bluuebunny/crossref_metadata_2025 huggingface dataset:
Biosynthesis and function of LFA-3 in human mutant cells deficient in phosphatidylinositol-anchored proteins.
Authors: Hollander N , Selvaraj P , Springer T A
Published: December 2022

Mutants that lack expression of phosphatidylinositol (PI)-anchored proteins were derived from the human B lymphoblastoid JY cell line. It was demonstrated that unlike wild-type cells, which normally express both a transmembrane and a PI-linked form of LFA-3 glycoprotein, the mutant cells expressed only the transmembrane form of LFA-3. [3H]Ethanolamine was not incorporated into LFA-3 of mutant cells, indicating that the anchor moiety was entirely missing. Blockade of normal biosynthesis of the PI-anchored form led to accumulation of two intermediates that may have intact and truncated polypeptide chains. The truncated LFA-3, which was not attached to the cell membrane, was secreted by mutant cells into culture supernatants. A possible division of adhesion function between the two forms of LFA-3 was studied by using the JY cell lines as targets for CTL. Wild-type and mutant JY cells formed conjugates with CTL and were subsequently lysed to a similar extent. In addition, wild-type and mutant JY cells stimulated CTL proliferation to the same extent. Antibody-blocking experiments demonstrated a predominant role for the CD2/LFA-3 pathway in interaction of both wild-type and mutant cells with CTL. Because E exclusively express only the PI-linked LFA-3 form, and this form is known to mediate cell adhesion, the present results indicate that the two distinct membrane-anchored LFA-3 forms are each capable of mediating adhesion. A possible division of signaling functions between the two forms of LFA-3 is under investigation.
Leukotriene C4 production from human eosinophils in vitro. Role of eosinophil chemotactic factors on eosinophil activation.
Authors: Tamura N , Agrawal D K , Townley R G
Published: December 2022

We studied the role of naturally occurring eosinophil chemotactic factors on leukotriene (LT)C4 production from highly purified (87.1 +/- 2.4%) normodense eosinophils. Platelet activating factor (PAF) directly induced LTC4 production from eosinophils in a dose (10(-9) to 10(-5) M) and a time-dependent manner. PAF (10(-5) M) induced 0.74 +/- 0.08 ng of LTC4 production/10(6) eosinophils. However, lyso-PAF, eosinophil chemotactic factor of anaphylaxis, and LTB4 failed to induce LTC4 production within the tested range. Furthermore, the pre-incubation of eosinophils with 5 micrograms/ml of cytochalasin B did not alter the chemotactic factor-induced LTC4 production. When eosinophils were stimulated by the submaximal concentration (1 microgram/ml) of calcium ionophore A23187, the pre-incubation of eosinophils with 10(-6) M or 10(-5) M of PAF, or 10(-5) M of eosinophil chemotactic factor of anaphylaxis significantly enhanced LTC4 production up to 163.9 +/- 17.5% (p less than 0.05), 279.2 +/- 32.9% (p less than 0.01) and 165.2 +/- 21.2% (p less than 0.05) of the control, respectively. However, the pre-incubation with lyso-PAF or LTB4 failed to enhance A23187-induced LTC4 production. The pre-incubation of eosinophils with phosphatidyl serine also failed to enhance A23187-induced LTC4 production. However, the direct stimulation of protein kinase C by PMA enhanced the submaximal concentration of A23187-induced LTC4 production from eosinophils up to 179.5 +/- 20.9% (p less than 0.05) of the control. Our findings indicate that PAF and ECF-A work not only as chemotactic factors but also induce a functionally active state of eosinophils probably through their post-receptor mechanisms, and contribute to the inflammatory processes.
Mechanism of human monocyte activation via the 40-kDa Fc receptor for IgG.
Authors: MacIntyre E A , Roberts P J , Abdul-Gaffar R , O'Flynn K , Pilkington G R , Farace F , Morgan J , Linch D C
Published: December 2022

It is shown that a mAb specific for the human 40-kDa FcR (FcRII) leads to activation of human monocytic cells but that extensive cross-linking of the receptor is required. Calcium mobilization can be induced in immature monocytic cells (undifferentiated U937 cells) and peripheral blood monocytes with an intact IgG1 anti-FcRII antibody (CIKM5) but not by F(ab')2 fragments of this antibody. The intact antibody can bind in a tripartite manner by its two F(ab') sites and its Fc-binding site whereas the F(ab')2 fragments of this antibody can only bind in a divalent fashion. A rise in intracellular free calcium ion concentration occurs when F(ab')2 fragments are cross-linked with F(ab')2 anti-mouse Ig indicating that more extensive cross-linking of FcRII is required rather than an obligatory requirement for an Fc-FcRII interaction. Calcium mobilization in response to intact or cross-linked F(ab')2 fragments of CIKM5 is associated with superoxide production only in IFN-gamma-primed peripheral blood monocytes and IFN-gamma differentiated U937 cells indicating that the activation signal produced via FcRII is inadequate to fully stimulate non-"primed" cells. A second mAb reactive with FcRII (2E1) does not cause calcium mobilization in monocytes or U937 cells, and partially blocks the effects of CIKM5. 2E1 also blocks CIKM5 superoxide production in IFN-gamma-primed monocytes and differentiated U937 cells. This may be explained in part by the fact that 2E1 is an IgG2a antibody and can only participate in bipartite binding with FcRII. When 2E1 is cross-linked with F(ab')2 anti-mouse Ig there is a small calcium response. This does not cause superoxide generation in IFN-primed monocytes but does do so in IFN-gamma differentiated U937 cells. FcRII is also expressed on granulocytes and some B cells but the effects of cross-linking the receptor on these cells differ from those seen in monocytes.
Defective thymic education of L3T4+ T helper cell function in graft-vs-host mice.
Authors: Fukuzawa M , Via C S , Shearer G M
Published: December 2022

Our study investigates the effect of a prior graft-vs-host (GVH) reaction on the subsequent ability of irradiated, bone marrow-re-populated mice to develop T cell function. The results indicate that such GVH-bone marrow transplanted (BMT) mice do not generate CTL responses to trinitrophenyl-modified syngeneic cells (TNP-self), but do generate strong CTL activity to H-2 alloantigens. This selective deficiency in TNP-self CTL response potential appeared as early as 10 days after GVH, and required both L3T4+ and Lyt-2+ donor T cells. The in vitro addition of either soluble Th factors or L3T4-enriched spleen cells from normal mice circumvented the defect in the TNP-self response in GVH-BMT mice. These results indicate that T effector function was not defective, and instead suggest a Th defect. Cell depletion and antibody-blocking, as well as IL-2 production experiments, indicate that the Th defect was selective for L3T4+ Th population and not for Lyt-2+ Th population. This defect in L3T4 Th function is not accounted for by the approximate twofold reduction in L3T4 cell numbers in GVH-BMT mice, because IL-2 production and CTL generation to L3T4-dependent Ag were at least eightfold below control levels. Rather, defective L3T4 Th function appears to be the consequence of a GVH-induced defect in thymic maturation because the defect was corrected in vivo by a neonatal parental thymus graft before irradiation and bone marrow transplantation. This system may be useful for elucidating the role of the thymus in the maturation of Th cells. Our findings raise the possibility that impaired development of T cell function occurring in marrow grafted patients who have undergone a GVH reaction could be partly due to a GVH-induced thymic defect.
The involvement of human tumor necrosis factors-alpha and -beta in the mixed lymphocyte reaction.
Authors: Shalaby M R , Espevik T , Rice G C , Ammann A J , Figari I S , Ranges G E , Palladino M A
Published: December 2022

The influence of recombinant human TNF-alpha and -beta (rHuTNF-alpha and -beta) in a human mixed lymphocyte reaction (MLR) was investigated. The addition of 1000 U/ml of either cytokine at the initiation of culture caused up to a sixfold increase in [3H]thymidine incorporation by responder cells. Furthermore, it was found that endogenous HuTNF-alpha is produced after allogeneic cell interaction and can be detected in the MLR supernatant within 1 h of culture initiation. The results also show that, in the absence of exogenous HuTNF-alpha, antibodies to rHuTNF-alpha can cause a significant inhibition of the MLR. These observations indicate the importance of TNF-alpha in allogeneic cell interaction and raise considerations for the use of antibodies, or other antagonists, to TNF-alpha as regulators of disease states associated with cell-mediated immune reactions.
Modulation of stimulus-dependent human platelet activation by C-reactive protein modified with active oxygen species.
Authors: Miyazawa K , Kiyono S , Inoue K
Published: December 2022

C-reactive protein (CRP) is one of the most characteristic acute phase proteins which appear in the serum during certain inflammatory diseases. We report here that human CRP acquired the ability to augment platelet reactivity when treated with an Fe2+ (Cu2+)-ascorbate system. CRP modified by such treatment showed no appreciable activation of platelets in the absence of platelet activators such as platelet-activating factor, thrombin, or ADP. However, in the presence of the modified-CRP, irreversible activation of platelets occurred with sub-optimal doses of platelet-activating factor and other stimulatory agents for platelets. CRP without any treatment did not show any modulating activity. Each component of the Fe2+-ascorbate system was required for modification of CRP, suggesting that CRP was modified through an oxidative process. The modification of the CRP structure was confirmed by the change in the fluorescence spectrum of 8-anilino-1-naphthalene sulfonate complexed with CRP, the increased susceptibility of CRP to proteolytic enzymes and the altered reactivity to anti-CRP mAb. We also found an inactivating system for the modified CRP in plasma. The modified human CRP did not show any modulating activity toward rabbit platelets, suggesting that the activity is species specific.
Human IFN-gamma production is inhibited by a synthetic peptide homologous to retroviral envelope protein.
Authors: Ogasawara M , Cianciolo G J , Snyderman R , Mitani M , Good R A , Day N K
Published: December 2022

A synthetic 17 amino acid peptide (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane proteins was investigated for its influence on the in vitro production of IFN-gamma from human peripheral mononuclear cells. The results showed that CKS-17 coupled to a carrier protein, BSA, inhibited production of IFN-gamma in a dose-dependent manner. Controls, consisting of BSA, which had undergone the coupling procedure or neurotensin coupled to BSA in an identical manner as CKS-17, showed no such inhibition. Reduction in IFN-gamma production could not be attributed to decreased viability of cells, delay of IFN-gamma production or to involvement of suppressor cells. Moreover, inhibition of IFN-gamma production was not related to the inhibition of DNA synthesis. The inhibition appeared to be a direct effect of CKS-17 on IFN-gamma-producing cells. Kinetic studies revealed that this suppression occurred when CKS-17 was introduced to the culture concurrent with or within 48 h after introduction of IFN inducers. Preincubation experiments showed that the presence of CKS-17 in the culture medium was not necessary to exert its inhibitory effect. These results suggest that a portion of retroviral envelope proteins possess important immunomodulatory actions.
Two regions within the human IL-2 gene promoter are important for inducible IL-2 expression.
Authors: Williams T M , Eisenberg L , Burlein J E , Norris C A , Pancer S , Yao D , Burger S , Kamoun M , Kant J A
Published: December 2022

We have examined regulatory domains of the human IL-2 gene promoter by transfection and transient expression of rDNA constructs in which the chloramphenicol acetyl transferase gene shows T cell-specific inducible expression and cyclosporin A-mediated inhibition when placed downstream of 587 bp of the human IL-2 5'-flanking region. A series of 5'-deletion constructs transfected into the Jurkat T lymphoid line demonstrates that a region encompassing 370 bp 5' of the transcription start site is sufficient for inducible chloramphenicol acetyl-transferase expression. Further dissection of this region with internal deletion (linker-scanner) mutants revealed that portions of at least two discrete regions from -42 to -169 and -289 to -361 bp relative to the transcription start site are critical for inducible expression of the IL-2 gene. T cell-specific expression of wild-type and mutant IL-2 promoter constructs could be increased severalfold by the insertion of an upstream SV40 enhancer. With use of a battery of IL-2 promoter constructs, we could not identify subregions within IL-2 5'-flanking sequences which are crucial for cyclosporin A inhibition of the IL-2 gene or deletion of which resulted in loss of T cell-specific expression, suggesting that such functions may be mediated at pre-transcriptional levels.
Biochemical and structural properties of a Hodgkin's disease-related membrane protein.
Authors: Nawrocki J F , Kirsten E S , Fisher R I
Published: December 2022

The HeFi-1 mAb recognizes a membrane protein on Hodgkin's disease cells and on a limited number of other human cells that are either tumorigenically transformed or virally activated. Herein biochemical and structural analyses of the HeFi-1 reactive membrane protein (HRMP) were done to identify its potential importance in cellular transformation in the Hodgkin's disease cell line L428, in the T cell lymphoma line HuT 78, and in several EBV-transformed lymphoblastoid cell lines. Immunoprecipitation studies demonstrated that the mature form of the HRMP had an apparent Mr of 120 kDa in tumor cells and 116 kDa in the EBV-transformed cell lines and that it was phosphorylated at both serine and tyrosine residues in all cell lines tested. The precursor to the HRMP is an 86-kDa core protein that, after processing by high mannose N-linked glycosylation, migrates with an apparent Mr of 90 kDa. This protein is then further processed to the mature 120-kDa HRMP in part by O-linked glycosylation, the addition of sialic acid residues, and by the conversion of N-linked oligosaccharides from the high mannose to the complex type. Detectable amounts of the 90-kDa molecule can be found in the membrane and, although this protein can be phosphorylated in vitro, it is not phosphorylated in intact cells. The combined results of this study suggest that the HRMP is involved in cellular metabolism and show that an unusual amount of post-translational processing of the 90-kDa precursor results in the formation, and perhaps phosphorylation, of the mature 120-kDa HRMP.
Specific endotoxic lipopolysaccharide-binding proteins on murine splenocytes. II. Membrane localization and binding characteristics.
Authors: Lei M G , Morrison D C
Published: December 2022

We have characterized the binding of LPS to an 80-kDa LPS-binding protein detected by an LPS photoaffinity probe to be present on murine splenocytes. Specific binding of LPS to the 80-kDa protein is directly proportional to LPS concentration at low concentrations of LPS and is saturable at high concentrations of LPS. Binding is inhibited by both homologous and heterologous underivatized LPS as well as by polysaccharide-free lipid A, indicating a specificity for the biologically active component of LPS. Analysis of the kinetics of binding indicate a time-dependent increase over the first 15 min, but increases are not detected after this time. Binding of LPS to the 80-kDa LPS-binding protein is reduced but still readily detectable at 4 degrees C in the presence of azide. The presence of the 80-kDa LPS-binding protein in an isolated cytoplasmic membrane fraction of murine splenocytes as well as its release from intact splenocytes by octylglucoside suggest that this LPS-binding protein is membrane localized. The results are consistent with, but do not establish unequivocally, the identity of the 80-kDa LPS-binding protein as a specific membrane receptor for lipid A.
Methylation in the 5' region of the murine beta Fc gamma R gene regulates the expression of Fc gamma receptor II.
Authors: Bonnerot C , Daëron M , Varin N , Amigorena S , Hogarth P M , Even J , Fridman W H
Published: December 2022

In order to identify possible mechanisms regulating the expression of Fc gamma RII, we have examined the methylation status of the beta Fc gamma R gene in a panel of Fc gamma RII (+) and (-) cells belonging to several different lineages. We used beta 1 cDNA probes, derived from beta Fc gamma R gene transcripts which encode murine Fc gamma RII molecules. We found that all CCGG sequences detected with these probes were methylated in the genomic DNA of the Fc gamma RII-(-) cells. By contrast, two CCGG sites were found to be selectively unmethylated in the DNA of all Fc gamma RII(+) cells tested. These sites could be assigned to the region of the 5' end of the beta Fc gamma R gene. Besides, the treatment of Fc gamma RII(-) thymoma cells BW5147 with 5-azacytidine induced a hypomethylation of the beta Fc gamma R gene concomitantly with the transcription of that gene as seen by Northern blotting and the expression of functional Fc gamma RII. Conversely, the DNA-methylating agent ethyl methanesulfonate completely reversed the phenotype of the 5-azacytidine-treated cells to that of the Fc gamma RII(-) BW5147 parent cells. In ethyl methanesulfonate-treated cells, the beta Fc gamma R gene was remethylated and the corresponding transcript was no more detectable. We conclude that the methylation of a specific 5' segment of the beta Fc gamma R gene regulates the expression of Fc gamma RII in murine T cells, B cells, mast cells, and macrophages, possibly by controlling the gene transcription.
Cloning and expression of the H chain V region of antibody OVB3 that reacts with human ovarian cancer.
Authors: Gallo M G , Chaudhary V K , Fitzgerald D J , Willingham M C , Pastan I
Published: December 2022

Hybridoma OVB3 produces an antibody (IgG2b kappa) that reacts with an Ag present on the surface of almost all human ovarian carcinomas. An EcoRI fragment of genomic DNA containing the rearranged H chain V region of monoclonal OVB3 was cloned from a lambda gtWES library and then sub-cloned into a pGEM4 vector. To show that the cloned DNA sequence did encode the V region of a functional antibody, the DNA fragment was inserted into plasmid pSV-VNP gamma SNase in place of VNp to produce pSV-VOVB3 gamma SNase. This plasmid was then transfected into variant OVB3 hybridoma cells, which no longer produced the H chain of antibody OVB3, and functional antibody was secreted by the recipient cells. The recombinant chimeric antibody bound to ovarian cancer cells and contained Staphylococcus aureus nuclease activity, proving that a functional V region gene had been cloned.
Acquisition of Ig isotype diversity after bone marrow transplantation in adults. A recapitulation of normal B cell ontogeny.
Authors: Velardi A , Cucciaioni S , Terenzi A , Quinti I , Aversa F , Grossi C E , Grignani F , Martelli M F
Published: December 2022

To gain insight into the prolonged susceptibility to infections noted after allogeneic bone marrow transplantation (BMT), multiple parameters of the humoral immune system were serially monitored in ten bone marrow recipients. IgM B cells appeared in the circulation 2 to 4 mo after engraftment. During the first 6 mo, the IgM B cells expressed low levels of CD21 (C3d/EBV receptors) and were largely CD38+. IgG and IgA B cells were also found to coexpress surface IgM and IgD, indicating that they may be involved in a process of isotype switch. These features are characteristic of neonatal B cells. To explore the pattern of Ig isotype switch, the emergence of plasma cell precursors for each of the four IgG subclasses was examined by culturing blood lymphocytes with PWM or LPS and enumerating bone marrow plasma cells. A marked IgG2 and IgG4 plasma cell deficiency and a relative increase in IgG1 and IgG3 plasma cells were detected both in vitro and in vivo. Serum IgG2 and IgG4 levels were deficient for more than 18 mo after BMT, elevated IgG1 levels accounting for the normal or increased levels of total IgG. The data suggest that a selective unresponsiveness to polysaccharide Ag and IgG2 subclass deficiency may contribute to the late bacterial infections in BMT recipients. These features of gradual development of the humoral immune system in adults undergoing successful marrow engraftment reproduce some of the maturational steps that occur during normal B cell ontogeny over the first 1 to 2 yr of life.
Specific endotoxic lipopolysaccharide-binding proteins on murine splenocytes. I. Detection of lipopolysaccharide-binding sites on splenocytes and splenocyte subpopulations.
Authors: Lei M G , Morrison D C
Published: December 2022

Experiments have been carried out using a unique radio-iodinated, disulfide-reducible, photoactivatable LPS derivative (ASD-LPS) to detect specific LPS-binding proteins on murine splenocytes. Fractionation of LPS-photo-cross-linked, reduced, and solubilized splenocyte extracts on two-dimensional polyacrylamide gels has allowed the identification of an 80-kDa LPS-binding protein with approximate pI of 6.5. This LPS-binding protein is present on partially purified populations of splenic B lymphocytes, T lymphocytes, and macrophages. It is also the dominant LPS-binding protein on the murine 70Z/3 B cell line and the YAC-1 and EL4 T cell lines but is not detectable on the undifferentiated murine Sp2/0 myeloma cell line. Of potential importance is the fact that the 80-kDa protein appears to be indistinguishable when photolabeled extracts of splenocytes from the C3HeB/FeJ (lpsn) and LPS-nonresponder C3H/HeJ (lpsd) mice are compared.
Triggering of cytotoxic T lymphocytes and NK cells via the Tp103 pathway is dependent on the expression of the T cell receptor/CD3 complex.
Authors: Fleischer B , Sturm E , De Vries J E , Spits H
Published: December 2022

The expression and function of the T cell activation molecule Tp103 on human cloned cytotoxic CD3+ and CD3- cells were studied. All in vitro growing CD3+ and CD3- clones expressed Tp103 regardless of their phenotype and the expression of a CD3-associated TCR complex. Whereas the CD2 pathway was functional in all these clones, only CD3-expressing clones could be triggered via Tp103 to kill target cells. In contrast, both CD2 and Tp103 pathways were suppressed after modulation of the TCR complex with anti-CD3 mAb. This indicates that the function of Tp103 but not of CD2 is dependent on the expression of a functional Ag receptor on cytotoxic T cells. Furthermore, modulation of the Ag receptor induces a state of unresponsiveness in cytotoxic T cells that cannot be attributed to just the removal of the CD3/TCR complex from the cell membrane.
Analysis of the molecular basis of HLA-A3 recognition by cytotoxic T cells using defined mutants of the HLA-A3 molecule.
Authors: Jelachich M L , Cowan E P , Turner R V , Coligan J E , Biddison W E
Published: December 2022

The structure-function relationships in human class I HLA molecules have been examined by the analysis of two T cell-defined subtypes of HLA-A3 (A3.1 and A3.2). These subtypes differ by two amino acid residues that are located at positions 152 (GluA3.1 vs ValA3.2) and 156 (LeuA3.1 vs GlnA3.2). By the methods of site-directed mutagenesis and DNA-mediated gene transfer, mammalian cell transfectants have been produced that express only one of the above A3.2 amino acid residues at either position 152 or position 156. Previous studies using murine transfectants have shown that A3.1- and A3.2-expressing cells can be distinguished by A3.1-restricted type A influenza virus-specific CTL and A3.2-allospecific CTL and have implied that amino acid position 152 plays a key role in this specificity. To test whether these results were a function of the virus specificity, the alloantigen, or the cell type expressing the class I molecules, we have tested the recognition of human and murine cell transfectants by A3.1-restricted, A/JAP/305/57 and B/Ann Arbor-specific CTL and by A3.1- and A3.2-allospecific CTL. The results indicate that the Glu at position 152 is critical for recognition by all of the A3.1-restricted CTL populations tested and 15 of 16 of the A3.1-allospecific CTL populations tested. The A3.1 Leu at position 156 was sufficient for recognition by only one A3.1-allospecific CTL line. Substitution of the charged Glu residue for the polar Gln at position 156 of A3.2 affected recognition of some but not all A3.2-alloreactive CTL. These data demonstrate that the structural basis for epitopes that are recognized by almost all CTL that discriminate between A3.1 and A3.2 is primarily the amino acid at position 152. The implications of these data for Ag presentation and CTL recognition are discussed.
Expression of the J11d marker on peripheral T lymphocytes of MRL-lpr/lpr mice.
Authors: Seth A , Pyle R H , Nagarkatti M , Nagarkatti P S
Published: December 2022

MRL-lpr/lpr (lpr) mice spontaneously develop massive lymphadenopathy resulting from the expansion of a unique population of Thy-1+ cells which are CD4- and CD8- (double negative) and the nature of which is not clear. The antibody J11d has been shown to define a differentiation Ag found on immature thymocytes but not on mature and functional peripheral CD4+ or CD8+ T cells. To analyze the possible relationship between the lpr double-negative T cells and the thymocytes, we investigated the simultaneous expression of J11d and Thy 1 Ag on the double-negative lpr lymph node cells by using two-color immunofluorescent staining technique. We observed that lpr mice at 3 to 4 weeks of age, before the onset of lymphadenopathy, did not have significant numbers (less than 4%) of J11d+ T cells in the periphery, similar to the number found in the control MRL +/+ mice. However, with increasing age of approximately 8 to 10 weeks and coinciding with the appearance of lymphadenopathy, a significant number (approximately 35%) of J11d+ Thy-1+ cells started appearing in the periphery of lpr mice and was maintained until the mice died at 20 to 24 weeks of age. The J11d+ T cells belonged to the abnormal double-negative T cell pool, inasmuch as J11d+ CD4+ or J11d+ CD8+ cells were absent in the lymph nodes of 20-wk-old lpr mice. Furthermore, 20-wk-old lpr mice demonstrated increased numbers (approximately 41%) of double-negative T cells in the thymus, a significant proportion of which were J11d+. In contrast, the 20-wk-old +/+ mice or 4-wk-old lpr mice had only 4% double-negative T cells in the thymus. The present study suggests that a significant number of peripheral double-negative T cells of lpr mice bear the immature thymic differentiation Ag J11d. The possibility that the accumulation of double-negative T cells results from abnormal peripheralization of double-negative J11d+ thymocytes, before complete differentiation into CD4+ or CD8+ T cells, is discussed.
IL-4 regulates differentiation and proliferation of human precursor B cells.
Authors: Hofman F M , Brock M , Taylor C R , Lyons B
Published: December 2022

The mechanism by which precursor and pre-B cells undergo differentiation is unclear; however, it is known that growth factors play an important role in this maturation process. The lymphokine, IL-4 has been shown to increase expression of class II Ag on B cells and induce B cell proliferation. In the murine system, IL-4 induced differentiation of precursor B cells into pre-B cells. In order to analyze growth factors on B cell development we have established an in vitro culture system for human bone marrow cells. We found that in the presence of IL-4, normal human precursor and pre-B cells can be induced to differentiate in the absence of cell proliferation with four days of culture. Furthermore, IL-4 depressed proliferation induced by supernatant from a T cell line. The differentiation was measured by an increase in both the number of cytoplasmic mu and surface IgM-positive cells. The effect of IL-4 on precursor and pre-B cell differentiation was detected as soon as 14 h of exposure to the lymphokine in the absence of an adherent feeder layer. These data suggest that IL-4 directly affects the differentiation process of normal human precursor and pre-B cells, and may antagonistically affect cell proliferation.
Immunoglobulin heavy H chain isotypes in a teleost fish.
Authors: Lobb C J , Olson M O
Published: December 2022

Three mouse mAb were derived which identified different populations of the approximately 700,000-Da tetrameric Ig of channel catfish (Ictalurus punctatus). Immunoprecipitation analyses using the three mAb in various combinations showed that the catfish Ig population identified by each mAb was antigenically distinct. Each Ig population contained both classes of catfish L chains (F and G). Solid-phase binding assays indicated that the mAbs preferentially identified H chains rather than L chains. Comparative peptide mapping analyses of the H chains defined by each mAb indicated that H chains were different, although each had the same apparent mass (approximately 70,000 Da). This structural distinction was not based upon allotypic variation because the analysis of the serum from individual catfish showed the presence of each H chain in fish examined. Therefore, the different H chains represent different isotypes; each representing approximately 20% of the total serum Ig. There is at least one undefined additional H chain isotype found in the serum Ig population. Partial sequence information was obtained on the first variable framework region (FR1) of two of the three H chain isotypes. These results indicated that the variable region associated with each isotype was heterogeneous; two and sometimes three different residues were represented in the majority of positions. The sequence information also showed that the FR1 of one H chain was distinct from the other; there was less than a 50% match of the primary residues. The analyses suggest that the V region genes which code for the FR1 are differently associated with each H chain isotype. Finally, the relative levels of the three H chain isotypes were monitored during the temporal immune response of individual catfish to the dinitrophenyl hapten. These results indicated that one H chain isotype was preferentially expressed early in the immune response (approximately 3 wk) and remained the predominantly expressed isotype during the humoral immune response.
Specific H chain junctional diversity may be required for non-T15 antibodies to bind phosphorylcholine.
Authors: Feeney A J , Clarke S H , Mosier D E
Published: December 2022

The secondary antibody response of mice to phosphorylcholine (PC) shows a markedly different clonal profile than the primary response. In particular, the T15 antibodies that dominate the primary response are a minor part of secondary IgG antibodies, whereas 511 and 603 antibodies become a more prominent part of the PC-specific secondary response. These three anti-PC families differ only in L chain usage. We partially sequenced the IgH chain mRNA of a series of secondary T15 and 511 hybridomas to determine the role of somatic mutation and affinity maturation in these changes in clonal profile. None of the sequenced T15 antibodies showed somatic mutations or affinity increases. In contrast, all of the 511 antibodies had extensive somatic mutation and most had significantly increased affinity for nitrophenyl-PC. The failure of T15-expressing B cells to contribute to the secondary IgG response thus is likely to be explained by their inability to undergo (or tolerate) substantial somatic mutation and affinity maturation. We also noted that all 511 antibodies sequenced by us or others had an extra amino acid encoded at the VH-D junction by either N region addition or diversity of VH-D joining. Published sequences also show a 603 family-specific change at the VH-D junction. The frequency with which these changes, which appear obligate for PC binding, occur may determine the under-representation of these clonotypes in the primary anti-PC response. The affinity maturation in 511 antibodies after somatic mutation appears to account for their expansion in the secondary response.
Histamine release from human basophils by synthetic block co-polymers composed of polyoxyethylene and polyoxypropylene and synergy with immunologic and non-immunologic stimuli.
Authors: Atkinson T P , Smith T F , Hunter R L
Published: December 2022

Co-polymers composed of polyoxyethylene and polyoxypropylene have been shown previously to trigger histamine release from mouse peritoneal mast cells; this property quantitatively is directly related to the ionophorous ability of these compounds to cause a functional exchange of intracellular K+ for extracellular Na+ across the cell membrane. We investigated the effect of an inflammatory copolymer, T130R2, on human basophils. The data demonstrate that T130R2 can cause calcium-dependent histamine release from human basophils in vitro. Further, at concentrations that do not cause histamine release, this co-polymer markedly augments release by suboptimal concentrations of the lectin Con A or anti-IgE antibody and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate but not the calcium ionophore A23187. Thus, these co-polymers induce mediator release from cells of both rodents and humans. In both instances it is likely that calcium-dependent cell triggering is the result of an influx of sodium ions with concomitant depolarization of the transmembrane potential. In common with the calcium ionophore A23187, the co-polymer T130R2 has the ability to synergize with stimuli which trigger the IgE receptor as well as those which directly activate the cellular calcium- and phospholipid-dependent protein kinase.
A newly characterized HLA-DP beta-chain allele. Evidence for DP beta heterogeneity within the DPw4 specificity.
Authors: Lair B , Alber C , Yu W Y , Watts R , Bahl M , Karr R W
Published: December 2022

cDNA clones corresponding to the DPw4 alpha- and DPw4 beta-chains were isolated from a cDNA library prepared from a DPw4 homozygous cell line, their nucleotide sequences were determined, and the corresponding amino acid sequences were deduced. This DPw4 alpha-chain is identical to the conserved DP alpha-chains from DPw4 and DPw2 haplotypes, although the DPw4 beta-chain (referred to as DPw4b beta) differs from all reported DP beta-chain sequences. The DPw4b beta-chain differs from the reported DPw4 beta sequence (referred to as DPw4a beta) at three amino acid positions in the first domain (36, 55, and 56). The DPw4b beta-chain sequence differs from the DPw2 beta-chain sequence only at position 69 in the first domain, suggesting that the lysine at position 69 in DPw4b beta and the glutamic acid at position 69 in DPw2 beta contribute to the epitopes that define "DPw4-ness" and "DPw2-ness," respectively. In addition, the patterns of sequence identities and differences among the DPw4b beta-, DPw4a beta-, DPw2 beta-, and DPw3 beta-chains suggest that the DPw4b beta sequence arose via a gene conversion event or a point mutation. The I-LR1 mAb, which was previously found to bind only to DPw2, DPw3, and DR5 molecules, binds to an L cell transfectant expressing the DPw4 alpha:DPw4b beta molecule. The DPw4b beta sequence provides the first evidence for structural heterogeneity within the DPw4 specificity.
Remethylation at sites 5' of the murine Lyt-2 gene in association with shutdown of Lyt-2 expression.
Authors: Carbone A M , Marrack P , Kappler J W
Published: December 2022

We have used hybridomas made by fusing the Lyt-2- AKR thymoma, BW5147, to Lyt-2+ SJL/J lymph node cells to study the regulation of Lyt-2 expression. Fusions of this type yielded hybridomas, the majority of which failed to express Lyt-2. In the minority of hybridomas that did express surface Lyt-2, expression was transient and greatly diminished in terms of molecules of Lyt-2 per cell. Lack of Lyt-2 expression was not due to loss of the gene encoding this cell surface molecule; rather, negative regulation of Lyt-2 appeared to be at the level of transcription (i.e., no Lyt-2 transcripts were detected in these hybridomas). We have shown that the Lyt-2 gene is undermethylated in normal Lyt-2+ T cells, whereas the gene is heavily methylated in Lyt-2- liver cells and in BW5147. Loss of Lyt-2 expression in (BW5147 x Lyt-2+ SJL/J lymph node cell) hybridomas was associated with remethylation of DNA within the Lyt-2 gene and at sites 5' of the Lyt-2 gene.
Production of a fibronectin-associated lymphokine by cloned mouse T cells.
Authors: Godfrey H P , Canfield L S , Kindler H L , Angadi C V , Tomasek J J , Goodman J W
Published: December 2022

Azobenzenearsonate-specific cloned mouse T cells able to transfer delayed hypersensitivity reactions in vivo produced macrophage agglutination factor (MaggF) after stimulation with mitogen or antigen in vitro. Mitogen (Con A) elicited MAggF production directly from T cells. Responses to Ag were Ag-specific, required syngeneic accessory cells in addition to T cells, and were independent of T cell fine specificity for azobenzenearsonate. Mouse MAggF shared a number of biochemical and immunochemical properties with the fibronectins (FN): 1) high Mr similar to that of plasma FN; 2) binding to gelatin, heparin, and polyclonal antibodies and mAb specific for cellular and plasma FN; 3) inhibition of activity in solution by monoclonal anti-human FN directed against plasma FN gelatin-binding domain; and 4) action on peritoneal exudate macrophages mediated through a FN-receptor cross reactive with one on human monocytes. MAggF production required active protein synthesis and was associated with significant increases in gelatin-binding immunoreactive FN (Mr 440 kDa on immunoblotting) in culture supernatants and T cell lysates. Metabolically labeled peptides could be precipitated by anti-FN from culture supernatants of activated T cells. Stimulated cultures contained significantly more cells with immunohistologically demonstrable cytoplasmic FN than unstimulated control cultures. We suggest that T cell FN is a distinct species of cellular FN which may play an important role in mediating delayed hypersensitivity inflammatory reactions in vivo.
Human recombinant IL-1 alters glucocorticoid receptor function in Reuber hepatoma cells.
Authors: Hill M R , Stith R D , McCallum R E
Published: December 2022

Exposure of Reuber hepatoma cells (RHC) to 30 and 300 fM human rIL-1 (hurIL-1) for 4 h significantly decreased cytosolic glucocorticoid binding. Scatchard analysis indicated that the 30 and 300 fM doses of hurIL-1 significantly decreased the Bmax (maximum number of available binding sites), but did not alter the Kd (affinity of the glucocorticoid receptor for ligand). The decrease in cytosolic glucocorticoid binding, expressed relative to cytosol protein, did not result from increased intracellular protein in hurIL-1-treated RHC. In addition, the receptor binding reaction in RHC treated with 300 fM hurIL-1 could be resolved only by computer application of a three-parameter model. Sucrose density gradient ultracentrifugation analysis confirmed significantly less untransformed (8 to 10S) receptor-ligand complexes in hurIL-1-treated RHC, which is biologically significant because hurIL-1 (300 fM) also inhibited the glucocorticoid induction of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK). Altered transformation of the receptor-ligand complex, a possible mechanism of action for hurIL-1-mediated inhibition of PEPCK induction, was examined. However, receptor transformation, verified by in vitro activation by high salt (0.3 M KCl) of glucocorticoid receptor-ligand complexes and subsequent sucrose density gradient ultracentrifugation analysis, was not affected by hurIL-1. Furthermore, cytoplasmic glucocorticoid binding, determined in intact cell dexamethasone uptake experiments, was decreased in hurIL-1-treated RHC. The decrease in cytoplasmic glucocorticoid binding was reflected subsequently in decreased nuclear binding. The results support our hypothesis that, during acute infection and inflammation, mediators alter metabolic pathways in the liver by interfering with glucocorticoid action.
IL-6/BSF-2 functions as a killer helper factor in the in vitro induction of cytotoxic T cells.
Authors: Okada M , Kitahara M , Kishimoto S , Matsuda T , Hirano T , Kishimoto T
Published: December 2022

rIL-6/B cell stimulatory factor 2 was found to augment CTL generation from mature as well as immature human T cells stimulated with UV-treated allogeneic cells. rIL-6 also acted on peanut agglutinin-positive murine thymocytes and Lyt-2-positive splenic T cells to give rise to CTL. rIL-6 alone could not induce CTL generation, the presence of IL-2 during the early phase of culture period was found to be essential for the IL-6 activity in the induction of CTL. The effect of rIL-6 was not mediated by the induction of IL-2 inasmuch as rIL-6 did not augment IL-2 production in MLC and anti-IL-2 antibody could not neutralize IL-6 activity. rIL-6 augmented CTL generation even when added 72 h after the initiation of cultures. The enhancing activity of rIL-6 could be neutralized with anti-IL-6 antibodies even when added 72 h after the initiation of cultures. The present data indicate that IL-6 acts in the late phase of CTL generation.
The characterization of four monoclonal antibodies specific for mouse IL-5 and development of mouse and human IL-5 enzyme-linked immunosorbent.
Authors: Schumacher J H , O'Garra A , Shrader B , van Kimmenade A , Bond M W , Mosmann T R , Coffman R L
Published: December 2022

Four rat mAb directed against mouse IL-5 have been characterized by their ability to remove and neutralize mouse IL-5 activity in various bioassays. All four mAb absorbed IL-5 activity from solution. Although all were able to neutralize mouse IL-5 bioactivity, two were significantly more effective. These two were also able to neutralize the activity of mouse IL-5 delivered to B cells during a cognate-linked interaction with a Th cell clone. A two-site sandwich ELISA specific for mouse IL-5 was developed by using pairs of mAb. The mouse IL-5 ELISA is capable of detecting natural or mouse rIL-5 in supernatants, crude bacterial lysates, and high concentrations of mouse serum, and has a detection limit of less than 20 pg. Two of these antibodies cross-reacted with and neutralized human rIL-5, and one of these was used for development of an ELISA for human IL-5.
A rabbit Ig lambda L chain C region gene encoding C21 allotopes.
Authors: Duvoisin R M , Hayzer D J , Belin D , Jaton J C
Published: December 2022

Southern blot analyses of germ-line DNA obtained from rabbits expressing lambda chains of C7 and/or C21 allotypes were performed with a rabbit C lambda region-specific probe; a 12-kbp EcoRI- and a 2-kbp BamHI-hybridizing fragment were detected only in the DNA from rabbits expressing the C21 allotype. The 12-kbp EcoRI fragment was cloned and shown to contain two C lambda region-encoding genes in the same orientation. Each is preceded by a J lambda gene segment. Nonamer-12-bp spacer-heptamer recombination signal sequences were found 5' of each J lambda segment, and splicing signals were identified at the 3' ends of the J lambda segments and the 5' ends of the corresponding C lambda genes. The C lambda 5 gene, which exhibits a sequence identical with that found in several cDNA clones, is carried by the 2-kbp BamHI fragment missing from the genomic DNA of rabbits which do not express the C21 allotype. The second C lambda gene, C lambda 6, lies 3' of C lambda 5, in a 1.6-kbp BamHI fragment which is present in genomic DNAs of all tested rabbits, irrespective of their phenotype. Its sequence is identical with that found in one cDNA clone and differs from that of C lambda 5 in 17 base positions resulting in four amino acid substitutions. A fragment of a cDNA, with a J-C region sequence identical with that encoded by the J lambda 5-C lambda 5 gene pair, was subcloned into a plasmid expression vector. The resulting polypeptide product could be specifically immunoprecipitated by anti-C21 but not anti-C7 alloantisera, showing that some, if not all, C21 allotopes are encoded by the C lambda 5 gene. In contrast, the C lambda 6 gene product was not precipitable, either by anti-C7 or by anti-C21 alloantisera, although it was readily immunoprecipitated by a goat anti-rabbit lambda chain antiserum.
Disruption of the internal thioester bond in the third component of complement (C3) results in the exposure of neodeterminants also present on activation products of C3. An analysis with monoclonal antibodies.
Authors: Hack C E , Paardekooper J , Smeenk R J , Abbink J , Eerenberg A J , Nuijens J H
Published: December 2022

Hydrolysis of the internal thioester bond in native C3 is thought to be a key event in initiating the alternative pathway of C activation, because the resulting C3(H2O) acquires "C3b-like" properties. Therefore, disruption of the internal thioester bond is probably accompanied by conformational changes in the C3 molecule. In this study, we demonstrate that such conformational changes indeed occur; 7 of the 19 mAb raised against C3 or C3 activation products recognized epitopes exposed on C3(H2O) but not on native C3. One of these epitopes is located on the C3a part, three on the C3c part, and another three on the C3d,g part. Because the 7 mAb bound equally well to C3 incubated either with MgCl2 or with methylamine (which primarily disrupts the thioester), the conformational changes detected by the mAb apparently occur after disruption of the thioester. Furthermore, the epitopes were also present on the corresponding C3 activation products. Immunoblotting experiments revealed that the epitopes for the three anti-C3d,g mAb were located on the C3d part, C-terminal to the thioester. The epitopes for 2 of the 3 anti-C3c mAb were located on the C-terminal alpha-chain fragment of C3c. Thus, this study provides immunochemical evidence for the biologic resemblance between C3(H2O) and C3 activation products. Implications of these findings for the activation process of C3 are discussed.
Regulation of complement factor H synthesis in U-937 cells by phorbol myristate acetate, lipopolysaccharide, and IL-1.
Authors: Minta J O
Published: December 2022

The capacity of the human monocyte cell line U-937 to synthesize complement factor H was examined. The kinetics of secretion of factor H into cell culture supernatant were followed by a sensitive solid phase RIA capable of measuring 0.1 ng of protein. Daily secretion of factor H was low and almost linear and was approximately 3 ng of factor H per 10(6) cells. Factor H synthesis was inhibited by cycloheximide but returned to the levels seen in untreated cultures after removal of the inhibitor. LPS and IL-1 both effected a time- and dose-dependent enhancement of factor H synthesis. Induction of U-937 cells with PMA to differentiate into macrophage-like cells also resulted in increased factor H synthesis. RIA of cell lysates, immunofluorescence microscopy, as well as FACS analysis all revealed that factor H Ag was also associated with the U-937 cell membrane. The population of U-937 cells bearing membrane-associated factor H was decreased from 77 to 43% after stimulation for 48 h with LPS (1 microgram/ml). [35S]Methionine metabolic labeling and SDS-PAGE analysis of factor H immunoprecipitates from unstimulated and stimulated culture supernatants and cell lysates demonstrated a major polypeptide, m.w. 150,000, and a minor component, m.w. 42,000. Western blot analysis of factor H in fresh normal plasma also detected both 150,000 and 42,000 m.w. factor H proteins. This is in agreement with the recent demonstration of a 4.4- and 1.8-kb mRNA for factor H in human liver. These data demonstrate that U-937 cells synthesize factor H that is structurally and antigenically similar to factor H in normal plasma. The exact nature of the membrane-bound factor H and its functions and mechanism of attachment to the cell membrane remain to be elucidated.