an identical epitope on the TCR complex in the presence or absence of initial proliferation, lymphokine secretion, or IL-2R expression, and that binding to the same epitope can result in varying long term effects on T cell function.
Oral tolerance in experimental autoimmune encephalomyelitis. III. Evidence for clonal anergy.
Authors: Whitacre C C , Gienapp I E , Orosz C G , Bitar D M
Published: December 2022

We have recently reported that experimental autoimmune encephalomyelitis (EAE) can be suppressed by the oral administration of myelin basic protein (MBP). The oral introduction of 20 mg MBP together with a trypsin inhibitor results in inhibition of EAE clinical signs, decreased CNS histopathologic changes and dramatically reduced MBP-specific proliferative responses in fed and challenged Lewis rats. In the present study, we have investigated the mechanism underlying MBP-induced oral tolerance in EAE. Neither lymphoid cells (lymph node cells, spleen cells, Peyer's patch lymphocytes, thymocytes) nor humoral elements derived from tolerant donors were capable of transferring the tolerance to naive recipients. Moreover, lymphoid cells obtained from orally tolerant donors exhibited a marked decrease in their capacity to transfer EAE to naive recipient rats, even after in vitro activation with MBP or Con A. We observed that EAE could be readily transferred into orally tolerant rats using MBP-specific encephalitogenic T cell lines. In vitro cell mixing studies showed that the proliferation of lymphocytes from MBP-sensitized donors was not inhibited by the addition of lymphoid cells from tolerant donors, arguing against the role of a suppressor cell. Investigation of MBP-stimulated lymphokine production showed that both IL-2 and IFN-gamma levels were substantially decreased in spleen and lymph node cell cultures from MBP-fed rats compared to vehicle-fed control animals. Furthermore, limiting dilution analyses revealed that MBP-fed rats exhibited a profound decrease in MBP-reactive, IL-2-secreting lymphocytes relative to control animals. Thus, because lymphocytes from MBP-fed rats neither proliferate nor secrete IL-2 or IFN-gamma in response to MBP and we can find no compelling evidence for the role of suppressor cells, we propose that the oral administration of MBP results in a state of clonal anergy.
Tumor necrosis factor-alpha dependent cytotoxicity of human skin mast cells is enhanced by anti-IgE antibodies.
Authors: Benyon R C , Bissonnette E Y , Befus A D
Published: December 2022

Mast cells dispersed from human skin and purified by density-gradient centrifugation were cytotoxic toward the mouse fibrosarcoma cell line WEHI-164. Skin mast cells were not cytotoxic toward the NK cell-sensitive cell line K562. Killing of WEHI-164 occurred over a prolonged (greater than 18 h) period of incubation with mast cells and was effectively inhibited by polyclonal antibodies and mAb against TNF-alpha suggesting that this cytokine plays an important role in mast cell-mediated cytotoxicity. Whereas lysates of rat peritoneal mast cells exhibited cytotoxicity toward WEHI-164, this was not found with lysates of unstimulated skin mast cells suggesting that TNF-alpha is not stored preformed in the latter. Killing of WEHI-164 cells by skin mast cells was enhanced by anti-IgE and there was a significant correlation between histamine release and cytotoxicity after activation with this stimulus. We conclude that human skin mast cells are a potential source of TNF-alpha and suggest that these cells, particularly after activation, might contribute to the synthesis of this multifunctional cytokine in inflammatory sites.
CD11b and CD11c antigens are rapidly increased on human natural killer cells upon activation.
Authors: Werfel T , Witter W , Götze O
Published: December 2022

A brief incubation with PMA or other secretagogues has been reported to enhance the expression of C3 receptors on myeloid cells. We now observed increases up to threefold in the expression of the CD11b/CD18 Ag (CR3) and the CD11c/CD18 (CR4, p150,95) Ag after 30-min incubation with PMA on a subpopulation of PBL. The majority of these cells was CD56+ and CD16+. Isolated NK cells retained their ability to respond to PMA with increased CD11b and CD11c membrane Ag expression. Preincubation of the cells with cycloheximide did not abrogate the effects of PMA. Other membrane molecules on lymphocytes (CD11a, CD35, CD45, CD45R0, CD56) were not modulated by PMA. Purified C5a, FMLP, or LPS increased CR3 on myeloid cells but not on lymphocytes. In contrast, cell activation by K562 cells led to an augmentation of the CD11b Ag expression on CD56+ lymphocytes but not on other lymphocytes or monocytes. This increase was inhibitable by CD11a mAb. Rapid increases of CD11b and CD11c Ag on the membrane of NK cells may be of biologic significance because many functions have been attributed to these molecules.
Induction of T cell CD7 gene transcription by nonmitogenic ionomycin-induced transmembrane calcium flux.
Authors: Ware R E , Hart M K , Haynes B F
Published: December 2022

The CD7 molecule is a 40-kDa member of the Ig superfamily that has structural homology to the murine Thy-1 molecule and is acquired early in human T cell ontogeny. Previous studies have demonstrated that expression of the CD7 molecule is markedly up-regulated during T cell activation. In this study, we have studied the signals required for CD7 up-regulation on human T cells. We found that nonmitogenic amounts of ionomycin selectively and maximally up-regulated T cell CD7 on mature (peripheral blood) T cells after 24 h. Whereas CD7 expression was increased 78 +/- 25% by 0.5 microM ionomycin, expression of CD25 (IL-2R alpha), class II MHC, 4F2, transferrin receptor, CD2, CD3, CD4, CD5, and CD8 molecules was not increased. Ionomycin-induced CD7 surface expression was associated with peak increases in CD7 mRNA after 4 to 6 h. Transcriptional analysis and CD7 mRNA half-life determination revealed the increase in CD7 mRNA was the result of increased CD7 gene transcription 1 h after ionomycin stimulation and was not due to prolongation of CD7 mRNA half-life. The up-regulation of surface CD7 expression by ionomycin was dependent on extracellular calcium and did not require the activation of T cell tyrosine protein kinase. Mitogenic CD2 and CD3 mAb as well as stimulation of T cells by PHA also up-regulated CD7 expression. CD7 up-regulation by ionomycin was transient (24 to 72 h) and inhibitable by cyclosporin A, whereas CD7 up-regulation by PHA was sustained over 5 to 7 days and was significantly less inhibitable by cyclosporin A. These data demonstrate that induction of a transmembrane calcium flux generates signals that lead to CD7 gene transcription.
Characterization of the autoantigen calreticulin.
Authors: Rokeach L A , Haselby J A , Meilof J F , Smeenk R J , Unnasch T R , Greene B M , Hoch S O
Published: December 2022

Anti-Ro/SS-A antibodies are commonly found in the sera of patients with Sjögren's syndrome and SLE. These antibodies also occur in the mothers of children with neonatal lupus and congenital heart block. Ro/SS-A is a ribonucleoprotein complex whose cellular function remains unknown. To study its cellular function and to characterize its immunoreactivity, we have used an oligonucleotide designed after the published amino terminal sequence of a putative 60-kDa Ro/SS-A autoantigen to isolate its cDNA. This cDNA encodes a polypeptide that is the human homologue of calreticulin, a calcium binding protein of the endoplasmatic reticulum. The encoded polypeptide also shows a 64.4% identity with RAL-1, an Ag of the river blindness pathogen Onchocerca volvulus. Contrary to the data published by other authors, our results indicate that calreticulin is not a Ro/SS-A autoantigen. Moreover, we show that anticalreticulin autoantibodies occur in the sera of patients with SLE and patients with onchocerciasis.
Utilization of V kappa families and V kappa exons. Implications for the available B cell repertoire.
Authors: Kalled S L , Brodeur P H
Published: December 2022

A molecular cloning approach was used to determine the relative utilization of 2 individual V kappa 21 genes, 13 V kappa gene families, and the 4 functional J kappa gene segments among splenic B cells of nonimmunized BALB/c mice. Based on the observed frequency of individual V kappa gene expression, we estimate that the mouse genome encodes 150 to 180 functional V kappa genes, and we suggest that most functional V kappa exons are expressed at comparable frequencies in the preimmune antibody repertoire. In contrast, clear differences in J kappa segment utilization were observed, J kappa 4 being consistently underrepresented with respect to the other J kappa segments.
IgM and IgG but not cytokine secretion is restricted to the CD27+ B lymphocyte subset.
Authors: Maurer D , Fischer G F , Fae I , Majdic O , Stuhlmeier K , Von Jeney N , Holter W , Knapp W
Published: December 2022

In a recent study we reported that CD27 is expressed on a subpopulation of human B lymphocytes and presented circumstantial phenotypic evidence that CD27 expression may be acquired late during B cell differentiation. Here we present functional data showing that, after in vitro stimulation, CD27+ but not CD27- B cells secrete large amounts of both IgM and IgG. Using double immunofluorescence staining of CD27 and IgD, three functionally different B cell subsets representing distinct stages of B cell differentiation can be isolated: 1) the CD27- IgD+ B cells, which do not secrete appreciable Ig; 2) the CD27+IgD+ B cells, which exclusively secrete IgM; and 3) the CD27+IgD- B cells, which comprise the IgG-producing cells. Furthermore, costimulation of CD27- B cells with low m.w. B cell growth factor, in the presence or in the absence of a CD40 mAb, does not induce these cells to become Ig-secreting cells. Although CD27- B cells hardly secrete Ig of any isotype in response to Staphylococcus aureus+IL-2, these cells proliferate vigorously and express the IL-2R alpha chain (CD25) under these stimulatory conditions. Furthermore, both CD27- and CD27+ B cells are capable of producing similar amounts of IL-6 and TNF-alpha. Taken together, these findings indicate that CD27 is a unique non-Ig surface marker discriminating naive from primed B lymphocytes. Furthermore, the capacity to proliferate and to secrete the B cell differentiation factors IL-6 and TNF-alpha already exists at an early B cell differentiation stage at which the cells lack CD27 expression and are not induced to produce Ig.
Generation of B cell memory and affinity maturation. Induction with Th1 and Th2 T cell clones.
Authors: Rizzo L V , DeKruyff R H , Umetsu D T
Published: December 2022

We used an adoptive transfer system and CD4+ T cell clones with defined lymphokine profiles to examine the role of CD4+ T cells and the types of lymphokines involved in the development of B cell memory and affinity maturation. Keyhole limpet hemocyanin (KLH)-specific CD4+ Th2 clones (which produce IL-4 and IL-5 but not IL-2 or IFN-gamma) were capable of inducing B cell memory and affinity maturation, after transfer into nude mice or after transfer with unprimed B cells into irradiated recipients and immunization with TNP-KLH. In addition, KLH-specific Th1 clones, which produce IL-2 and IFN-gamma but not IL-4 or IL-5, were also effective in inducing B cell memory and high affinity anti-TNP-specific antibody. The induction of affinity maturation by Th1 clones occurred in the absence of IL-4, as anti-IL-4 mAb had no effect on the affinity of the response whereas anti-IFN-gamma mAb completely blocked the response. Th1 clones induced predominantly IgG2a and IgG3 antibody, although Th2 clones induced predominantly IgG1 and IgE antibody. We thus demonstrated that some Th1 as well as some Th2 clones can function in vivo to induce Ig synthesis. These results also suggest that a single type of T cell with a restricted lymphokine profile can induce both the terminal differentiation of B cells into antibody secreting cells as well as induce B cell memory and affinity maturation. Moreover, these results suggest that B cell memory and affinity maturation can occur either in the presence of Th2 clones secreting IL-4 but not IFN-gamma, or alternatively in the presence of Th1 clones secreting IFN-gamma but not IL-4.
In vivo deposition of myosin-specific autoantibodies in the hearts of mice with experimental autoimmune myocarditis.
Authors: Neumann D A , Lane J R , Wulff S M , Allen G S , LaFond-Walker A , Herskowitz A , Rose N R
Published: December 2022

Experimental autoimmune myocarditis (EAM) is elicited in certain strains of mice by immunizing with mouse cardiac myosin. Concomitant with the onset of myocardial inflammation is the induction of circulating IgG antibodies to myosin. To further examine the role of myosin in disease, both EAM-susceptible (A/J) and EAM-resistant (B10.A) mice were immunized with myosin emulsified in CFA and examined for myocardial inflammation and IgG deposition. Myocarditis was common in susceptible, but not resistant strain mice. IgG deposition was extensive in A/J mice, but modest in B10.A mice, when compared to controls given adjuvant alone. Localization was independent of inflammatory or necrotic lesions. A spot ELISA indicated that antimyosin IgG antibody-secreting cells were present in the myocardial infiltrate and likely contributed to antibody localization. Antibody was eluted from the hearts of immunized animals and found to react strongly with normal heart tissue by indirect immunohistochemistry. This reactivity was not completely absorbed by skeletal muscle, indicating that some of the antibody was heart-specific. Western immunostaining demonstrated that eluates from immunized A/J and B10.A mice possessed anti-myosin antibody activity; similar reactivity was not observed in eluates from control mice of either strain. Comparison of heart reactivity with syngeneic and allogeneic tissue suggests that although myosin immunization elicits homologous antibody in both strains, each may recognize distinct epitopes. These findings strongly suggest that cardiac myosin or a myosin-like determinant is expressed on the surface of normal mouse myocytes.
Identification of IL-7-dependent bone marrow-derived Thy-1-B220- lymphoid cell clones that rearrange and express both Ig and T cell receptor genes.
Authors: Takai Y , Sakata T , Iwagami S , Tai X G , Kita Y , Hamaoka T , Sakaguchi N , Yamagishi H , Tsuruta Y , Teraoka H
Published: December 2022

Bone marrow stromal cell lines and lymphoid cell lines were co-established from the Whitlock-Witte type of long term liquid cultures of MRL/1 and C57BL/10 (B10) (Thy-1.1) bone marrow cells. The present study investigates the immunologic nature of parental and cloned lymphoid cell lines. Both strains of parental lines and their clones did not grow alone but proliferated on the monolayers of co-established parental stromal cell lines from a syngeneic or alternative strain. When various lymphokines or cytokines were tested for their capacity to support the growth of these lymphoid cell clones, only IL-7 could substitute for the growth-promoting function of stromal cells. These IL-7-dependent clones expressed neither Thy-1 nor B220 Ag. However, all of them from two strains were found to rearrange synchronously H chain of Ig as well as gamma chain of TCR genes. Some of the clones transcribed a mature size of IgH mRNA. Co-expression of mRNA for lambda 5 but not for IgL chain (kappa, lambda) genes resulted in the generation of cell surface mu chain in these clones. Other clones expressed a smaller size of IgH mRNA without exhibiting surface mu chain. Irrespective of the differences in IgH rearrangements and its mRNA expression, a mature size TCR gamma mRNA was detected in all of the clones. Thus, these results demonstrate the existence of untransformed (IL-7-dependent) immature lymphoid cells rearranging both Ig and TCR genes. Their unique features concerning cell surface markers (B220- mu+), specific growth factor requirement, and various modes of Ig/TCR gene rearrangements are discussed in the context of early lymphoid development.
Comparison of GK1.5 and chimeric rat/mouse GK1.5 anti-CD4 antibodies for prolongation of skin allograft survival and suppression of alloantibody production in mice.
Authors: Rashid A , Auchincloss H , Sharon J
Published: December 2022

GK1.5 is a rat mAb that recognizes the mouse CD4 Ag. It has been shown to deplete CD4+ cells in vivo and to be immunosuppressive. To evaluate the effect of the C region of this antibody in achieving cell depletion, chimeric antibodies, each having the rat GK1.5 V regions and one of the four mouse IgG C region isotypes, were compared with the native rat antibody. The chimeric antibodies and the native antibody were tested for their ability to mediate in vitro C-dependent cytotoxicity, in vivo cell depletion, and prolongation of allogeneic skin graft survival and suppression of alloantibody production. In vitro C-dependent cytotoxicity assays revealed that rat IgG2b and the chimeric antibodies containing mouse IgG2a, mouse IgG2b, and mouse IgG3 were effective in lysing CD4+ lymphocytes whereas mouse IgG1 was ineffective. In vivo studies of CD4+ cell depletion showed that mouse IgG2a, rat IgG2b, and mouse IgG2b were effective isotypes, mouse IgG1 was less effective, and mouse IgG3 did not deplete CD4+ cells. A correlation was found between the ability of an isotype to deplete CD4+ cells in vivo and its ability to prolong the survival of skin allografts and to suppress alloantibody production. The nondepleting mouse IgG3 was ineffective in these assays. Overall the most effective mouse isotype was IgG2a which was as effective as rat IgG2b. These results indicate 1) that syngeneic isotypes of mAb can cause cell depletion and consequently the prolongation of allograft rejection and suppression of alloantibody production; 2) that not all isotypes are equally effective; and 3) that the ability of a given isotype to deplete cells in vivo does not correlate with its ability to mediate C-dependent lysis in vitro. Our results are consistent with the hypothesis that in vivo depletion of cells is mediated by opsonization and binding through the FcR.
Induction of arthritis with monoclonal antibodies to collagen.
Authors: Terato K , Hasty K A , Reife R A , Cremer M A , Kang A H , Stuart J M
Published: December 2022

mAb were developed from DBA/1 mice immunized with chick type II collagen. A total of 69 IgG antibodies was isolated and characterized. The majority (36%) reacted with a CNBr-derived peptide CB11 previously identified as containing a major immunogenic and arthritogenic epitope(s). Seven of the antibodies reactive with CB11 crossreacted strongly with mouse type II collagen. These were administered to DBA/1 mice in an attempt to induce arthritis. Individual antibodies were able to induce mild lesions consisting of minimal synovial proliferation but not overt arthritis. However, a combination of antibodies induced severe arthritis with marked destruction of articular cartilage. The minimal effective combination consisted of three antibodies. Arthritis developed within 48 to 72 h after injection of the antibodies and persisted for the duration of the observation period of 3 wk. Antibody levels were measured at intervals and persisted for the 3 wk observation period although at diminishing levels. Competitive binding assays demonstrated that each of the effective antibodies bound independently suggesting that some spatial or quantitative relationship was important possibly related to their ability to activate complement.
Regulation of the effector stages of experimental autoimmune encephalomyelitis via neuroantigen-specific tolerance induction. II. Fine specificity of effector T cell inhibition.
Authors: Tan L J , Kennedy M K , Miller S D
Published: December 2022

Ag-specific tolerance induced by the i.v. administration of splenocytes coupled with mouse spinal cord homogenate, containing a mixture of myelin Ag, dramatically inhibits development and expression of clinical and histologic signs of both active and adoptive forms of relapsing experimental autoimmune encephalomyelitis (R-EAE) in the SJL/J host. Here we examined the dose-dependency, route of tolerogen administration, and fine neuroantigen specificity of inhibition of adoptive R-EAE. Expression of clinical R-EAE induced by a polyclonal population of bovine myelin basic protein (MBP)-specific effector T cells was dramatically inhibited in a dose-dependent manner following the i.v., but not s.c. or i.p., injection of MBP-coupled splenocytes. The exquisite Ag specificity of the inhibition was evident by the observation that splenocytes coupled with intact bovine MBP or species variants of MBP homologous with bovine MBP within the major encephalitogenic region (amino acids 84-104), but not with proteolipid protein or mouse kidney homogenate, were able to suppress disease expression. Splenocytes coupled with the MBP84-104 peptide, containing a nested set of the major SJL/J encephalitogenic epitopes, completely inhibited peptide-specific T cell responses, but only partially inhibited the expression of disease transferred by T cells specific for intact MBP, suggesting the participation of T cell responses specific for additional MBP determinants in disease pathogenesis. However, splenocytes coupled with previously identified minor SJL/J encephalitogenic epitopes (MBP91-104 or MBP17-27), or with the Lewis rat major encephalitogenic epitope (MBP68-86), did not suppress disease expression. Collectively, the results demonstrate that MBP84-104-specific T cells and T cells specific for an as yet unidentified MBP epitope(s) contribute to the pathology of R-EAE. In addition, the results demonstrate that peptide-specific tolerance induction appears to have potential for the treatment of T cell-mediated inflammatory diseases.
Tolerance to host minor histocompatibility antigens after allogenic bone marrow transplantation. Specific donor-host unresponsiveness is maintained by peripheral tolerizing cells.
Authors: Brochu S , Roy D C , Perreault C
Published: December 2022

The development of graft-host tolerance after allogeneic bone marrow (BM) transplantation is more demanding than the acquisition of self-tolerance because both donor-derived mature T cells and immature thymocytes encounter host Ag. The mechanism involved in tolerization of mature T cells, contained in unmanipulated BM, remains undefined. In previous experiments, we showed in vivo unresponsiveness to host minor histocompatibility Ag (MiHA) in immunocompetent chimeras obtained after MiHA-incompatible BM transplantation. In this study, we wanted to determine: first, what was the specificity of this graft-host unresponsiveness, and second, whether peripheral cells were involved in tolerization? LP recipients were irradiated (9, 5 Gy), injected with 10(7) undepleted BM cells from B10 donors and studied 100 to 150 days later. (B10-->LP) chimeras were immunized in vivo and restimulated in vitro with cells displaying one or multiple incompatible MiHA. In bulk culture experiments, chimeras demonstrated specific CTL unresponsiveness to host MiHA but responded normally to third party MiHA. In limiting dilution analysis conditions, chimeras showed a profound deficit, but not a complete absence of anti-host CTL precursor. Studies with congenic stimulators/targets showed that graft-host tolerance was induced against both immunodominant (e.g., H-3.2) and nonimmunodominant (e.g., H-8.2) MiHA although at the CTL precursor level, it was more complete against the former. Furthermore, chimera spleen cells inhibited the generation of CTL activity against host- and donor-type MiHA but not against third party Ag. This specific suppressor activity was not T cell dependent, and was mediated by radiosensitive cells that are not found in freshly explanted organs from normal mice. Taken together, our results suggest that peripheral tolerization can be a remarkably efficient process to maintain tolerance to MiHA after BM transplantation. Thus, peripheral tolerizing mechanisms may contribute not only to the induction of tolerance to Mls superantigens or to the product of transgenes (if expressed at high levels) but also to a wide array of MiHA.
Molecular characterization of the WC1 antigen expressed specifically on bovine CD4-CD8- gamma delta T lymphocytes.
Authors: Wijngaard P L , Metzelaar M J , MacHugh N D , Morrison W I , Clevers H C
Published: December 2022

Although gamma delta T lymphocytes were identified several years ago, the functional importance of these cells remains to be established. gamma delta T cells of ruminants are unique in two respects. First, they are present at much higher levels compared to man and rodents. Second, ruminant CD4-CD8- gamma delta T cells uniquely express a 220 kD surface Ag recognized by a panel of mAb, recently clustered as WC1. WC1 has been most extensively studied in sheep with the use of the mAb T19. Here, we report on the isolation of a full length cDNA clone, encoding the WC1 Ag, from a COS cell cDNA expression library prepared from a bovine gamma delta T cell line. The protein encoded by the pWC1 cDNA clone was reactive with the bovine mAb CC15 and IL.A29, and with T19. The cDNA clone consisted of 4475 bp and contained a single long open reading frame of 1436 amino acids. The pWC1 cDNA clone encoded a type 1 integral membrane protein with an extracellular domain consisting of 11 scavenger receptor cysteine-rich-repeats with homology to CD5 and CD6. Southern blotting suggested that the bovine genome contained multiple sequences highly related to the isolated WC1 cDNA. Furthermore, WC1-like sequences were present in the genomes of all mammals tested including mouse and man. The molecular characterization of the WC1 Ag as reported here provides a starting point for the definition of its role in gamma delta T cell biology.
Redox status of cells influences constitutive or induced NF-kappa B translocation and HIV long terminal repeat activity in human T and monocytic cell lines.
Authors: Israël N , Gougerot-Pocidalo M A , Aillet F , Virelizier J L
Published: December 2022

We have tested the hypothesis that cellular activation events occurring in T lymphocytes and monocytes and mediated through translocation of the transcription factor NF-kappa B are dependent upon the constitutive redox status of these cells. We used phenolic, lipid-soluble, chain-breaking antioxidants (butylated hydroxyanisole (BHA), nordihydroquairetic acid, or alpha-tocopherol (vitamin E) to show that peroxyl radical scavenging in unstimulated and PMA- or TNF-stimulated cells blocks the functions depending on NF-kappa B activation. BHA was found to suppress not only PMA- or TNF-induced, but also constitutive, HIV-enhancer activity concomitant to an inhibition of NF-kappa B binding activity in both lymphoblastoid T (J.Jhan) and monocytic (U937) cell lines. This was also true for KBF (p50 homodimer) binding activity in U937 cells. Secretion of TNF, the product of another NF-kappa B-dependent gene, was abolished by BHA in PMA-stimulated U937 cells. The anti-oxidative effect of BHA was accompanied by an increase in thiol, but not glutathione, content in stimulated and unstimulated T cell, whereas TNF stimulation itself barely modified the cellular thiol level. Oxidative stress obtained by the addition of H2O2 to the culture medium of J.Jhan or U937 cells could not by itself induce NF-kappa B activation. These observations suggest that TNF and PMA do not lead to NF-kappa B activation through induction of changes in the cell redox status. Rather, TNF and PMA can exert their effect only if cells are in an appropriate redox status, because prior modification toward reduction with BHA treatment prevents this activation. It appears that a basal redox equilibrium tending toward oxidation is a prerequisite for full activation of transduction pathways regulating the activity of NF-kappa B-dependent genes.
Molecular interactions mediating T-B lymphocyte collaboration in human lymphoid follicles. Roles of T cell-B-cell-activating molecule (5c8 antigen) and CD40 in contact-dependent help.
Authors: Lederman S , Yellin M J , Inghirami G , Lee J J , Knowles D M , Chess L
Published: December 2022

In lymphoid follicles, CD4+ T lymphocytes provide contact-dependent stimuli to B cells that are critical for the generation of specific antibody responses in a process termed Th function. The CD4+ T cell-restricted surface activation protein, 5c8 Ag (T-BAM), has recently been shown to be a component of the contact-dependent helper signal to B cells. To further dissect this process, we utilized a Jurkat T cell lymphoma clone, termed D1.1, that constitutively expresses T-BAM and activates peripheral B cells to express surface CD23 in a contact-dependent mechanism that is inhibited by mAb anti-T-BAM (5c8). Similar to its effect on peripheral B cells, Jurkat D1.1 activates B cells from lymphoid organs, as well as a B cell lymphoma clone, RAMOS 266,4CN 3F10 (RAMOS 266), to up-regulate surface CD23. Interestingly, mAb to the B cell surface molecule, CD40 (mAb G28-5 and B-B20), inhibit D1.1 induced activation of RAMOS 266 and peripheral and lymphoid B cells. In contrast, mAb to CR2 or the adhesion molecules, LFA1, LFA3, or ICAM-1, have little effect. The inhibitory effect of anti-CD40 mAb on B cell activation induced by D1.1 is specific because anti-CD40 potentiates, rather than inhibits, the up-regulation of CD23 on B cells induced by rIL-4. Moreover, cross-linking CD40 molecules by anti-CD40 mAb bound to Fc gamma RII+ (CD32) L cells induces B cell CD23 expression. In vivo, T-BAM-expressing cells are CD4+ T cells that are restricted to lymphoid organs and are localized in the mantle and centrocytic zones of lymphoid follicles and the spleen periarteriolar lymphoid sheath in association with CD40+ B cells. Taken together, these data demonstrate that T-BAM on T cells and CD40 on B cells are involved in contact-dependent T-B help interactions that occur in lymphoid follicles.
Salmonella typhimurium porins stimulate platelet-activating factor synthesis by human polymorphonuclear neutrophils.
Authors: Tufano M A , Tetta C , Biancone L , Iorio E L , Baroni A , Giovane A , Camussi G
Published: December 2022

Porins, a family of hydrophobic proteins located in the outer membrane of cell-wall of Gram-negative bacteria, were shown to stimulate the synthesis and release of platelet-activating factor (PAF), a 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine mediator of inflammation and endotoxic shock produced by polymorphonuclear neutrophils. PAF synthesis was independent either from contamination by LPS or generation of TNF. Experiments with labeled precursors demonstrated that PAF was synthesized via the remodeling pathway that involves acetylation of 1-O-alkyl-sn-glyceryl-3-phosphorylcholine generated from 1-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase A2 (PLA2) activity. Porins, indeed, induced a sustained PLA2-dependent mobilization of [14C]arachidonic acid that was inhibited by p-bromodiphenacylbromide. p-Bromodiphenacylbromide, an inhibitor of PLA2, also blocked PAF synthesis by preventing the mobilization of 2-lyso-PAF, the substrate for PAF-specific acetyltransferase. The addition of 2-lyso-PAF restored PAF synthesis. The activity of acetyl CoA:2-lyso-PAF acetyltransferase was transiently increased in porin-stimulated PMN and the [3H]acetyl group was incorporated in the synthetized PAF after cell preincubation with [3H]acetyl CoA. The activation of PAF synthesis by porins as well as its release were dependent on extracellular Ca2+. Porins by forming trans-membrane channels determined a sustained influx of 45Ca2+ into the cytosol. As shown by inhibitors of Ca(2+)-calmodulin complexes, calmodulin mediated the Ca(2+)-dependent activation of enzymes involved in PAF synthesis.
Taxol increases steady-state levels of lipopolysaccharide-inducible genes and protein-tyrosine phosphorylation in murine macrophages.
Authors: Manthey C L , Brandes M E , Perera P Y , Vogel S N
Published: December 2022

Taxol, a microtubule stabilizing agent, exhibits promise in the treatment of breast and ovarian tumors. Recently, this novel drug has been shown to activate murine macrophages to express TNF-alpha and to down-regulate TNF-alpha receptors, activities shared by bacterial LPS. Our study sought to determine if taxol could regulate gene expression in murine macrophages and to examine further the ability of taxol to generate an LPS-like signal. Toward this end, the ability of taxol to induce TNF-alpha mRNA and five other genes (IL-1 beta, IP-10, D3, D7, and D8) associated with LPS-activation of macrophages was examined by Northern blot analysis. Taxol alone (1-30 microM) induced murine C3H/OuJ macrophages to secrete bioactive TNF-alpha and express increased levels of each of the six genes under investigation. The magnitude and the kinetics of induction of each gene closely resembled that seen with Escherichia coli K235 LPS. Macrophages from LPS-hyporesponsive C3H/HeJ mice, however, failed to induce detectably any of the genes in response to taxol, despite being sensitive to the microtubule stabilizing effects of taxol as determined by immunofluorescence microscopy. The gene induction activity of taxol was in marked contrast to an alternative macrophage activator, heat killed Staphylococcus aureus, which induced a distinct gene profile in C3H/OuJ macrophages and which was equally active in C3H/OuJ and C3H/HeJ macrophages. These data are consistent with an ability of taxol to generate an LPS-like signal, possibly through a common signaling intermediate. As a first step toward identifying signal responses shared by taxol and LPS, we have shown that taxol, as shown previously for LPS, rapidly induces the tyrosine phosphorylation of a 41- and 42-kDa protein.
Human serum amyloid P component oligomers bind and activate the classical complement pathway via residues 14-26 and 76-92 of the A chain collagen-like region of C1q.
Authors: Ying S C , Gewurz A T , Jiang H , Gewurz H
Published: December 2022

Serum amyloid P component (SAP) was polymerized using the cleavable cross-linker 3,3'-dithio-bis-(sulfo-succinimidylpropionate) to study its interaction with the C system. Dimers and trimers, but no larger oligomers, were observed; the trimers retained native SAP immunoreactivity (except for one calcium-dependent epitope) without displaying neo-SAP epitopes. The SAP trimers bound strongly to C1q, at the level of the collagen-like region (CLR). SAP bound to synthetic C1q A chain peptides 14-26 and 76-92, and these peptides inhibited the binding of SAP trimers to the CLR. When incubated in dilute human serum, SAP trimers consumed total C and C4, but not alternative pathway, hemolytic activities. Consumption of C4 by SAP trimers was inhibited by C1q A chain peptide 14-26. Thus, SAP oligomers bind C1q and activate the classical C pathway via the collagen-like region of C1q, at sites located within residues 14-26 and/or 76-92 of the C1q A chain.
Follicular dendritic cells and B cell costimulation.
Authors: Burton G F , Conrad D H , Szakal A K , Tew J G
Published: December 2022

Ag-bearing follicular dendritic cells (FDC) are found throughout secondary lymphoid tissues in close association with rapidly proliferating germinal center B lymphocytes. We reasoned that FDC might provide costimulatory signals that would enhance the ability of Ag to stimulate B cell proliferation in the germinal centers. To test this, FDC were cultured with B cells activated by a slg-dependent (goat anti-mouse mu conjugated to dextran (anti-mu-dex)) or -independent (LPS) pathway and their proliferation was measured by using [3H]thymidine incorporation. The addition of FDC markedly augmented B cell proliferation in a dose-dependent fashion. Depletion of FDC from cultures abrogated the increased proliferation. Addition of highly purified FDC obtained from cell sorting resulted in B cell costimulation, whereas addition of other sorted cells was without effect. The FDC accessory activity was apparent over the entire culture period and over a wide range of either polyclonal B cell activator. When B cells and activators were cultured in the absence of FDC, only about one fourth of the cells remained viable after 3 days. In contrast, virtually all cells in cultures containing FDC, B cells, and activator were viable. Cultures containing FDC and B cells from nude mice proliferated normally in the presence of anti-mu-dex plus rIL-4, implying that IL-4 provides adequate T cell help in this system. The costimulatory activity of the FDC could not replace either the anti-mu-dex or IL-4 in this system and was not MHC restricted. These data support the concept that FDC not only provide Ag but also facilitate B cell proliferation by means of other costimulatory interactions that contribute to make the microenvironment in the germinal center favorable for B cells to proliferate.
Killer cell recruitment and renewal capacity of purified cytolytic and noncytolytic human peripheral blood natural killer cell subsets.
Authors: Lebow L T , Jewett A , Bonavida B
Published: December 2022

The inability to isolate NK precursors at different stages of development has impeded understanding of the processes involved in NK maturation. The present studies utilize a flow cytometric technique that enables the isolation of operationally defined cell subsets, lytic (killers) and nonlytic conjugate-forming (binders), and nonconjugate-forming (free) cells, within NK-enriched preparations to test whether these might represent cells in different stages of NK development. To characterize both the steps involved in NK maturation and the cells responsible for IL-2 induced proliferation, these purified subsets were analyzed for their killer cell recruitment and renewal capacity. After a 2-h exposure to IFN-alpha or IL-2, induction of lytic function was developed only in the binder subset as detected in the single cell assay. Neither enhancement of killer cell recycling nor induction of binding function among the subsets was observed. However, after an 18-h culture period, with or without rIL-2, killer cells preferentially expressed activation Ag CD69 (Leu-23) and the IL-2R alpha-chain, TAC (CD25). In addition, all cells in contact with K562 targets displayed enhanced expression of these Ag. Killer cells also showed an enhanced capacity to proliferate in response to rIL-2 in a 6-day [3H]TdR incorporation assay. Additional irradiated K562 targets enhanced the proliferative capacity of all the subsets, with only a marginal effect on sorted free cells. Nevertheless, sorted free cells, in addition to binders, developed potent binding and lytic function when tested in the single-cell assay after 4 days of IL-2 culture. The lytic activity of killers was reduced, as compared with freshly isolated killers. The results are consistent with a two-step model for NK maturation, involving the acquisition of lytic function before proliferative capacity, and specific triggering of killer cells through interaction with target cells for induction of a proliferative competent state.
Uncoupling of early signal transduction events from effector function in human peripheral blood neutrophils in response to recombinant macrophage inflammatory proteins-1 alpha and -1 beta.
Authors: McColl S R , Hachicha M , Levasseur S , Neote K , Schall T J
Published: December 2022

Macrophage inflammatory proteins-1 (MIP-1) alpha and beta are members of the C-C branch of the platelet factor 4 superfamily of cytokines, recently designated the "chemokine" superfamily. It has been suggested that the major cellular targets for the biologic activities of the C-C chemokines are the mononuclear leukocytes. However, the original designation of murine MIP-1 proteins as inflammatory mediators was based on suggestions that they activated neutrophil functions such as chemotaxis, the respiratory burst, and degranulation. In this study, we have evaluated the ability of human (Hu) MIP-1 alpha and beta to affect purified human neutrophil function. Although both rHuMIP-1 alpha and -1 beta stimulated significant calcium mobilization in human monocytes, only HuMIP-1 alpha exerted a detectable effect on neutrophils. HuMIP-1 alpha stimulated a small, dose-dependent increase in intracellular calcium, which was accompanied by a simultaneous change in right-angle light scatter, the latter indicating induction of shape change. While the effect of HuMIP-1 alpha on calcium mobilization in neutrophils was small when compared with that elicited by IL-8 or Gro alpha, it had similar characteristics to that by other receptor-dependent neutrophil agonists in that it was dependent on pertussis toxin-sensitive G proteins and on both mobilization of calcium from intracellular sources as well as influx from the extracellular environment. In addition, stimulation of neutrophils with HuMIP-1 alpha led to desensitization to subsequent additions of HuMIP-1 alpha. The stimulatory effect of HuMIP-1 alpha on neutrophil calcium mobilization and shape change was not coupled to other standard measures of neutrophil effector function. For instance, neither HuMIP-1 alpha nor -1 beta had any detectable stimulatory effect on the Na+/H+ antiport, degranulation, actin polymerization, or chemotaxis. Moreover, although HuMIP-1 alpha binding could easily be measured on monocytes or monocytic cell lines, the number of sites were too few to characterize on neutrophils by the same technique. Taken together, these results show that neither HuMIP-1 alpha nor -1 beta stimulate significant neutrophil activation and support the concept that the biologic effects of members of the C-C branch of the platelet factor 4 superfamily are not primarily directed toward neutrophils.