bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of LFA-1 and VLA-4 in the adhesion of cloned normal and LFA-1 (CD11/CD18)-deficient T cells to cultured endothelial cells. Indication for a new adhesion pathway.
Authors: Vennegoor C J , van de Wiel-van Kemenade E , Huijbens R J , Sanchez-Madrid F , Melief C J , Figdor C G
Published: December 2022

Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.
Chimeric and humanized antibodies with specificity for the CD33 antigen.
Authors: Co M S , Avdalovic N M , Caron P C , Avdalovic M V , Scheinberg D A , Queen C
Published: December 2022

L and H chain cDNAs of M195, a murine mAb that binds to the CD33 Ag on normal and leukemic myeloid cells, were cloned. The cDNAs were used in the construction of mouse/human IgG1 and IgG3 chimeric antibodies. In addition, humanized antibodies were constructed which combined the complementarity-determining regions of the M195 antibody with human framework and constant regions. The human framework was chosen to maximize homology with the M195 V domain sequence. Moreover, a computer model of M195 was used to identify several framework amino acids that are likely to interact with the complementarity-determining regions, and these residues were also retained in the humanized antibodies. Unexpectedly, the humanized IgG1 and IgG3 M195 antibodies, which have reshaped V regions, have higher apparent binding affinity for the CD33 Ag than the chimeric or mouse antibodies.
Developmental regulation of D beta reading frame and junctional diversity in T cell receptor-beta transcripts from human thymus.
Authors: George J F , Schroeder H W
Published: December 2022

During fetal life, both Ig and TCR-gamma delta repertoires are enriched for a specific set of Ag receptors with limited diversity. In order to test the hypothesis that diversification of the human TCR-beta repertoire also follows a developmental program, we examined TCR-beta DJ and VDJ transcripts from fetal and adult thymi. A consistent bias for D beta 1.1 and J beta 1.1 was present in DJ transcripts amplified from 8-wk gestation thymi. Although it is possible to splice and translate D beta 1.1 in all three reading frames, 8-wk gestation fetal DJ transcripts were enriched for D beta 1.1 spliced to J beta 1.1 in reading frame one. Preference for D beta 1.1 reading frame one and for use of J beta 1.1 dwindled with increasing gestational age. Reading frame bias was not affected by the insertion of non-germ-line-encoded nucleotides (N regions) or by the site of gene splicing. In contrast, choice of reading frame and use of J beta 1.1 appeared random in VDJ transcripts from the same 8-wk gestation and adult thymic samples. Among the occasional VDJ transcripts that contained D beta 1.1-J beta 1.1 rearrangements, use of D beta reading frame one was rare. Up to 75% of DJ and VDJ transcripts at 8-wk gestation lacked N regions. Both the percentage of DJ and VDJ transcripts with N regions and the average number of inserted nucleotides per transcript increased with gestational age. Primarily as a result of enhanced N region addition, both the length and sequence diversity of the VDJ junctions increased markedly from 8-wk gestation to adult life with a concomitant increase in the potential size of the TCR-alpha beta repertoire. Terminal deoxynucleotidyl-transferase (TdT), the enzyme associated with N region addition, has not been detected in thymus before 19 wk of gestation. However, we were able to amplify TdT mRNA from all fetal thymi examined. This suggests that TdT may be expressed as early as 8 wk gestation, although at low levels. The early fetal thymus appears enriched for a population of thymocytes that produce minimal quantities of TdT, express a special set of TCR-beta DJ transcripts, and generate a TCR-beta repertoire with limited diversity. These findings support the hypothesis that limitations in the diversity of the TCR repertoire imposed at the time of gene rearrangement contribute to the immaturity of the fetal immune response.
A stromal cell-specific monoclonal antibody augments the stromal cell-dependent B lymphopoiesis.
Authors: Kaisho T , Nagasawa T , Kishimoto T , Kikutani H
Published: December 2022

We produced the mAb R25 against the stromal cell line ST2, which could support B lymphopoiesis in vitro. R25 enhanced the ability of ST2 to support B lymphopoiesis and precipitated molecules of 110 and 120 kDa (p110/120) from ST2 cell lysates. p110/120 were also expressed on other stromal and fibroblast cell lines but not on freshly isolated bone marrow hematopoietic cells, spleen cells, and lymphoid cell lines. However, R25 had no or weak effect on the stromal cell-dependent myelopoiesis. Even under conditions in which bone marrow cells were separated from stromal cells with a membrane filter, R25 could augment the stromal cell-dependent B lymphopoiesis. However, R25 did not induce the increase of IL-7 mRNA of ST2 cells. Taken together, these findings suggest that the stromal cell surface molecules p110/120 are involved in the stromal cell-dependent B lymphopoiesis and that certain soluble factors distinct from IL-7 may contribute to the p110/120-mediated B cell generation.
Intracellular events involved in CD5-induced human T cell activation and proliferation.
Authors: Alberola-Ila J , Places L , Cantrell D A , Vives J , Lozano F
Published: December 2022

In this report we describe a novel pathway of human T cell activation and proliferation involving the CD5 surface Ag. The CD5-specific Cris1 mAb induces by itself monocyte-dependent proliferation of PBMC. Among a panel of CD5-specific mAb (Leu1, OKT1, LO-CD5a, F101-1C5, and F145GF3), only the F145GF3 mAb shared this property with Cris1. The analysis of the biochemical pathway involved in this activation showed the lack of detectable hydrolysis of inositol phosphates or early increments in the intracellular cytosolic calcium concentration, after triggering cells with the mitogenic CD5 mAb. However, stimulation with CD5 induces activation of protein kinase C, as measured by phosphorylation of a specific peptide substrate (peptide GS), which can be inhibited by a pseudosubstrate peptide inhibitor. Stimulation with CD5 mAb induces also tyrosine kinase activity, with a substrate pattern that differs from that induced after triggering lymphocytes through the TCR-CD3 complex. On the other hand the IL-2/IL-2R pathway seems involved in the CD5-mediated proliferation of PBMC because anti-IL-2R-specific mAb inhibits CD5-induced proliferation, and stimulation with mitogenic CD5 mAb induces production of IL-2 and expression of IL-2R alpha and beta chains. Therefore, the triggering of the CD5 Ag can induce IL-2- and monocyte-dependent human T cell proliferation by a biochemical pathway that differs, at least in the first stages, from the one that mediates TCR-CD3 complex-induced T cell activation.
Evidence for functional heterogeneity of rat CD4+ T cells in vivo. Differential expression of IL-2 and IL-4 mRNA in recipients of cardiac allografts.
Authors: Papp I , Wieder K J , Sablinski T , O'Connell P J , Milford E L , Strom T B , Kupiec-Weglinski J W
Published: December 2022

The in vivo relevance of functional dichotomy of CD4+ Th clones was studied by analyzing the induction of mRNA encoding for Th1- (IL-2) and Th2- (IL-4) specific lymphokines in a model of accelerated (24 h) cardiac allograft (Tx) rejection in presensitized rats. The polymerase chain reaction-assisted screening of total cellular RNA from cardiac Tx of otherwise untreated sensitized recipients has revealed sequential lymphokine mRNA expression, with the peak of IL-2 mRNA (6-12 h) preceding that for IL-4 mRNA, which was maximal at the time of actual Tx loss (24 h). Both IL-2 and IL-4 transcripts could be readily detected by polymerase chain reaction analysis in the spleens during the course of accelerated rejection. Treatment of prospective cardiac Tx recipients with BWH-4, a mouse anti-rat CD4 mAb, abrogated rejection at 24 h and prolonged cardiac Tx survival to ca. 11 days, coinciding with significantly diminished IL-2 mRNA expression. In contrast, CD4 targeted therapy preserved intra-Tx and splenic transcription of the IL-4 gene. Spleen lymphocytes from mAb-conditioned recipients separated by magnetic microspheres into phenotypically distinct subpopulations, showed differential induction of IL-2 and IL-4 mRNA. Thus, IL-2 mRNA was at most very weakly expressed, whereas IL-4 transcription was strongly induced both in CD4+ T cells and its OX-22- subset. This study demonstrates the induction of IL-4 mRNA in situ in the rat system, describes discordant elaboration of IL-2 and IL-4 mRNA in untreated/anti-CD4 mAb-treated cardiac Tx recipients, and identifies OX-22- CD4+ T cells as the IL-4 mRNA producers. Thus, these results provide evidence for functional heterogeneity of rat CD4+ T cells in vivo, as defined by divergent mRNA lymphokine transcription profiles.
Differential expression of transforming growth factor-beta 1, -beta 2, and -beta 3 by glioblastoma cells, astrocytes, and microglia.
Authors: Constam D B , Philipp J , Malipiero U V , ten Dijke P , Schachner M , Fontana A
Published: December 2022

The type beta transforming growth factors (TGF) are potent regulators of the growth and functions of lymphocytes and macrophages. Recently the human glioblastoma cell line 308 was shown to produce TGF-beta 2. The relevance of this finding was evaluated further by comparing human glioblastoma cells with their nontransformed animal counterpart, astrocytes, with regard to the production of the three TGF-beta isoforms observed so far in mammals. In this report astrocytes are demonstrated to secrete also TGF-beta 2 and to express TGF-beta 1, -beta 2, and -beta 3 mRNA in vitro. In contrast, cultured murine brain macrophages release TGF-beta 1 and are positive for TGF-beta 1 mRNA only. Glia cell-derived TGF-beta 1 and -beta 2 are detected in latent form whereas both latent and active TGF-beta are identified in the supernatant of three human glioblastoma cell lines tested. These cell lines, however, show heterogeneity in regard to the isoform of TGF-beta expressed but share with astrocytes the inability to release TGF-beta 3. Provided production and activation of latent TGF-beta occur in vivo, astrocytes and microglia may then be expected to exert regulatory influences on immune mediated diseases of the central nervous system.
Identification and characterization of membrane cofactor protein of human spermatozoa.
Authors: Cervoni F , Oglesby T J , Adams E M , Milesifluet C , Nickells M , Fenichel P , Atkinson J P , Hsi B L
Published: December 2022

Membrane cofactor protein (MCP) regulates C activation by serving as a cofactor for the cleavage of C3b and C4b by the serine protease factor I. An MCP-like molecule on the inner acrosomal membrane of human spermatozoa has been characterized. Three mAb and a rabbit polyclonal antibody against MCP recognized the sperm protein. On SDS-PAGE, it migrated as a single band with a molecular mass of 38,000 and 44,000 Da under nonreducing or reducing conditions, respectively. The molecular mass was 10,000 to 20,000 Da less than the two forms of MCP expressed on others cells. The electrophoretic pattern, by one- and two-dimensional gel analysis, and the isoelectric point profile (4.5 to 5.0) of the sperm protein were similar among multiple individuals. In contrast to MCP of other cells, digestion with endoglycosidases did not alter either the m.w. or the pI of the protein, suggesting that it is a poorly or nonglycosylated form of MCP. The solubilized sperm protein bound C3 with broken thioester bond to Sepharose and possessed cofactor activity for factor I-mediated cleavage of C3 with the broken bond. A mAb that blocks the regulatory function of MCP inhibited the cofactor activity of the sperm lysate. Thus, the sperm protein is an antigenic and functional homologue of MCP but has the distinct structural features of a lower m.w. and an apparent lack of glycosylation. MCP may play an essential role in the survival of the acrosome-reacted spermatozoa by modulating C activation in the female genital tract.
Structure and mapping of the gene encoding mouse high affinity Fc gamma RI and chromosomal location of the human Fc gamma RI gene.
Authors: Osman N , Kozak C A , McKenzie I F , Hogarth P M
Published: December 2022

We describe the isolation and characterization of the gene encoding the mouse high affinity Fc receptor Fc gamma RI. Using a mouse cDNA Fc gamma RI probe four unique overlapping genomic clones were isolated and were found to encode the entire 9 kb of the mouse Fc gamma RI gene. Sequence analysis of the gene showed that six exons account for the entire Fc gamma RI cDNA sequences including the 5'- and 3'-untranslated sequences. The first and second exons encode the signal peptide; exons 3, 4, and 5 encode the extracellular Ig binding domains; and exon 6 encodes the transmembrane domain, the cytoplasmic region, and the entire 3'-untranslated sequence. This exon pattern is similar to Fc gamma RIII and Fc epsilon RI but differs from the related Fc gamma RII gene which contains 10 exons and encodes the b1 and b2 Fc gamma RII. Southern blot analysis had shown that the mouse Fc gamma RI gene is a single copy gene with no RFLP in inbred strains of mice, but analysis of an intersubspecies backcross of mice showed that unlike other mouse FcR genes which are on mouse chromosome 1 the locus encoding Fc gamma RI, termed Fcg1, is located on chromosome 3. Interestingly, the Fcg1 locus is located near the end of a region with known linkage homology to human chromosome 1. Analysis of human x rodent somatic cell hybrid cell lines indicates that the human FCG1 locus encoding the human Fc gamma RI maps to chromosome I and therefore possibly linked to other FcR genes on this chromosome. These results suggest that the linkage relationships among these genes in the human genome are not preserved in the mouse.
Thymocytopoiesis is maintained by blood-borne precursors throughout postnatal life. A study in parabiotic mice.
Authors: Donskoy E , Goldschneider I
Published: December 2022

This study was designed to resolve the long standing controversy as to whether hematogenous precursors or resident intrathymic precursors maintain thymocytopoiesis in postnatal life. The kinetics of thymic chimerism was examined over a 21-wk period in 105 pairs of nonirradiated, Thy-1-alloantigen-disparate, parabiotic B10.S mice. Thymic chimerism reached reached maximum levels of 25% in the Thy-1b and 16% in the Thy-1a partners (mean 20.5% and 9.8%, respectively) 6 to 7 wk after parabiotic union. Most of the donor-origin thymocytes were located in the thymus cortex and expressed high levels of Thy-1 Ag and terminal deoxynucleotidyl transferase. Thymic chimerism did not reach 50% because circulating prothymocytes, unlike peripheral T cells, do not distribute randomly in the blood of parabiotic partners. This was shown in irradiated pairs of Thy-1-alloantigen-identical parabiotic mice, one member of which was injected i.v. with Thy-1-alloantigen-disparate bone marrow cells. Only 10% of the total donor-origin prothymocyte activity was detected in the opposite parabiont 72 h later. Similarly, the level of thymic chimerism in nonirradiated, Thy-1-alloantigen-disparate parabionts closely corresponded to the donor: host ratio of prothymocytes in the blood (i.e., between 10 and 20%), as determined directly by an intrathymic adoptive transfer assay. The continued dependence of thymic chimerism on blood-borne precursors was formally demonstrated by surgically separating mice 9 wk after parabiosis. Twelve weeks later, thymic chimerism was 50 to 80% lower in the separated parabionts than in sham-operated controls. The possible role of "stress" in initiating or maintaining thymic chimerism during parabiosis appeared to be excluded by the existence of similar kinetics of thymocytopoiesis (including the onset of thymic involution) in parabiotic and nonparabiotic mice; and by the occurrence of similar levels of thymic chimerism in adrenalectomized and nonadrenalectomized parabiotic mice. Thus these experiments demonstrate that the maintenance of thymocytopoiesis in postnatal life, as in prenatal life, is primarily dependent upon blood-borne prothymocytes, and that intrathymic precursors are replaced at an average rate of 2 to 3%/day.
Growth arrest of human B lymphocytes is accompanied by induction of the low molecular weight mammalian heat shock protein (Hsp28).
Authors: Spector N L , Samson W , Ryan C , Gribben J , Urba W , Welch W J , Nadler L M
Published: December 2022

A large number of protein and molecular markers have been identified that delineate the early stages of human B cell activation and proliferation. In contrast, few if any molecules are transiently expressed precisely as activated B cells stop proliferating and undergo growth arrest. We demonstrate that the low molecular weight heat shock protein (hsp28) exhibits unique induction kinetics that specifically demarcates this interval. After mitogenic activation of unstimulated splenic B cells, hsp28 protein and phosphorylation transiently increase coinciding precisely with the peak of cellular proliferation and the onset of growth arrest. Although most neoplastic B cells constitutively express hsp28, three cell lines were identified that were hsp28-. No differences in phenotype or growth kinetics were detected between hsp28+ and hsp28- neoplastic B cells demonstrating that hsp28 expression is not essential for cell growth. However, when treated with phorbol ester or heat shock, these hsp28- cell lines synthesize hsp28 followed by the onset growth arrest. The consistency with which hsp28 induction transiently delineates the interval from peak proliferation to the onset of growth arrest suggests hsp28 itself is likely to be involved in regulating this process.
IL-2 and a contact-mediated signal provided by TCR alpha beta + or TCR gamma delta + CD4+ T cells induce polyclonal Ig production by committed human B cells. Enhancement by IL-5, specific inhibition of IgA synthesis by IL-4.
Authors: van Vlasselaer P , Gascan H , de Waal Malefyt R , de Vries J E
Published: December 2022

To study the role of T cells in T-B cell interactions resulting in isotype production, autologous purified human splenic B and T cells were cocultured in the presence of IL-2 and Con A. Under these conditions high amounts of IgM, IgG, and IgA were secreted. B cell help was provided by autologous CD4+ T cells whereas autologous CD8+ T cells were ineffective. Moreover, CD8+ T cells suppressed Ig production when added to B cells cocultured with CD4+ T cells. Autologous CD4+ T cells could be replaced by allogeneic activated TCR gamma delta,CD4+ or TCR alpha beta,CD4+ T cell clones with nonrelevant specificities, indicating that the TCR is not involved in these T-B cell interactions. In contrast, resting CD4+ T cell clones, activated CD8+, or TCR gamma delta,CD4-,CD8- T cell clones failed to induce IL-2-dependent Ig synthesis. CD4+ T-B cell interaction required cell-cell contact. Separation of the CD4+ T and B cells by semiporous membranes or replacement of the CD4+ T cells by their culture supernatants did not result in Ig synthesis. However, intact activated TCR alpha beta or TCR gamma delta,CD4+ T cell clones could be replaced by plasma membrane preparations of these cells. Ig synthesis was blocked by mAb against class II MHC and CD4. These data indicate that in addition to CD4 and class II MHC Ag a membrane-associated determinant expressed on both TCR alpha beta or TCR gamma delta,CD4+ T cells after activation is required for productive T-B cell interactions resulting in Ig synthesis. Ig production was also blocked by mAb against IL-2 and the IL-2R molecules Tac and p75 but not by anti-IL-4 or anti-IL-5 mAb. The CD4+ T cell clones and IL-2 stimulated surface IgM-IgG+ and IgM-IgA+, but not IgM+IgG- or IgM+IgA- B cells to secrete IgG and IgA, respectively, indicating that they induced a selective expansion of IgG- and IgA-committed B cells rather than isotype switching in Ig noncommitted B cells. Induction of Ig production by CD4+ T cell clones and IL-2 was modulated by other cytokines. IL-5 and transforming growth factor-beta enhanced, or blocked, respectively, the production of all isotypes in a dose-dependent fashion. Interestingly, IL-4 specifically blocked IgA production in this culture system, indicating that IL-4 inhibits only antibody production by IgA-committed B cells.
Suppression of experimental autoimmune uveitis in mice by induction of anterior chamber-associated immune deviation with interphotoreceptor retinoid-binding protein.
Authors: Hara Y , Caspi R R , Wiggert B , Chan C C , Wilbanks G A , Streilein J W
Published: December 2022

Immunization with bovine interphotoreceptor retinoid-binding protein induces autoimmune uveitis in B10.A mice. We have examined whether this soluble retina-specific Ag can induce anterior chamber-associated immune deviation when injected into the anterior chamber (AC) of the eye, and whether this deviant immune response has any effect on uveitis is susceptible mice. The results of these experiments indicate that interphotoreceptor retinoid-binding protein (IRBP) injected intracamerally altered the subsequent immune response of B10.A mice such that a) they were not able to develop IRBP-specific delayed hypersensitivity, nor (b) were they able to express significant autoimmune uveitis following a uveitogenic regimen. Moreover, spleen cells from mice that received IRBP in the AC suppressed uveitis when adoptively transferred into naive recipients. The splenic suppressor cells were able to prevent autoimmune uveitis in recipient mice when administered after the uveitogenic regimen. Most important, IRBP-specific splenic cells from mice treated with IRBP in the AC when injected into mice with established uveitis caused an abrupt cessation of the intraocular inflammation. The ability of intracamerally-injected soluble Ag to induce suppressor T cells that act on the efferent limb of the immune response suggests that the anterior-chamber-associated immune deviation phenomenon may have physiologic relevance in terms of preservation of the integrity of ocular tissue and renders this approach particularly suitable for treating already established experimental autoimmune diseases of this type. These results are discussed in terms of other methods that have been devised experimentally to suppress and prevent autoimmune uveitis and encephalomyelitis.
Gangliosides suppress tumor necrosis factor production in human monocytes.
Authors: Ziegler-Heitbrock H W , Käfferlein E , Haas J G , Meyer N , Ströbel M , Weber C , Flieger D
Published: December 2022

Both normal and malignant cells contain gangliosides as important cell membrane constituents that, after being shed, may influence cells of the immune system. We have studied the impact of gangliosides on the expression of TNF in blood monocytes and in the monocytic cell line Mono Mac 6. Although under standard culture conditions, bovine brain gangliosides (100 micrograms/ml) suppressed LPS-stimulated TNF production 5-fold in PBMC and 10-fold in Mono Mac 6 cells, suppression was more efficient under serum-free conditions. Looking at highly purified gangliosides, GD3, GD1a, GM3, GM2, and GM1 were all effective in reducing TNF production in PBMC, and in Mono Mac 6 by factor 10 to 50. The suppressive activity was lost in molecules, lacking the sugar moiety or the lipid moiety. Gangliosides appear to act at an early step of activation in that TNF transcripts were reduced and the mobilization of the nuclear factor kappa B was blocked. Furthermore, in time kinetics, gangliosides were effective for up to 30 min after addition of LPS, but not thereafter. However, the expression of the CD14 Ag, a receptor molecule for LPS-LPS binding protein complexes, was unaffected by gangliosides. Finally, when using Staphylococcus aureus or platelet activating factor as a stimulus, gangliosides were able to suppress TNF production in Mono Mac 6 cells by factor 5 to 10, as well. On the other hand, phorbol ester-induced production of O2- was similar in cells treated with and without gangliosides. Taken together, our data demonstrate that TNF gene expression in monocytes induced by different types of stimuli can be blocked by gangliosides at an early step of signal transduction.
Synthetic peptides of human CD4 enhance binding of Ig to monocyte/macrophage cells. I. Characterization and mapping studies.
Authors: Lenert P , Mehta R L , Zanetti M
Published: December 2022

Human T cell glycoprotein CD4 binds to class II MHC molecules and to HIV envelope protein gp120. We have shown that CD4 and synthetic peptides corresponding to amino acid residues 21-49 of the first extracellular domain of CD4, also bind Ig and, with greater avidity, antibody:Ag complex. We investigated the effect of CD4 synthetic peptides on the binding and uptake of human Ig by monocyte/macrophage U937 cells. We found that a synthetic peptide corresponding to amino acid residues 21-49 enhanced binding to U937 cells of both aggregated and nonaggregated Ig. The enhancement was concentration dependent, occurred both in normal and low ionic strength conditions, and varied with the time and the temperature of the preincubation step. The enhancement was maximal after preincubation for 3 h at 37 degrees C. A peptide concentration of 20 micrograms/ml was sufficient for optimal binding of both nonaggregated and aggregated Ig. CD4 peptide 21-49 also enhanced binding of Ig to Staphylococcus aureus protein A. These studies open a new perspective in the way monocyte/macrophage cells handle Ig, antibody:Ag or Id:anti-Id complex, in particular when present at threshold amounts in a nonprecipitating form.
N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) protects thymocytes from programmed cell death.
Authors: Ramakrishnan N , Catravas G N
Published: December 2022

Gamma-irradiation, glucocorticoid hormones, and calcium ionophores stimulate a suicide process in thymocytes, known as apoptosis or programmed cell death, that involves internucleosomal DNA fragmentation by a Ca(2+)- and Mg(2+)-dependent nuclear endonuclease. In this study we report that N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) blocked DNA fragmentation and cell death in thymocytes exposed to gamma-radiation, dexamethasone, or calcium ionophore A23187. WR-1065 protected the thymocytes from radiation-induced apoptosis when incubated with cells after irradiation but not before and/or during irradiation. WR-1065 inhibited Ca(2+)- and Mg(2+)-dependent DNA fragmentation in isolated thymocyte nuclei. Our results suggest that WR-1065 protects thymocytes from apoptosis by inhibiting Ca(2+)- and Mg(2+)-dependent nuclear endonuclease action.
Beta 2-adrenoceptor stimulation increases the number of antigen-specific precursor B lymphocytes that differentiate into IgM-secreting cells without affecting burst size.
Authors: Sanders V M , Powell-Oliver F E
Published: December 2022

Previous studies have shown that early addition of a beta 2-adrenergic agonist to whole splenocyte cultures immunized with SRBC induced an increase in the number of cells secreting Ag-specific antibody. Because of the low frequency of Ag-specific B lymphocytes in these cultures, it has been difficult to determine the cellular mechanism by which this increase is produced. To gain insight into this cellular mechanism, the present study was designed to evaluate the responsiveness of TNP-specific B lymphocytes cultured at both high density and limiting dilution with keyhole limpet hemocyanin (KLH)-specific, IL-4-producing Th lymphocytes, TNP-KLH, and the beta 2-adrenergic agonist, terbutaline. The results showed that a maximal twofold increase in both the number of anti-TNP IgM-secreting cells and the amount of anti-TNP IgM secretion occurred in terbutaline-exposed lymphocytes after 5 days of bulk culture. This response occurred in a concentration-dependent manner and was inhibited by concomitant culture with beta-adrenoceptor antagonists. No appreciable change was measured in the level of either IgG1 secretion in terbutaline plus Ag-exposed bulk cultures or MHC class II expression on terbutaline plus Ag-exposed TNP-specific B lymphocytes as compared with Ag alone. These data raised the possibility that beta 2-adrenoceptor stimulation induced either the differentiation of a larger proportion of TNP-specific B lymphocyte precursors into anti-TNP IgM-secreting cells, or the extensive proliferation of a constant number of TNP-specific B lymphocyte precursors, or both. Limiting dilution results showed that beta 2-adrenoceptor stimulation induced a twofold increase in the number of TNP-specific B lymphocyte precursors that differentiated into anti-TNP IgM-secreting cells, without affecting the number of anti-TNP IgM-secreting cells produced by each precursor clone.
Regions of the CD4 molecule not involved in virus binding or syncytia formation are required for HIV-1 infection of lymphocytes.
Authors: Hasunuma T , Tsubota H , Watanabe M , Chen Z W , Lord C I , Burkly L C , Daley J F , Letvin N L
Published: December 2022

Cell surface-expressed CD4 binds to the envelope glycoprotein of HIV-1 and mediates syncytia formation through interacting with membrane expressed HIV-1 gp120. Further possible roles of the CD4 molecule in the process of cell infection by HIV-1 remain poorly understood. In our study we describe two mAb that recognize the V3/V4 domain of the CD4 molecule. Although these mAb do not inhibit gp120-CD4 binding or HIV-1-induced syncytia formation, they inhibit HIV-1 infection of human PBL. These findings suggest that discrete, definable domains of the CD4 molecule may be involved in interactions after HIV-1 envelope binding that lead to virus entry into the cell.
Local T helper cell signals by lymphocytes infiltrating intraocular tumors.
Authors: Ksander B R , Acevedo J , Streilein J W
Published: December 2022

Tumor-infiltrating lymphocytes from mice bearing minor histoincompatible tumor cells in the anterior chamber (AC) or subconjunctival (SCon) space of the eye have been shown to contain large numbers of tumor-specific precursor cytotoxic T cells. Because SCon tumors eventually acquire directly cytotoxic, tumor-specific T cells and are rejected by their hosts and because AC tumors never acquire cytotoxic effector cells and are not rejected, we have examined tumor-infiltrating lymphocytes from both types of ocular tumors for the capacity to secrete lymphokines in response to in vitro stimulation with tumor cells. The results indicate that T "helper" cells were able to infiltrate both SCon and AC tumors. In the former, T cells capable of secreting IL-2 and IL-4 were found whereas in the latter only IL-2-secreting T cells were detected. These findings implicate a defect in local delivery of appropriate T cell help as the reason why AC tumors are not rejected. The failure of AC tumor-bearing mice to destroy their tumors correlates not only with defective delivery of local help but with a systemic inability to produce tumor-specific T cells that can secrete IL-2 and IL-4. Because these mice also generate down-regulatory T cells that suppress the expression of tumor-specific delayed hypersensitivity, they appear to have an immunologically mediated block in T helper cell differentiation which renders them unable to generate either T helper 1 or T helper 2 cells. This immunologic abnormality is discussed in terms of tumor rejection and the phenomenon of immunologic privilege.
CD2/LFA-3 ligation induces phospholipase-C gamma 1 tyrosine phosphorylation and regulates CD3 signaling.
Authors: Kanner S B , Damle N K , Blake J , Aruffo A , Ledbetter J A
Published: December 2022

Activation of T cells through the TCR/CD3 receptor complex with either specific Ag or antibody results in tyrosine phosphorylation of intracellular protein substrates and phosphatidylinositol-phospholipase C (PLC) signaling, leading to the generation of PI breakdown products and the mobilization of intracellular calcium. Stimulation of the T cell surface receptor CD2 similarly propagates early signals through phosphatidylinositol-PLC activation. Previous reports have shown that CD3 activation leads to tyrosine phosphorylation of the PLC isozyme PLC gamma 1. In this report, we investigated the potential similarity between CD3-induced signaling through PLC gamma 1 and that induced by CD2. We show that stimulation of CD2 receptors on T cells caused tyrosine phosphorylation of PLC gamma 1. Cross-linking of CD2 with CD3 receptors augmented the phosphorylation of PLC gamma 1 on tyrosine, whereas ligation of the CD45 tyrosine phosphatase with CD2 receptors prevented PLC gamma 1 tyrosine phosphorylation. T cells stimulated by ligation of CD2 with its counter-receptor in the form of a soluble LFA-3/Ig fusion protein cross-linked on the cell surface, resulted in a low, but detectable level of PLC gamma 1 phosphorylation with prolonged kinetics, whereas that induced by cross-linking with anti-CD2 was stronger but transient. Co-ligation of LFA-3/Ig with suboptimal concentrations of anti-CD3 resulted in profound augmentation of PLC gamma 1 tyrosine phosphorylation, mobilization of intracellular calcium and T cell proliferation. To explore the relationship between CD3- and CD2-stimulated signaling, T cells were desensitized through 1 h incubation with anti-CD3. CD3 receptor modulation potently down-regulated CD2-induced PLC gamma 1 tyrosine phosphorylation and calcium mobilization. In contrast, PMA or ionomycin treatment did not alter CD2-stimulated tyrosine phosphorylation of PLC gamma 1, suggesting that tyrosine kinase inhibition by CD3 receptor modulation was not caused by signaling events downstream of PLC gamma 1. Taken together, these results support the hypothesis that CD2 provides a potent co-stimulatory signal for CD3-induced T cell activation that is associated with tyrosine kinase(s) and PLC gamma 1.
Cytokine- and Ig-producing T cells in mucosal effector tissues: analysis of IL-5- and IFN-gamma-producing T cells, T cell receptor expression, and IgA plasma cells from mouse salivary gland-associated tissues.
Authors: Mega J , McGhee J R , Kiyono H
Published: December 2022

The present study has focused on the analysis of cytokine- and Ig-producing mononuclear cells (MC) that reside in the salivary glands and their associated tissues (SGAT) in the oral region. The SGAT are located under the mandibular area and consist of submandibular glands, periglandular lymph nodes, and cervical lymph nodes. MC were isolated from individual SGAT and examined for T cell subsets and TCR expression, in comparison with T cells obtained from other mucosa-associated and systemic tissues. Forty to fifty percent of MC in submandibular glands were CD3+ T cells, equally divided into CD4+ CD8- and CD4- CD8+ T cell subsets. On the other hand, the intestinal lamina propria and Peyer's patches possessed a approximately 2 to 3:1 ratio of CD4+ CD8- to CD4- T cells. A high frequency of CD4- CD8- (double negative) (DN) T cells (approximately 6 to 10%) was also isolated from submandibular glands. In contrast, approximately 70 to 90% of MC in periglandular lymph nodes and cervical lymph nodes were CD3+ T cells and like the peripheral lymph nodes consisted of fivefold higher numbers of CD4+ CD8- than CD4- CD8+ T cells, with low numbers of DN cells (less than 5%). When expression of gamma/delta and alpha/beta TCR was examined in individual T cell subsets of submandibular glands, the CD4- CD8+ and DN T cell fractions contained 25% and 100% gamma/delta TCR+ cells, respectively. On the other hand, essentially all CD4+ CD8- T cells in SGAT as well as CD4- CD8+ cells in periglandular lymph nodes and cervical lymph nodes were alpha/beta TCR+ T cells. When cytokine production was examined by using IFN-gamma- and IL-5-specific enzyme-linked immunospot assays, the CD3+ CD4+ CD8- T cells in submandibular glands contained T cells spontaneously producing IFN-gamma and IL-5. Further, IL-5 spot-forming cells (SFC) were two- to threefold greater in number, compared with IFN-gamma SFC. The periglandular lymph node T cells contained cytokine producing cells with a ratio of 2:1 for IL-5 and IFN-gamma SFC cells, whereas cervical lymph node T cells did not produce cytokines unless stimulated with T cell mitogens. When the isotype distribution of Ig-producing cells was examined among SGAT, submandibular glands contained large numbers of IgA-producing cells, with few IgM- and IgG-producing cells, a pattern similar to that of the lamina propria. Further, elevated numbers of IgA-secreting cells were also seen in periglandular lymph nodes but not in cervical lymph nodes.(ABSTRACT TRUNCATED AT 400 WORDS)
Linear epitope mapping of an Sm B/B' polypeptide.
Authors: James J A , Harley J B
Published: December 2022

Autoantibodies binding the Sm B/B' peptides are commonly associated with SLE. IgG antibodies binding overlapping octapeptides of Sm B/B' have been evaluated in 10 patients with anti-Sm and anti-nRNP precipitins, 5 patients with other autoimmune serology, and 4 normal human sera. Neither normal controls nor patients without an anti-Sm precipitin significantly bind any of the Sm B/B' octapeptides. All sera tested containing an anti-Sm precipitin strongly bind octapeptides from eight regions of the Sm B/B' sequence. Three of these eight regions share the same octapeptide sequences (PPPGMRPP) that are consistently the most immunoreactive octapeptides from Sm B/B'. Binding of the similar PPPGIRGP, as well as binding to deletion and substitution peptides, suggest that the motif PPPG(I,M) (R,K) appears to best define this binding. Interestingly, PAPGMRPP in the nRNP C peptide is as antigenic as PPPGMRPP and may provide a partial explanation for the cross-reactivity shown between Sm and nRNP autoantibodies. However, the sequence, PPPGMIPP, from nRNP A is not antigenic. These data define the linear sequence autoantigenicity of the Sm B/B' protein. They also demonstrate that the predominant autoimmune epitope is a proline-rich sequence from which limited variance is permitted before antigenicity is destroyed.
Characterization of proteoglycan-reactive T cell lines and hybridomas from mice with proteoglycan-induced arthritis.
Authors: Leroux J Y , Poole A R , Webber C , Vipparti V , Choi H U , Rosenberg L C , Banerjee S
Published: December 2022

Hyperimmunization with chondroitin sulfate-depleted fetal human cartilage proteoglycan (HFPG) leads to the development of peripheral arthritis and spondylitis in BALB/c mice. Chondroitin-sulfate-depleted adult human cartilage proteoglycan (HAPG) is much less effective at inducing arthritis. These observations suggest age differences in the presence of arthritogenic proteoglycan (PG) epitopes. Earlier studies from this laboratory have indicated an important role for PG-reactive T cells in the pathogenesis of this arthritis model. To investigate further the cellular immunity to PG in mice, two T cell lines, JY.A and JY.D, and two T cell hybridomas, TH5 and TH14, were isolated from mice with PG-induced arthritis and characterized. Two patterns of reactivity to PG emerged from the analysis of these T cells. One pattern, as demonstrated by the T cell line JY.D and the two T cell hybridomas, TH5 and TH14, was characterized by reactivity to HFPG, HAPG, chondroitin sulfate-depleted bovine cartilage PG, the G1 domain (hyaluronate binding region) of bovine cartilage PG and bovine link protein. The epitope(s) recognized by these T cells appear to be part of the homologous regions shared between the G1 domain and the link protein. The second pattern of reactivity, as demonstrated by the T cell line JY.A, was characterized by reactivity to HFPG but not to HAPG or the other PG Ag or bovine link protein. All the T cell lines and hybridomas had a CD4+, CD8- phenotype, possibly belonged to the TH1 subset (IL-2+, IL-4-), and were MHC class II restricted. These studies indicate that HFPG has T cell epitopes in common with HAPG (such as in the G1 domain) and different than those in HAPG. The significance of this data in terms of PG structure, changes with age, and induction of arthritis remains to be established.
Administration of recombinant IL-4 to humans regulates gene expression, phenotype, and function in circulating monocytes.
Authors: Wong H L , Lotze M T , Wahl L M , Wahl S M
Published: December 2022

As a multifunctional cytokine that can augment certain T cell responses, IL-4 is being evaluated currently as a possible therapeutic agent in the treatment of cancer patients. In this report, PBMC were isolated from such patients before and after IL-4 therapy and were analyzed for phenotypic and functional alterations. Differential blood counts showed that the relative percentages of lymphocytes and, to a lesser degree, monocytes decreased after treatment with IL-4. However, when phenotypically analyzed by FACS, monocyte numbers generally increased, whereas there was a decrease in lymphocyte numbers. Monocytes taken from patients before therapy and cultured with and without LPS exhibited normal patterns of monokine-specific (IL-1 beta and TNF-alpha) mRNA expression, as well as secretion of peptide. The addition of rIL-4 to these cultures, however, resulted in the down-regulation of both gene expression and peptide release. Although monocytes taken from post-therapy patients also displayed normal patterns of monokine gene expression and cytokine release after stimulation with LPS, they were no longer inhibited by the addition of exogenous rIL-4 in vitro. These data suggest that monocytes become refractory to the inhibitory effects of IL-4 after in vivo exposure to this cytokine. However, when these monocytes were examined for expression of CD14, CD32, and CD64, exogenous IL-4 was observed to decrease markedly the appearance of these markers, regardless of whether the cells were obtained before or after therapy. Furthermore, monocytes from post-therapy patients exhibited reduced production of PGE2 and superoxide anion, compared with cells obtained before therapy. This effect persisted in culture independent of the further addition of exogenous IL-4. These data suggest a dichotomy between the IL-4-dependent mechanisms that regulate monokine production and those that regulate certain other monocyte functions. The exact mechanisms that govern these two pathways are not known. These results have important clinical implications for the use of IL-4 as an immunotherapeutic agent, as well as providing insight into the physiologic role of IL-4.
Bacterial lipopolysaccharide up-regulates platelet-activating factor-stimulated Ca2+ mobilization and eicosanoid release in human Mono Mac 6 cells.
Authors: Aepfelbacher M , Ziegler-Heitbrock H W , Lux I , Weber P C
Published: December 2022

When human monocytic Mono Mac 6 cells were treated with bacterial LPS (10 ng/ml, 72 h), they showed an increase in phagocytic activity, superoxide anion production, and expression of monocyte/macrophage-associated cell surface Ag. In these more mature (LPS-treated) cells but not in untreated cells, platelet-activating factor (PAF) (100 nM) produced a three- to fourfold increase in cytosolic free Ca2+ concentration. The cytosolic free Ca2+ concentration increase was inhibited by the PAF receptor antagonist L-659,989 (10 microM) and by EGTA (2 mM), indicating receptor-dependent Ca2+ influx. Furthermore, L-659,989 (10 microM), as well as PAF (1 microM), inhibited specific [3H]PAF binding in LPS-treated but not in untreated cells. Consistent with these results, PAF (100 nM) stimulated release of arachidonic acid and thromboxane B2 only in LPS-treated cells, and this could be inhibited by L-659,989 (10 microM) and EGTA (2 mM). Our data indicate that LPS up-regulates PAF-induced Ca2+ influx, resulting in arachidonic acid and eicosanoid release in Mono Mac 6 cells.
Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages.
Authors: Fadok V A , Voelker D R , Campbell P A , Cohen J J , Bratton D L , Henson P M
Published: December 2022

During normal tissue remodeling, macrophages remove unwanted cells, including those that have undergone programmed cell death, or apoptosis. This widespread process extends to the deletion of thymocytes (negative selection), in which cells expressing inappropriate Ag receptors undergo apoptosis, and are phagocytosed by thymic macrophages. Although phagocytosis of effete leukocytes by macrophages has been known since the time of Metchnikoff, only recently has it been recognized that apoptosis leads to surface changes that allow recognition and removal of these cells before they are lysed. Our data suggest that macrophages specifically recognize phosphatidylserine that is exposed on the surface of lymphocytes during the development of apoptosis. Macrophage phagocytosis of apoptotic lymphocytes was inhibited, in a dose-dependent manner, by liposomes containing phosphatidyl-L-serine, but not by liposomes containing other anionic phospholipids, including phosphatidyl-D-serine. Phagocytosis of apoptotic lymphocytes was also inhibited by the L isoforms of compounds structurally related to phosphatidylserine, including glycerophosphorylserine and phosphoserine. The membranes of apoptotic lymphocytes bound increased amounts of merocyanine 540 dye relative to those of normal cells, indicating that their membrane lipids were more loosely packed, consistent with a loss of membrane phospholipid asymmetry. Apoptotic lymphocytes were shown to express phosphatidylserine (PS) externally, because PS on their surfaces was accessible to derivatization by fluorescamine, and because apoptotic cells expressed procoagulant activity. These observations suggest that apoptotic lymphocytes lose membrane phospholipid asymmetry and expose phosphatidylserine on the outer leaflet of the plasma membrane. Macrophages then phagocytose apoptotic lymphocytes after specific recognition of the exposed PS.