bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple nuclear factors interact with sequences within the J beta 2-C beta 2 intron of the murine T cell receptor beta-chain gene.
Authors: Mauxion F , Pray M G , Sen R
Published: December 2022

Identification of tissue-specific DNaseI hypersensitive sites in the TCR J beta 2-C beta 2 intron has suggested the presence of sequences involved in the regulation of gene expression. Therefore, we have searched for protein-DNA interactions within a 930-bp fragment derived from the J beta 2-C beta 2 intron by in vitro DNaseI protection experiments and electrophoretic mobility-shift assays. This analysis has revealed, in addition to a previously characterized NF-kappa B binding site, the presence of seven potential protein-DNA interaction sites within this fragment. Interestingly, they are clustered in the regions where in vivo T cell-specific nuclease hypersensitive sites have been previously identified. Binding sites for four potential transcription factors have been mapped precisely by methylation-interference experiments. Sequence comparisons show that one of them is homologous to the Y box present in the promoter regions of MHC class II genes. Identification of several protein-DNA interactions clustered within the J beta 2-C beta 2 intron and the presence of binding sites for two well-characterized transcription factors suggest a transcriptional regulatory function for this region.
Characterization of IL-2 receptor expression and function on murine macrophages.
Authors: Cox G W , Mathieson B J , Giardina S L , Varesio L
Published: December 2022

This study was designed to examine the expression and function of IL-2R on murine macrophages. We used a model system of murine macrophage cell lines (ANA-1 and GG2EE) that was established by infecting normal murine bone marrow-derived cells with the J2 (v-raf/v-myc) recombinant murine retrovirus. ANA-1 macrophages did not constitutively express detectable levels of mRNA for the p55, IL-2R alpha. However, a brief exposure to IFN-gamma was sufficient to induce IL-2R alpha mRNA in ANA-1 macrophages. Flow cytometric analysis indicated that ANA-1 macrophages expressed low constitutive levels of IL-2R alpha on their cell surface that were augmented after treatment of the cells with IFN-gamma. Affinity binding and cross-linking of [125I]IL-2 to ANA-1 macrophages demonstrated that IL-2R alpha and the p70-75, IL-2R beta were both present on ANA-1 macrophages constitutively. IFN-gamma increased the expression of IL-2R alpha on ANA-1 macrophages but did not increase the expression of IL-2R beta on these macrophages. Although IL-2 alone did not induce the tumoricidal activity of ANA-1 macrophages, IL-2 acted synergistically with IFN-gamma to induce macrophage tumoricidal activity. These data demonstrate the expression of IL-2R on murine macrophage cell lines and establish the role of IL-2 as a costimulator of macrophage-mediated tumoricidal activity.
IFN-gamma enhances endothelial activation induced by tumor necrosis factor but not IL-1.
Authors: Doukas J , Pober J S
Published: December 2022

Previous studies have established that different cytokines induce distinct patterns of activation in cultured endothelial cells (EC). Treatment of EC with either TNF or IL-1 causes transient induction of endothelial leukocyte adhesion molecule-1 (ELAM-1) and a sustained increase in intercellular adhesion molecule-1 (ICAM-1) expression. TNF but not IL-1 also increases class I MHC Ag expression. IFN-gamma, which by itself increases EC class I MHC and ICAM-1 but does not induce ELAM-1 expression, has been found to act synergistically with TNF to increase class I expression. In our study, we have further examined IFN-gamma effects on both TNF and IL-1 beta responses. In contrast to IFN-gamma plus TNF cotreatment, IFN-gamma up-regulation of class I MHC molecules is not augmented by cotreatment with IL-1 beta. IFN-gamma plus TNF cotreatment synergistically increases ICAM-1 expression by 24 h of cotreatment, whereas IFN-gamma plus IL-1 beta cotreated EC show at most additive increases. IFN-gamma increases TNF-induced ELAM-1 expression such that a greater number of EC express ELAM-1 at both 4 and 24 h in the presence of IFN-gamma plus TNF compared to cultures treated with TNF alone, although the maximal level of surface expression on individual cells is not increased. Inasmuch as these times represent pre- and post-peak expression time (6 h), respectively, IFN-gamma appears both to accelerate and to prolong transient ELAM-1 expression. Although similar interactions are seen with IL-1 beta, IFN-gamma has a consistently greater effect on TNF-induced compared to IL-1-induced ELAM-1 expression. We next explored the possible mechanism(s) of the synergy between IFN-gamma and TNF. IFN-gamma and TNF do not cooperatively enhance total protein synthesis. Unexpectedly, IFN-gamma and IL-1 beta combine to depress protein synthesis, which may contribute to the failure of these cytokines to positively interact. IFN-gamma and TNF cooperatively increase ELAM-1 mRNA at 6 h and ICAM-1 mRNA at both 6 and 24 h, whereas IFN-gamma and IL-1 show markedly less cooperative augmentation of these transcripts. We conclude that IFN-gamma enhances TNF-induced EC activation by selectively and synergistically increasing synthesis of specific surface molecules, whereas IL-1-induced EC activation is largely unaffected by IFN-gamma.
Synergistic effect of IL-4 and IFN-gamma on the expression of polymeric Ig receptor (secretory component) and IgA binding by human epithelial cells.
Authors: Phillips J O , Everson M P , Moldoveanu Z , Lue C , Mestecky J
Published: December 2022

The expression of secretory component (SC), the epithelial receptor for polymeric Ig, was enhanced by the addition of human rIFN-gamma or rIL-4, as revealed by the binding of radiolabeled polymeric, J chain-containing IgA or anti-SC antisera to the human colonic adenocarcinoma epithelial cell line HT-29. In combination, these cytokines exhibited a synergistic effect, and the potentiating effect of IL-4 was inhibitable by polyclonal anti-IL-4 antisera. Because the binding of radiolabeled polymeric IgA (pIgA) to HT-29 cells was inhibited by unlabeled pIgA or a polyclonal anti-SC reagent, but not by IgG, monomeric IgA, or Fab alpha fragments, we conclude that the receptor involved in the increased binding of pIgA is indeed SC. These data suggest that the expression of SC on human epithelial cells and the subsequent binding of pIgA (produced in mucosal tissues and glands by subepithelial plasma cells) is regulated by lymphokines such as IL-4 and IFN-gamma that are presumably derived from T cells found in abundant numbers in these tissues. These findings demonstrate a novel pathway of interaction between T cell products and epithelial cells that may result in enhanced translocation of large amounts of locally produced pIgA through epithelial cells into external secretions.
Autoantibodies to thyroglobulin are encoded by diverse V-gene segments and recognize restricted epitopes.
Authors: Gleason S L , Gearhart P , Rose N R , Kuppers R C
Published: December 2022

Murine experimental autoimmune thyroiditis has been used as a model for human autoimmune thyroiditis. Experimental autoimmune thyroiditis is induced in mice by immunization with mouse thyroglobulin (Tg) in CFA. To characterize the antibodies to this autoantigen, we have studied the binding specificities and determined the nucleotide sequences of monoclonal anti-Tg antibodies. The specificities of the mAb for determinants on Tg varied extensively. Seven of 16 mAb showed reactivity to only mTg, 4 reacted to Tg from more than one species and four reacted to a variety of Ag. Many of the mAb were competitively inhibited by thyroid hormones, suggesting that they recognize the hormonogenic sites on the Tg molecule. The mAb could be divided into at least seven reactivity patterns based on reciprocal competitive inhibition studies, indicating that mTg contains at least seven antigenic regions. DNA sequence analysis of the mAb showed that a large number of V region gene segments encoded the H and L chains. No evidence for preferential use of any V region family or gene segment was found. Gene segments from the VH 7183, Q52, J558, and VH10 families were used by heavy chains, and the V kappa 1, 4, 8, 9, 19, and 21 families were used by kappa-chains. The results indicate that the antigenic epitopes on mTg elicit a very diverse autoantibody response that is derived from a large number of V region gene segments. Many of these autoantibodies show specific reactivity with mTg indicating they recognize species specific epitopes. The results suggest that clonal deletion of autoreactive Ab to certain self-epitopes may not occur.
IL-3 augments adhesiveness for endothelium and CD11b expression in human basophils but not neutrophils.
Authors: Bochner B S , McKelvey A A , Sterbinsky S A , Hildreth J E , Derse C P , Klunk D A , Lichtenstein L M , Schleimer R P
Published: December 2022

A number of natural and recombinant human cytokines have been tested for their ability to activate basophil and neutrophil adhesiveness for human umbilical vein endothelial cells in vitro. Coincubation of basophils and endothelial cell monolayers for 10 min with biologically relevant concentrations of rIL-1, natural IL-2, rIL-4, rIL-5, rIL-6, rIL-8, rGM-CSF, and rIFN-gamma had no effect on basophil adhesiveness. In contrast, rIL-3 induced basophil adhesiveness for endothelial cells (optimal at 1 ng/ml: 144 +/- 18% of control adherence (mean +/- SEM); control basophil binding, 13 +/- 3%, n = 9, p less than or equal to 0.05). This increase in adhesiveness was similar in magnitude to that induced by an optimal concentration of a known potent inducer of basophil adhesiveness (1 microM FMLP, 164 +/- 15% of control adherence, n = 9). Under these experimental conditions, the effects of rIL-3 occurred at concentrations of 0.1 to 30 ng/ml, were partially dependent on calcium, and were not accompanied by histamine release. Fixation experiments demonstrated that the effect of rIL-3 was directed against the basophil rather than the endothelial cell. Neither rIL-3 nor the other cytokines tested had any effect on the adherence of 51Cr-labeled neutrophils, even when tested simultaneously on cells from the same donors. Under experimental conditions that permitted histamine release, no correlation was seen between the ability of rIL-3 (0.3 to 300 ng/ml) to induce histamine release or enhance adhesiveness (n = 8). mAb blocking experiments demonstrated a role for both CD11 and CD18 adherence glycoproteins in basophil adherence induced by rIL-3, and indirect immunofluorescence and flow cytometric analysis revealed that rIL-3 treatment led to rapid and sustained increases in cell surface expression of CD11b antigens on basophils but not neutrophils (e.g., after 10 min: 217 +/- 29 vs 91 +/- 11% of control mean fluorescence intensity, p less than 0.05). However, no correlation was seen between the magnitude of changes in CD11b expression and changes in adhesion when tested simultaneously. These results suggest that local production of IL-3 during allergic reactions in vivo may selectively promote basophil activation, adhesion to endothelium, and recruitment to extravascular sites of inflammation.
Antiproliferative effect of IFN-gamma on proliferation of mouse connective tissue-type mast cells.
Authors: Takagi M , Koike K , Nakahata T
Published: December 2022

Mouse rIFN-gamma suppressed clonal growth of tissue-type mast cells (CTMC) separated from mouse peritoneal cells (greater than or equal to 98% purity) in methylcellulose culture containing IL-3 + IL-4 in a dose-dependent fashion. The IFN-gamma-mediated suppression was also observed both in serum-free culture and in single-cell culture. In addition, mAb to mouse IFN-gamma neutralized the suppressive effect of IFN-gamma on CTMC proliferation supported by the combination of IL-3 + IL-4. These observations suggest a direct antiproliferative effect of IFN-gamma on CTMC. No suppression of CTMC proliferation was observed when CTMC were washed with alpha-medium after 24 h of preincubation with IL-3 + IFN-gamma and then cultured in methylcellulose medium containing IL-3 + IL-4. IFN-gamma had suppressive effects on CTMC after preincubation with IL-3 + IL-4. These preincubation studies suggest that IFN-gamma exerts a reversible inhibitory effect on CTMC which have entered into a proliferative state by IL-3 + IL-4, and has neither cytotoxic nor irreversible suppressive effects on resting CTMC in cultures containing IL-3 alone. CTMC proliferation after stimulation with the cross-linking of cell-bound IgE by specific Ag in the presence of IL-3 was also suppressed by IFN-gamma. By contrast, proliferation of CTMC induced by the combination of 12-O-tetradecanoyl phorbol-13-acetate + IL-3 was not affected by the addition of IFN-gamma to the culture. Based on these observations, we suggest that IFN-gamma antagonizes the colony inducing effect of IL-4 and IgE-Ag stimulation which act synergistically with IL-3 on CTMC.
Neutrophil adherence receptors (CD 18) in ischemia. Dissociation between quantitative cell surface expression and diapedesis mediated by leukotriene B4.
Authors: Welbourn R , Goldman G , Kobzik L , Paterson I , Valeri C R , Shepro D , Hechtman H B
Published: December 2022

Neutrophils and eicosanoid chemoattractants are centrally involved with ischemia-reperfusion (I/R) injury. The CD 18 complex of adhesive glycoproteins, readily up-regulated by chemoattractants in vitro, is required for polymorphonuclear leukocyte (PMN) adherence to endothelium. This study tests whether CD 18 is up-regulated by ischemia in vivo and its role in mediating PMN diapedesis. Anesthetized rabbits underwent 3 h of bilateral hindlimb tourniquet ischemia (n = 16). Ten min after tourniquet release, levels of plasma leukotriene (LT)B4 increased to 390 +/- 62 pg/ml (mean +/- SE), higher than 134 +/- 26 pg/ml in control rabbits (n = 13, p less than 0.01). Aliquots of plasma were added to whole blood from normal rabbits (n = 6) for flow cytometric analysis of neutrophils with the CD 18 mAb R 15.7. Addition of I/R plasma failed to demonstrate an increase in surface expression of CD 18. Similarly, no CD 18 up-regulation was observed in vivo upon reperfusion in ischemic animals pretreated with mAb R 15.7 (n = 3). However, I/R plasma when introduced into plastic chambers taped atop dermabrasion sites in normal rabbits (n = 12) resulted in diapedesis, measured by the accumulation after 3 h of 1130 +/- 125 PMN/mm3 in the chambers relative to 120 +/- 31 PMN/mm3 with control plasma (p less than 0.01). Diapedesis in response to I/R plasma was abolished by pretreatment with mAb R 15.7 (less than 5 PMN/mm3, n = 6), was reduced by U 75,302, an LTB4 receptor antagonist (253 +/- 101 PMN/mm3, n = 6) (both p less than 0.01) and was not protein synthesis dependent. These results demonstrate that PMN diapedesis in response to I/R plasma is exclusively dependent upon the CD 18 glycoprotein complex by an LTB4-dependent mechanism, despite the fact that CD 18 is not up-regulated on circulating PMN in ischemia. These data indirectly indicate the functional importance of conformational changes of CD 18 in determining PMN adhesion.
Marked difference in the in vivo antitumor efficacy between two immunotoxins targeted to different epitopes of common acute lymphoblastic leukemia antigen (CD10). Mechanisms involved in the differential activities of immunotoxins.
Authors: Luo Y , Seon B K
Published: December 2022

In the present study, two isotype-matching mAb, SN5d and SN5, which are directed toward two distinctively different epitopes of common acute lymphoblastic leukemia Ag (CD10) but show a very similar binding affinity to leukemia cells, were compared for their in vivo antitumor activity after conjugated to ricin A chain (RA). Our recently established nude mouse model carrying an ascitic tumor of NALM-6 human pre-B leukemia cells was used as the tumor model. A marked difference was observed in the in vivo antitumor efficacy between SN5d-RA and SN5-RA; SN5d-RA was much more effective than SN5-RA. Several experiments were carried out to gain information concerning the mechanisms involved in the different antitumor efficacy of the two immunotoxins. Although naked (unconjugated) mAb SN5d was much less effective than SN5d-RA conjugates in the in vivo tumor suppression, mAb SN5d was more effective than mAb SN5 in the in vivo tumor suppression. Additionally, marked differences were found between SN5d and SN5 in the induction of antigenic modulation and in the regulation of Ag biosynthesis and expression. Binding of SN5 to NALM-6 leukemia cells caused strong antigenic modulation (down-regulation of Ag expression) and strongly down-regulated Ag biosynthesis and cell surface expression of new Ag. In contrast, binding of SN5d to NALM-6 leukemia cells caused little modulation of overall cell surface expression of common acute lymphoblastic leukemia Ag; the decrease of old Ag by endocytosis after binding to mAb SN5d was compensated by newly exocytosed cell-surface expressed Ag. The present results appear to reveal a novel mechanism which regulates cytotoxic activities of antibodies and immunoconjugates.
Suppressor cell clones specific for ultraviolet radiation-induced tumors. Function and surface proteins.
Authors: Trial J , McIntyre B W
Published: December 2022

Lymphocytes were cloned from animals bearing UV-induced skin tumors. These cells were I-J+, CD4-, CD8-, and had become growth factor independent. Extracts, but not supernatants, of these clones suppressed primary immune reactions in vitro against UV-induced tumors, but not methylcholanthrene-induced tumors. The cells therefore had the functional characteristics of afferent suppressor T cells directed against a common Ag on UV-induced tumors. Surface iodination of the clones revealed an extremely low level expression of molecules that might be TCR or related molecules.
Mechanisms accounting for lymphocytic alveolitis in hypersensitivity pneumonitis.
Authors: Trentin L , Migone N , Zambello R , di Celle P F , Aina F , Feruglio C , Bulian P , Masciarelli M , Agostini C , Cipriani A
Published: December 2022

Hypersensitivity pneumonitis (HP) is a lung disorder characterized by an exaggerated accumulation of CD8+ T lymphocytes in the pulmonary parenchyma. To investigate the mechanisms accounting for the T cell alveolitis taking place in the lung of HP patients and their pattern of growth, cells recovered from the bronchoalveolar lavage (BAL) of seven patients were evaluated for: 1) the expression of activation markers, including IL-2R (p55 and p75 subunits), HLA-DR and VLA-1 Ag; 2) the ability of IL-2 and IL-4 to induce in vitro proliferation; 3) the capability to synthesize and release IL-2 by determining the levels of IL-2 in BAL cell-free supernatants and by evaluating the presence of mRNA transcripts for IL-2; and 4) the molecular configuration of the beta- and gamma-genes of the TCR. This study demonstrates that a high number of BAL lymphocytes recovered from the lungs of HP patients express activation markers including the p75 chain of IL-2R, VLA-1, and HLA-DR Ag. These cells express the CD3+,CD8+,CD16-,CD56+ phenotype and proliferate in vitro in the presence of IL-2 but do not release this cytokine. Furthermore, IL-2 transcripts could not be detected in BAL resting T lymphocytes. No proliferation was observed in the presence of IL-4. The analysis of the configuration of the TCR beta- and gamma-genes showed a polyclonal pattern, with the exception of one case in which extra bands were observed following digestion with BamHI and EcoRI restriction enzymes. Taken together, our data suggest that the IL-2 system may play a central role in the mechanisms accounting for lymphocytic alveolitis in HP patients. Although the pattern of growth is usually polyclonal, such polyclonal recruitment seems to be biased toward cells that have rearranged and possibly expressed particular V beta or V gamma genes, thus leading to the hypothesis that the events that take place in the lung of these patients may occasionally elicit an oligoclonal expansion of the cells proliferating in lung parenchyma.
Anti-CD3 antibody induces rapid expression of cytokine genes in vivo.
Authors: Scott D E , Gause W C , Finkelman F D , Steinberg A D
Published: December 2022

Anti-CD3 antibody was administered to mice i.v. and the kinetics of spleen cell cytokine mRNA expression studied by Northern analysis. Untreated mice and mice receiving control antibody had low or undetectable amounts of mRNA for c-fos, c-myc, IL-2, IL-4, and IFN-gamma. After injection of anti-CD3 antibody, substantial increases in all were found. Induction of c-fos was detected at 10 min and of c-myc at 30 min after injection. IL-2, IL-4, and IFN-gamma mRNA were induced by 30 min and reached peak levels at 60 min. Thereafter, IL-2 and IL-4 mRNA declined, whereas IFN-gamma mRNA persisted. The induced cytokine mRNA was not observed in athymic nu/nu mice nor in normal spleen cells from which T cells had been depleted in vitro. The early in vivo induction of IL-4 mRNA contrasts with prior in vitro studies in which IL-4 production was difficult to detect after primary stimulation. To assess the possibility that many T cells had been preprimed in vivo, germfree mice were compared with conventional mice and no differences in cytokine mRNA were found. These data show that T cell-dependent IL message production can be induced rapidly in vivo without prepriming and that the cytokine messages induced after anti-CD3 antibody administration do not suggest a predominance of either Th1 or Th2 type cells.
First T cell receptor alpha gene rearrangements during T cell ontogeny skew to the 5' region of the J alpha locus.
Authors: Thompson S D , Pelkonen J , Hurwitz J L
Published: December 2022

Fetal, neonatal, and early postnatal thymi were assessed for TCR J alpha gene rearrangements. Gene probes spanning the distance from 5' to 3' regions of the J alpha locus were used to determine the approximate location of gene rearrangements within hybridomas representing each of the early thymocyte populations. The predominant location of rearrangements was within the 5' region of the J alpha locus. Among the several cells in which rearrangements were found on only one chromosome, the one rearrangements was always in the 5' region. When two rearrangements were found, the rearrangements on homologous chromosomes were usually in the same region. The overall pattern among thymocytes was in great contrast to that previously observed among hybridomas derived from stimulated adult spleen cells within which rearrangements fell mostly to the 3' side of the alpha-locus. Results reveal the nonrandom nature of the TCR-alpha gene rearrangement event and may reflect an incidence of multiple V-J alpha joining events on each chromosome during T cell development in vivo. Due to the fact that most mature cells bear two J alpha joins, the allelic exclusion of alpha-chains cannot be explained by a mechanism whereby a functional rearrangement on one chromosome inhibits subsequent rearrangement on the second. Instead allelic exclusion may rely on a low frequency of productive vs nonproductive rearrangement events and an incompatibility between multiple alpha- and beta-protein pairs.
Clonal anergy of memory B cells in epitope-specific regulation.
Authors: Galelli A , Charlot B
Published: December 2022

Immunization of carrier (keyhole limpet hemocyanin (KLH)) primed mice with the hapten-carrier TNP-KLH induces specific suppression for the IgG anti-TNP-response without interfering with the response to epitopes on the carrier molecule. To examine the status of hapten-specific memory B cells from suppressed mice, highly enriched populations of TNP-specific memory B cells were purified from the spleen of TNP-KLH (control) or KLH/TNP-KLH (suppressed) immunized mice and tested in vitro for their ability to respond to TD or TI (TNP-KLH, TNP-LPS) antigenic challenge in presence of a KLH-specific Th cell line. Similar numbers of TNP-specific B cells with the characteristics of memory B cells were obtained from control and suppressed mice. TNP-specific B cells from suppressed mice could be triggered to IgG production by TNP-LPS but had an impaired ability to differentiate into IgG-secreting cells in response to TNP-KLH. This impaired IgG response to TNP-KLH was not due to an active suppression by a subset of TNP-specific B cells, or to an impedence of memory cells to a class switching but to an intrinsic memory B cell defect. TNP-specific B cells from suppressed mice were as efficient as memory B cells from control mice to present TNP-KLH to KLH-specific Th cells and to proliferate in response to T cell help. Our data support the view that the effector mechanism of epitope specific regulation does not interfere with the development of hapten-specific memory B cells but that these cells have an intrinsic defect that prevents their differentiation into active IgG antibody secreting cells in response to a T-dependent antigenic challenge.
Activation of tyrosine phosphorylation in human T cells via the CD2 pathway. Regulation by the CD45 tyrosine phosphatase.
Authors: Samelson L E , Fletcher M C , Ledbetter J A , June C H
Published: December 2022

In this study we compare the effect of CD3 and CD2 ligation on tyrosine kinase activation in human peripheral blood T cells. Using antiphosphotyrosine antibody to detect tyrosine phosphorylation of cellular substrates, we demonstrate that mAb stimulation of either CD3 or CD2 results in tyrosine phosphorylation of the TCR-zeta chain and 135- and 100-kDa proteins. However, differences are observed between CD3 and CD2 ligation; only the former results in rapid tyrosine phosphorylation of 72-, 65-, and 40-kDa substrates. Co-aggregation of CD2 and CD45, a tyrosine phosphatase, results in inhibition of intracellular calcium elevation and T cell proliferation. We demonstrate in this study that this manipulation also inhibits polyphosphoinositide hydrolysis and tyrosine phosphorylation of the 100-kDa substrate. The failure of tyrosine phosphorylation of the 100-kDa substrate is specific in that phosphorylation of the 135-kDa protein is not inhibited. Similar results are observed when CD2 and CD45 are independently cross-linked rather than co-aggregated. The observation that CD45 cross-linking alters tyrosine phosphorylation of T cell substrates and effects polyphosphoinositide hydrolysis is further evidence that tyrosine phosphorylation regulates early events in T cell activation including, perhaps, phospholipase C activity.
Functional and biochemical characterization of mouse P40/IL-9 receptors.
Authors: Druez C , Coulie P , Uyttenhove C , Van Snick J
Published: December 2022

The existence of saturable and specific binding sites for mouse P40/IL-9 was demonstrated on a variety of factor-dependent T cell lines derived from Th clones by long term culture in the presence of P40-containing T cell supernatants. Scatchard transformation of the data obtained with one such line was consistent with the existence of a single class of receptors with a Kd of approximately 100 pM and a density of 3000/cell. P40 binding to these cells was followed by rapid internalization of the ligand. P40-receptors (P40-R)3 were also found on certain Th clones maintained in conventional cultures, especially after stimulation with Ag and APC. Only T cell clones that proliferated in response to P40 showed significant levels of binding, suggesting that the regulation of P40-R expression is an important element in the control of P40-responsiveness. In accord with this idea, fresh T cells, cytolytic T cell clones and a wide variety of other cells including B cells and fibroblasts, which do not proliferate in response to P40, showed no significant binding. However, P40-R were not restricted to a few unusual Th clones. They were also detected on several T cell tumors, on macrophages and on mast cell lines. The latter point is of particular interest in view of the mast cell growth factor activity recently ascribed to P40. Cross-linking studies with T-cell lines and mast cells indicated that the P40-R consists of a 64-kDa glycoprotein, the molecular mass of which is reduced to 54 kDa on treatment with N-glycosidase F.
Distinct adhesive properties of granulocytes and monocytes to endothelial cells under static and stirred conditions.
Authors: Kuijpers T W , Hakkert B C , van Mourik J A , Roos D
Published: December 2022

Adherence of neutrophils and monocytes to endothelium is an important event in the sequence of leukocyte responses to inflammatory disease. Double-color flow microfluorimetry analysis was used to determine neutrophil and monocyte adherence to suspended endothelial cells under stirred conditions. The static adherence to endothelial cell monolayers was simultaneously determined. In both assays, neutrophils behaved in a similar way. Basal adherence of neutrophils was very low. Activation by PMA or by the chemoattractants platelet-activating factor and FMLP induced rapid binding, primarily mediated by CR3. Nonactivated neutrophils showed CD18-dependent (lymphocyte function-associated Ag-1 and CR3) and CD18-independent adherence to endothelial cells when the endothelial cells were prestimulated with rIL-1 beta. In contrast to neutrophils, nonactivated monocytes adhered avidly to resting endothelial cells. This adherence was partly CD18 dependent and partly CD18 independent. Whereas monocyte adherence under stirred conditions strongly increased upon activation by PMA, a significant decrease in adherence was found under static conditions, which was prevented by cytochalasin B. This decrease was limited to a distinct CD18-independent binding mechanism, and absent under stirred conditions. We conclude that monocytes adhere with (at least) three binding mechanisms to endothelial cells, a CD18-dependent and two CD18-independent mechanisms.
Gene transcription during in vitro activation of human B lymphocytes with Staphylococcus aureus Cowan I strain.
Authors: Boumpas D T , Anastassiou E D , Tsokos G C , Thyphronitis G , Balow J E
Published: December 2022

With the use of nuclear transcription run-off assays we have determined the kinetics of transcriptional activity for the c-fos, c-myc, DR beta, DQ beta, (TfR), Blast-1, and Ig kappa genes in human tonsillar B lymphocytes of high to intermediate density after stimulation with Staphylococcus aureus Cowan I strain (SAC). Nuclei were isolated at various times after stimulation with SAC. Stimulation with SAC resulted in significant increase in the transcription of all of the genes. Maximum expression of the c-fos, transferrin receptor, DR beta, and DQ beta genes was obtained within 0.5 h after stimulation. Maximum expression of c-myc and Blast-1 was obtained within 3 h. Peak expression of Ig kappa was observed at 8 h. Transcriptional activities of all genes examined, except for the Blast-1 and Ig kappa, decreased rapidly after their respective peaks; they remained at higher levels than their base line. Inhibition of protein synthesis by cycloheximide did not significantly affect the kinetics of transcription of these genes. These data demonstrate the cascade of transcriptional activity of various genes associated with human B cell activation and indicate that the induction of their transcription is independent of de novo protein synthesis.
Expression and characterization of TCA3: a murine inflammatory protein.
Authors: Wilson S D , Kuchroo V K , Israel D I , Dorf M E
Published: December 2022

TCA3 is a cDNA originally isolated from activated T cells. Transcription of this gene has been shown to correlate with Ag-induced cellular activation of both T cells and mast cells. Based on the predicted amino acid sequence encoded by the cDNA, we previously proposed that TCA3 represents a cytokine. In this report we have used rDNA technology to express TCA3 in two mammalian cell lines. In both cases, TCA3 was expressed as a secreted molecule with an apparent molecular mass of 16 kDa. Digestion of the (rTCA3) with the enzyme N-glycanase revealed that approximately 8 kDa is caused by N-linked glycosylation. Intradermal injection of rTCA3 into mouse footpads resulted in a rapid swelling response. The sites of injection were characterized histologically by a local accumulation of neutrophils. These findings are discussed with particular attention to a family of related proteins, some of whose members also have inflammatory properties.
Prevalence and polymorphism of human VH3 genes.
Authors: Sasso E H , Van Dijk K W , Milner E C
Published: December 2022

To better understand heterogeneity of human VH genes, we have studied polymorphism among three homologous genes from VH3, the most complex human VH gene family. Sequence-specific oligonucleotide probes were used to identify the germ-line source of each gene. The fetally expressed gene 56p1, and the germ-line VH element 1.9III, were identified and shown to be distinct. Also identified was T5M10, a VH element that resembles 56p1 and 1.9III, and likely corresponds to the previously described germ-line gene, hv3005. The elements 56p1, 1.9III, and T5M10 were each polymorphic, and in 52 unrelated individuals, their prevalences were 62%, 92%, and 35%, respectively. Among individuals lacking the hybridization bands characteristic of 56p1, 1.9III, or T5M10, alternative RFLP or coding region sequence variants were not found. In the population studied, these three VH elements were present in five different combinations, and at least three haplotypes. The distribution of these VH elements indicated they are not alleles of a single locus, and 56p1 and 1.9III are probably not alleles of each other. These results suggest that polymorphism is commonplace among human VH3 genes, but that a limited number of conserved alleles dominate each locus. We conclude that heterogeneity in the VH germ-line repertoire may derive more from diversity of haplotypes than from diversity of alleles.
Murine monoclonal anti-idiotype antibody as a potential network antigen for human carcinoembryonic antigen.
Authors: Bhattacharya-Chatterjee M , Mukerjee S , Biddle W , Foon K A , Köhler H
Published: December 2022

Carcinomas of the gastrointestinal tract are not curable by standard therapies. Thus, new therapeutic approaches for this disease are needed. This study proposes the use of anti-Id mAb as Ag substitutes to induce anti-tumor immunity in gastrointestinal cancer patients. Recently, we have generated and characterized one monoclonal anti-Id antibody, designated 3H1 (Ab2), which mimics biologically and antigenically a distinct and specific epitope of the 180,000 m.w. carcinoembryonic antigen (CEA) primarily expressed in high density by human pancreatic and colonic tumor cells. This epitope is unique to CEA and not present on other CEA-related lower m.w. members of the Ag family also found on normal tissues. The antigenic determinant as defined by the mAb 8019 (Ab1) against which the Ab2, 3H1 was raised, is absent on normal adult tissues by immunoperoxidase staining and haematopoietic cells including granulocytes by flow cytometry analysis. Anti-Id (Ab2) 3H1 induced CEA-specific antibodies in mice and rabbits. The immune sera from both mice and rabbits competed with Ab1 for binding to the colon carcinoma cell line LS174T and inhibited the binding of radioiodinated Ab1 to Ab2. This indicates that anti-anti-Id (Ab3) in mice and rabbits share idiotopes with Ab1 (8019). Furthermore, monoclonal Ab3 that bind to CEA have been generated from mice immunized with 3H1. The Ab3 (both polyclonal as well as monoclonal) immunoprecipitated the same 180,000 m.w. CEA as Ab1 (8019) by Western blotting analysis and showed almost identical immuno-staining patterns as Ab1 on colonic adenocarcinoma tissue sections from several patients. Collectively these data suggest that Ab2 3H1 could potentially be used clinically as a network Ag for immunotherapy of patients with CEA positive tumors.
Intact antigen receptor-mediated generation of inositol phosphates and increased intracellular calcium in CD4 CD8 T lymphocytes from MRL lpr mice.
Authors: Budd R C , Winslow G , Inokuchi S , Imboden J B
Published: December 2022

The predominant T lymphocytes that accumulate in the peripheral lymphoid tissues of mice homozygous for the lpr gene bear the phenotype CD3+CD4-CD8-. By certain functional criteria these cells would appear to have impaired CD3-mediated signal transduction, in that they do not respond to alloantigen and produce little if any detectable IL-2 or other lymphokines. However, the signal pathway appears adequate for achieving other T cell functions, including induction of high affinity IL-2R, and thymic deletion. To clarify the basis of this seeming discrepancy, we examined transmembrane signal transduction in T cell subsets of lpr/lpr (lpr) and +/+ mice, as defined by increased [Ca2+]i and the generation of inositol phosphates (InsPs). Stimulation of lpr CD4-CD8- cells with anti-CD3 antibody produced prompt and sustained increases in the concentration of [C2+]i and in InsPs. Similar responses occurred in mature T cells from lpr and +/+ mice, except for the somewhat slower kinetics of their increased [Ca2+]i. In marked distinction to the anti-CD2-mediated response, Con A, even in high doses, could not stimulate any increase of [Ca2+]i in lpr CD4-CD8- cells, and only modest increases in InsPs. Mature T cells, whether of lpr or +/+ origin, yielded normal increased [Ca2+]i with Con A. The reason for the differences in signal transduction between anti-CD3 and Con A stimulation of lpr CD4-CD8- cells may relate to the absence of surface structures on these immature T cells that are required for activation by Con A but not by anti-CD3. The data demonstrate that the CD3 complex in lpr CD4-CD8- T cells can couple to phospholipase C to hydrolyze phosphoinositides. These activation properties of lpr CD4-CD8- T cells have interesting functional parallels to thymocytes at the time of thymic selection, as well as tolerance induction of mature T lymphocytes.
HLA-DR2a is the dominant restriction molecule for the cytotoxic T cell response to myelin basic protein in DR2Dw2 individuals.
Authors: Jaraquemada D , Martin R , Rosen-Bronson S , Flerlage M , McFarland H F , Long E O
Published: December 2022

The HLA-DR2 restriction of the T cell response to myelin basic protein (MBP) was studied using murine L cells transfected with DRalpha and either DR2a or DR2b beta-chain cDNA. DR2a and DR2b represent the two isotypic DRbeta chains expressed in DR2Dw2 haplotypes. Eleven MBP-specific cytolytic T cell lines derived from patients with multiple sclerosis were isolated. Two of these cell lines recognized MBP-pulsed DR2-expressing L cell transfectants and four of them could only recognize the L cells if the adhesion molecule ICAM-1 was expressed in addition to HLA-DR2. Five of the six lines were restricted by HLA-DR2a; one line recognized Ag in conjunction with DR2b, but only if ICAM-1 was coexpressed. The remaining five lines did not lyse MBP-pulsed L cells. The ability of the DR2b molecules on transfected cells to stimulate T cells was confirmed with DR2b-allospecific T cell clones. Although five MBP-specific lines were restricted by DR2a, they recognized different parts of the MBP molecule, as demonstrated by the presentation of shorter peptides. Thus, our results suggest that DR2a is a dominant restriction molecule in MBP-specific responses by DR2+ MS patients. The results also indicate that the reported heterogeneity in MBP epitopes recognized by DR2-restricted T cells, may not be due to the use of different restriction elements but rather to the binding of different MBP peptides to DR2a molecules.
Expression of the human IL-2 receptor on lymphocytes involves rapid turnover of its p55 alpha-subunit (Tac).
Authors: Sayar D , Ketzinel M , Gerez L , Silberberg C , Reshef A , Kaempfer R
Published: December 2022

Upon mitogenic stimulation, both mRNA encoding the p55 alpha-subunit (Tac) of the human IL-2R alpha and IL-2R alpha protein are induced and expressed in tonsil lymphocyte populations over several days. Using a quantitative dot-blot immunoassay for the IL-2R alpha subunit, a rapid disappearance of this polypeptide from cells is demonstrated in the presence of the translation inhibitor, cycloheximide. The half-life of IL-2R alpha subunit protein is 2 to 3 h. This decline in cell-associated IL-2R alpha subunit is matched by a rapid decline in IL-2R alpha on the cell surface and is not accompanied by any increase in soluble IL-2R alpha protein. Long term expression of the IL-2R on the cell surface is thus the result of continual synthesis and rapid breakdown of IL-2R alpha chains in the cell. Steady state expression of the IL-2R after an immune stimulus hence depends upon continuous expression of the IL-2R alpha subunit gene. Rapid turnover of the unstable alpha-subunit on the cell surface provides a novel mechanism for sensitive control of functional IL-2R expression.
Effects of IFN on human eosinophils in comparison with other cytokines. A novel class of eosinophil activators with delayed onset of action.
Authors: Valerius T , Repp R , Kalden J R , Platzer E
Published: December 2022

Extracellular killing is regarded as one of the main functions of eosinophils. Therefore, a cytotoxicity assay against antibody-coated Daudi-lymphoma cells was established to measure cytokine effects on peripheral blood eosinophils from healthy volunteers. Optimal time of exposure to cytokines and half optimal concentrations (EC50) were determined and the capability of various cytokines to enhance cytotoxicity of eosinophils was compared. Thus, after 24 h with cytokine, the highest activation of eosinophils was observed with recombinant human rhIFN-gamma (EC50 = 0.2 U/ml), followed by the known activators of eosinophils recombinant human granulocyte/macrophage CSF (rhGM-CSF), rhIL-3, and murine IL-5 (mIL-5). rhIFN-alpha and natural human IFN-beta (nhIFN-beta) enhanced cytotoxicity as well. On the other hand, in short term assays, eosinophils were not stimulated by IFN and the strongest stimulator was rhGM-CSF (EC50 = 0.2 U/ml), followed by rhIL-3, mIL-5, rhTNF, and rhIL-4. With rhTNF-alpha enhancement was more pronounced on freshly isolated eosinophils (EC50 = 0.6 U/ml) and declined with time. No significant stimulation was detected with rhG-CSF, rhIL-1 beta, rhIL-2, rhIL-6, and rhIL-8. On neutrophils, rhIL-8 enhanced cytotoxicity, but the stimulation was weak in relation to other neutrophil activators. Studies on the mechanism of cytotoxic activity revealed that cytotoxicity required opsonization of targets with specific antibody. FMF analysis was performed demonstrating that freshly isolated eosinophils express Fc-gamma RII (CD32), small amounts of Fc-gamma RIII (CD16), but not Fc-gamma RI (CD64). In experiments with blocking antibodies to Fc-gamma R cytotoxicity was restricted to Fc-gamma RII. Expression of Fc-gamma RII was not enhanced by rhGM-CSF, rhTNF-alpha, and mIL-5, but a significant increase in the number of positive cells was observed after incubation with rhIFN-gamma for 24 h (p less than 0.05). In addition, enhanced viability of eosinophils was observed when cultured in the presence of rhIFN-gamma, rhIFN-alpha, rhGM-CSF, and rhTNF-alpha, but not of rhG-CSF and rhIL-2. Thus, IFN appear to be another class of activators of eosinophils, characterized by their delayed type of action.
A molecular and immunochemical characterization of mouse CR2. Evidence for a single gene model of mouse complement receptors 1 and 2.
Authors: Molina H , Kinoshita T , Inoue K , Carel J C , Holers V M
Published: December 2022

The relationships between functional, biochemical, and genetic homologues of human and mouse C receptors 1 (CR1) and 2 (CR2) are incompletely understood. We have isolated and characterized a partial mouse CR2 cDNA clone and determined the exon-intron organization of the gene encoding it. Together they predict a form of mouse CR2 highly identical to the 15 short consensus repeat form of human CR2. Strong similarities in genomic organization and exon-intron junctions indicate that this mouse gene and human CR2 are evolutionary homologues. A polyclonal rabbit anti-mouse CR2 fusion protein, BRN-1, was prepared. BRN-1 immunoprecipitates bands of 155 to 160 kDa under nonreducing conditions in mouse CR2 expressing B cell lines. In mouse spleen a doublet of 155 kDa and 190 kDa under nonreducing and 165 and 205 kDa under reducing conditions is recognized by immunoprecipitation and Western blot analysis. Staphylococcus aureus V8 protease maps of these two proteins show many shared bands. Crossed immunoprecipitation using BRN-1 and 7E9, a previously described mAb reported to identify the 190-kDa mouse CR1 and a smaller 150-kDa protein, indicates that both antibodies react with the same proteins. Therefore, by using BRN-1 we have now linked the genetic mouse CR2 to its functional, biochemically characterized gene product. The observation that BRN-1 also recognizes a second 190-kDa mouse protein defined functionally as a homologue of human CR1, and that these proteins have very similar peptide maps, provides strong evidence that these two proteins are expressed by a single mouse CR2/CR1 transcription unit.
Mouse Ig coded by VH families S107 or J606 bind to protein A.
Authors: Seppälä I , Kaartinen M , Ibrahim S , Mäkelä O
Published: December 2022

Twenty-five monoclonal mouse Ig (5 IgA, 7 IgM, and 13 IgG1) were tested for binding to staphylococcal protein A. They were allowed to attach to protein A Sepharose column at pH 8.0 and were then eluted with a pH gradient from approximately 7.5 to 3.0. Five of them (IgM or IgA) did not bind. Ten came off with pH approximately 6. They were all IgG1, and were probably bound (weakly) via the Fc portion. The remaining 10 (3 IgA, 4 IgM, and 3 IgG1) were more firmly bound; they came off with pH-values ranging from 5.0 to 3.5. They all expressed VH genes of families J606 or S107, whereas all the 15 Ig that were not firmly bound expressed VH genes of six other families. The VL domains seem to be unimportant for protein A binding inasmuch as a firmly binding and a weakly binding IgG1 antibody share identical VK domains. VH sequences of protein A-binding and nonbinding Ig were compared. No likely peptide sequences were found that might make the ligand for protein A.
The subcellular distribution of platelet-activating factor in stimulated human neutrophils.
Authors: Riches D W , Young S K , Seccombe J F , Henson J E , Clay K L , Henson P M
Published: December 2022

Exposure of human peripheral blood neutrophils to a variety of phagocytic and soluble stimuli is known to induce the synthesis and secretion of platelet-activating factor (PAF), a unique ether-linked phospholipid. It has recently been observed in this laboratory, that whereas some PAF is secreted to the exterior of the cell, the majority of the newly synthesized PAF is retained intracellularly. This observation led us to investigate the subcellular distribution of intracellular PAF in stimulated human neutrophils, and to question the possible intracellular role of this molecule. Approximately 2 x 10(8) neutrophils were exposed to either the phagocytic stimulus, opsonized zymosan particles (25 particles/cell), the soluble stimulus, Ca2(+)-ionophore A23187 (5 micrograms/ml), or were left unstimulated for up to 30 min. After disruption, the cells were fractionated into nuclei, phagolysosomes, specific granules, azurophil granules, membranes, and cytosol. Fractions were analyzed for representative organellar markers, as well as for total protein, total phospholipid phosphorous, and PAF. In cells that had been exposed to opsonized zymosan particles, the majority of the PAF was localized to the phagolysosomal fraction, with lesser amounts being detected in the membranous and granular fractions of the cells. In neutrophils that had been exposed to A23187, the major portion of the PAF was detected in the membranous fractions with smaller amounts being seen in fractions corresponding to the specific granules. On the basis of these data, combined with the known physicochemical properties of PAF, it is speculated that the PAF detected at discrete intracellular locations in stimulated human neutrophils may play an important role in the endocytic and/or secretory functions of neutrophils.