bluuebunny/crossref_metadata_2025 huggingface dataset:
Human IgA and IgG F(ab')2 that bind to staphylococcal protein A belong to the VHIII subgroup.
Authors: Sasso E H , Silverman G J , Mannik M
Published: December 2022

Staphylococcal protein A (SPA) is a bacterial membrane protein that possesses, in addition to its Fc gamma-binding activity, a distinct specificity for the Fab region of some IgM, IgA, IgG, and IgE. The Fab site that binds to SPA has been localized to the V region of the Ig H chain. In a previous study of human monoclonal and polyclonal IgM, we demonstrated that binding to SPA was highly restricted to molecules of the VHIII subgroup, and that nearly all VHIII IgM were able to bind SPA. The present study examines the VH composition of SPA-binding and SPA-nonbinding fractions of purified human polyclonal IgA, and IgG F(ab')2 fragments. We found that 22% of the IgA and 15% of the IgG F(ab')2 bound to SPA-agarose. Analysis with VH subgroup-specific antisera indicated that the SPA-binding fraction of IgA was dominated by the VHIII subgroup, and the SPA-binding fraction of IgG F(ab')2 contained only VHIII molecules. Furthermore, substantial portions of the total VHIII protein in IgA and in IgG F(ab')2 bound to SPA. We conclude that Fab binding to SPA is both restricted to and highly prevalent among human VHIII molecules, regardless of Ig class. These results suggest that protein A is an Ig superantigen.
Occupancy of CD11b/CD18 (Mac-1) divalent ion binding site(s) induces leukocyte adhesion.
Authors: Altieri D C
Published: December 2022

The group of leukocyte integrins CD11a-c/CD18 coordinate disparate adhesion reactions in the immune system through a regulated process of ligand recognition. The participation of the receptor divalent ion binding site(s) in this mechanism of ligand binding has been investigated. As compared with other divalent cations, Mn2+ ions have the unique property to dramatically stimulate the adhesive functions of the leukocyte integrin CD11b/CD18 (Mac-1), expressed on myelo-monocytic cells. This is reflected in a three- to fivefold increased early monocyte adhesion (less than 20 min) to resting, unperturbed endothelial cells, and increased association of CD11b/CD18 with its soluble ligands fibrinogen and factor X. CD11b/CD18 ligand recognition in the presence of Mn2+ ions is specific, time and concentration dependent, and inhibited by anti-CD11b mAb. At variance with Ca(2+)-containing reactions where CD11b/CD18 functions as an inducible receptor activated by adenine nucleotides or chemoattractants, Mn2+ ions induce per se a constitutive maximal ligand binding capacity of CD11b/CD18, that is not further modulated by cell stimulation. Rather than quantitative changes in surface density, Mn2+ ions increase the affinity of CD11b/CD18 for its complementary ligands up to 10-fold, as judged by Scatchard plot analysis of receptor:ligand interaction under these conditions. Furthermore, monocyte exposure to Mn2+ ions induces the expression of activation-dependent neo-antigenic epitopes on CD11b/CD18, selectively recognized by mAb 7E3. These data suggest that in addition to cell-activating stimuli, favorable engagement of divalent ion binding site(s) can provide an alternative pathway to rapidly regulate the receptor affinity of leukocyte integrins.
Protein kinase C plays a role in the induction of tyrosine phosphorylation of lymphoid microtubule-associated protein-2 kinase. Evidence for a CD3-associated cascade that includes pp56lck and that is defective in HPB-ALL.
Authors: Nel A E , Hanekom C , Hultin L
Published: December 2022

Ligation of the CD3 receptor induces multiple signal transduction events that modify the activation state of the T cell. We have compared two lines that express biologically active CD3 receptors but differ in their biochemical activation pathways during ligation of this receptor. Jurkat cells respond to anti-CD3 with Ca2+ mobilization, PKC activation, induction of protein tyrosine phosphorylation, and activation of newly characterized lymphoid microtubule associated protein-2 kinase (MAP-2K). MAP-2K itself is a 43-kDa phosphoprotein that requires tyrosine phosphorylation for activation. Although ligation of the CD3 receptor in HPB-ALL could stimulate tyrosine phosphorylation of a 59- kDa substrate, there was no associated induction of [Ca2+]i flux, PKC, or MAP-2K activation. A specific PKC agonist, PMA, which bypasses the CD3 receptor, could, however, activate MAP-2K in HPB-ALL cells. This implies that defective stimulation of PKC by the CD3 receptor is responsible for its failure to activate MAP-2K in HPB-ALL. The defect in PKC activation is likely distal to the CD3 receptor as A1F14- failed to activate MAP-2K in HPB-ALL but was effective in Jurkat cells. The stimulatory effect of PMA on MAP-2K activity in HPB-ALL was accompanied by tyrosine phosphorylation of this kinase which implies that PKC may, in some way, regulate tyrosine phosphorylation of MAP-2K. A candidate for this role is pp56lck which underwent posttranslational modification (seen as mobility change on SDS-PAGE) during anti-CD3 and PMA stimulation in Jurkat or PMA treatment in HPB-ALL. There was, in fact, exact coincidence between induction of PKC activity, posttranslational modification of lck and tyrosine phosphorylation/activation of MAP-2K. Lck kinase activity in an immune complex kinase assay was unchanged during PMA treatment. An alternative explanation is that modification of lck may alter its substrate profile. We therefore looked at the previously documented ability of PKC to dissociate lck from the CD4 receptor and found that PMA could reduce the stoichiometry of the lck interaction with CD4 in HPB-ALL and to a lesser extent in Jurkat cells. These results imply the existence of a kinase cascade that is initiated by PKC and, in the course of which, lck and MAP-2K may interact.
Human T cell clone identifies a potentially protective 54-kDa protein antigen of Toxoplasma gondii cloned and expressed in Escherichia coli.
Authors: Saavedra R , de Meuter F , Decourt J L , Hérion P
Published: December 2022

Protective immunity against Toxoplasma gondii is recognized to be cell mediated and IFN-gamma is considered to be the major mediator of resistance. Thus, protective Ag of the parasite must induce IFN-gamma-producing T cells. In order to identify such Ag, we have constructed a T. gondii cDNA library in the cloning/expression vector lambda gt11, screened this library with a pool of sera of immune donors, and further screened the set of selected recombinant Ag using, as probe, a T. gondii-reactive T-cell clone (TCC) derived from an infected/immune individual and producing a high level of IFN-gamma. One recombinant Ag was shown to induce TCC proliferation and was characterized. The corresponding mature T. gondii Ag has an apparent molecular mass of 54 kDa and the sequence of the cDNA clone suggests that it is membrane associated. The epitope defined by the TCC on this Ag was found to be present in three Toxoplasma strains independently of their phenotype (virulent or cyst forming). Recognition of this Ag by the TCC was shown to be restricted by HLA-DPw4, the most frequent allele in the Caucasian population (approximately 40%). The use of this Ag as a vaccine component is proposed.
Induction of the immune response to Legionella pneumophila cytolysin by monoclonal anti-idiotypic antibodies.
Authors: Spitsin S V , Drobyshevskaya E I , Barkhatova O I , Belyi Y F , Tartakovsky I S , Nesterenko V G
Published: December 2022

A panel of mAb (IgG1, IgG3, IgM) against Legionella pneumophila cytolysin (CL)-protease of 37 kDa was obtained. Subtyping of L. pneumophila strains of serogroup 1 by using mAb against CL (mAb-CL) was carried out. The results of comparative analysis of the specificity of mAb-CL and the panel of mAb kindly provided by Dr. J. M. Barbaree (Centers for Disease Control, Atlanta, GA) allowed us to recommend mAb-CL to be used as a diagnostic tool to reveal the pathogenicity of L. pneumophila strains of serogroup 1. Hybridomas were also raised in a syngenic system which produced anti-idiotypic mAb (mAb2) against anti-CL mAb B6/1. The Ab2 belonged to Ab2 gamma type: 1) Ab2 reacted with B6/1 Id only, 2) Ab2 inhibited the interaction of B6/1 Ab1 with CL, and 3) CL inhibited the reaction of Ab2 with Ab1. The use of Ab2 allowed us to show that B6/1 Id is expressed in 4 to 32% of serum antibodies during the primary and secondary immune responses of BALB/c mice to CL. Ab2 induced the production of anti-anti-idiotypic antibodies (Ab3) in BALB/c mice, and some of them reacted with CL. Thus, we have demonstrated the possibility of inducing an antibody response to CL (one of the main L. pneumophila pathogenic factors) in intact syngenic mice with anti-idiotypic antibodies.
Human recombinant migration inhibitory factor activates human macrophages to kill Leishmania donovani.
Authors: Weiser W Y , Pozzi L M , David J R
Published: December 2022

A recombinant form of the first lymphokine to be discovered, migration inhibitory factor (rMIF) was obtained from COS-1 cells transfected with a cDNA library from a human T cell hybridoma (6). rMIF has an amino acid sequence unrelated to that of any known protein. To learn more about the biology of MIF, we tested its ability to effect the survival of Leishmania donovani in macrophages. We found that rMIF activates blood monocyte-derived macrophages in vitro to suppress the growth of and kill these intracellular parasites. The anti-leishmanial effect (ranging from 50 to 77% reduction of parasites) is maximal when macrophages have been incubated with rMIF 48 to 72 h before infection and is similar to that seen with macrophages activated by IFN-gamma. Of interest, whereas the activation of human macrophages by IFN-gamma is inhibited by IL-4 and not enhanced by LPS, the activation by rMIF is enhanced by LPS but is not inhibited by IL-4. The data presented here demonstrate that rMIF is a potent activator of macrophages and is likely to be critical in cell-mediated immune host defenses.
Influence of a V kappa 8 L chain transgene on endogenous rearrangements and the immune response to the HA(Sb) determinant on influenza virus.
Authors: Carmack C E , Camper S A , Mackle J J , Gerhard W U , Weigert M G
Published: December 2022

A rearranged murine V kappa 8/J kappa 5 L chain gene that codes for the L chain of most antibodies generated in the primary response of BALB/c mice to the antigenic site, Sb, of the hemagglutinin (HA) molecule of influenza virus A/PR/8/34 (PR8) has been cloned. Three transgenic lines were generated by microinjecting the gene. Lines Ga and L each contain a single copy of the transgene whereas line Gb contains three complete copies. Mice of the Ga lineage showed increased V kappa 8-specific mRNA levels only in spleen, but not in nonlymphoid organs and therefore displayed apparently normal lymphoid-specific regulation of the Ig transgene. B cell hybridomas generated from these mice were analyzed for rearrangements of endogenous V kappa genes. Greater than 90% of the C kappa alleles were retained in germ-line configuration in the Ga line, compared with only 0 to 18% in the L line. Thus, a wide variation in the frequency of endogenous rearrangements is seen among mice of different lineages using the same transgene construct. None of more than 150 hybridomas derived from LPS-stimulated splenic B cells of Ga mice exhibited HA-binding activity although they expressed the transgene and, in most cases, excluded endogenous V kappa rearrangements. In contrast, a large fraction of hybridomas isolated after primary immunization with PR8 were HA(Sb)-specific. This indicated that the transgene was functional but formed HA-specific antibodies with a more restricted set of H chains than previously hypothesized. The primary anti-HA response to immunization with PR8 was diminished in all lines compared with normal mice except for a slightly accelerated but transient burst of anti-HA antibody formation in two out of three lines (Ga and Gb). This early response in G lineage mice was largely specific for HA(Sb) and thus appeared to be composed of transgene-expressing antibodies. No differences in serum titers were observed in the secondary anti-HA responses to booster inoculation with PR8 between transgenic and normal mice.
Analysis of anti-tumor antibodies in mice and rabbits induced by monoclonal anti-idiotope antibodies.
Authors: Chattopadhyay P , Starkey J , Raychaudhuri S
Published: December 2022

Eight different mouse monoclonal anti-idiotope antibodies (mAb2) generated against a mouse monoclonal anti-human melanoma proteoglycan Ag (MPG) antibody (mAb1), MEM136, were tested for their ability to induce anti-MPG responses in mice and rabbits. All Ab2 were idiotypically cross-reactive and combining site-specific as demonstrated by competitive cross-inhibition studies and their ability to inhibit the binding of MEM136 to the melanoma cells, Colo38. However, only two Ab2, IM32 and IM06, were able to induce specific anti-TAA-specific (Ab1') responses in rabbits. When IM32 and IM06 were tested in allogeneic stains of mice for the induction of anti-MPG responses, only IM32 produced an Ab1' response. In mice, the Ab3 response induced by IM32 is idiotypically cross-reactive with its Ab1. Furthermore, the IM32-induced murine Ab3 and MEM136 recognized a similar MPG epitope on the melanoma cells because the Ab3 inhibited the binding of MEM136 to melanoma cells. The Ab3 induced by IM32 and IM06 in rabbits also recognized a similar epitope as the Ab1. In rabbits, the Ab3 response induced by IM32 and IM06 were idiotypically cross-reactive with each other. However, additional studies indicated that the majority of Ab3 induced by IM32 were IM32 Id-specific and lacked IM06 idiotopes. Further experimentation indicated that IM32-induced rabbit Ab3 were biologically active as demonstrated by the ability of the Ab3 to inhibit melanoma cell invasion in a Matrigel assay.
Monoclonal antibody against actin cross-reacts with the Thy-1 molecule and inhibits lymphocyte function-associated antigen-1 dependent cell-cell interaction of T cells.
Authors: Nishimura T , Takeuchi Y , Gao X H , Urano K , Habu S
Published: December 2022

To assess the molecular mechanisms involved in cell-cell interactions of T cells, we produced a mAb that could block PMA-induced homotypic T cell aggregation. The established mAb (TN14 mAb) showed an inhibitory effect on T cell aggregation as large as that of mAb against lymphocyte function-associated Ag-1. TN14 mAb showed a strong reactivity with cell surface molecules on T cells, whereas it did not react with any cell surface Ag on macrophages, B cells, bone marrow cells, or WEHI 164 cells, which were used for immunization. However, all cell lysates could react with TN14 mAb in Western blotting analysis, indicating that TN14 mAb recognized some cytoplasmic protein. From biochemical analysis, TN14 mAb was demonstrated to react with the 43-kDa cytoskeletal protein actin. Moreover, from an immunoprecipitation study, TN14 mAb was demonstrated to cross-react with the Thy-1 molecule on T cells. The binding activity of TN14 mAb with the Thy-1 molecule on T cells was strongly blocked by the addition of purified actin, indicating that TN14 mAb recognized an actin-related epitope of the Thy-1 molecule. TN14 mAb blocked not only T cell aggregation but also cell-mediated cytotoxicity by CTL. These results strongly support the hypothesis that the Thy-1 molecule, a member of the Ig superfamily, may play an important role in cell-cell interactions of T cells.
Evaluation of monophosphoryl lipid A (MPL) as an adjuvant. Enhancement of the serum antibody response in mice to polysaccharide-protein conjugates by concurrent injection with MPL.
Authors: Schneerson R , Fattom A , Szu S C , Bryla D , Ulrich J T , Rudbach J A , Schiffman G , Robbins J B
Published: December 2022

Concurrent injection of monophosphoryl lipid A (MPL) in saline or as an oil-in-water emulsion enhanced both the primary and secondary serum antibody responses to the capsular polysaccharide (CP) components of seven conjugates: the enhanced responses were Ag-specific. In contrast, MPL did not enhance the serum antibody response to five of the six unconjugated CP. MPL and trehalose dimycolate injected concurrently with the unconjugated Vi CP of Salmonella typhi (Vi) enhanced the serum antibody response to that Ag. MPL further enhanced the Vi antibody levels when injected with conjugates of this CP. The serum antibody responses to Pseudomonas aeruginosa exotoxin A, used as the carrier protein for the Staphylococcus aureus types 5 and 8 conjugates, were also enhanced by MPL. MPL in oil-in-water emulsion was generally more effective than when administered in saline.
Acylation of cell-associated IL-1 by palmitic acid.
Authors: Bakouche O , Moreau J L , Lachman L B
Published: December 2022

To determine whether membrane-associated IL-1 is palmitylated, we labeled LPS-activated human monocytes with [3H]palmitic acid. The plasma membranes were isolated, and the membrane proteins extracted and analyzed simultaneously by SDS-PAGE-autoradiography and Western blot analysis from the same gel. When the monocytes were labeled with [3H]palmitate, 23- and 31-kDa bands were visualized, for membrane-associated IL-1 and its precursor, respectively. The 31- and 23-kDa bands were excised from several gels and rehydrated and analyzed again by SDS-PAGE, autoradiography, and Western blot analysis. The 23- and 31-kDa bands appeared again by both methods. To further investigate membrane-associated IL-1 acylation, human monocytes were labeled with [3H]palmitate, the plasma membranes isolated, and the membrane proteins extracted by CHAPS detergent. Immunoprecipitation of isolated membrane proteins using anti-IL-1 antibodies revealed two bands of 23 and 31 kDa after autoradiography. The studies demonstrate that both membrane-associated IL-1 and the IL-1 precursor are acylated with palmitic acid.
Human eosinophil cytotoxicity-enhancing factor. II. Multiple forms synthesized by U937 cells and their relationship to thioredoxin/adult T cell leukemia-derived factor.
Authors: Balcewicz-Sablinska M K , Wollman E E , Gorti R , Silberstein D S
Published: December 2022

Recently, our laboratory reported the purification and partial amino acid sequence of a 10-kDa eosinophil cytotoxicity-enhancing factor (ECEF) polypeptide from the U937 cell source. This cytokine enhanced human eosinophil antibody-dependent cytotoxic function by greater than 200% and was half-maximally effective at a concentration of approximately 1 ng/ml. In this study, we describe the conditions required for ECEF synthesis and the use of rabbit antibody raised to 10-kDa ECEF to investigate the existence of related polypeptide species. Unstimulated U937 cells released an immunoreactive 14-kDA species. Cells stimulated with 7.5 micrograms/ml of LPS also released a 13-kDa species. Cells stimulated with 400 ng/ml of PMA also synthesized a 10-kDa species (equivalent in size to the form we had purified). This 10-kDa species remained primarily cell associated, but detectable amounts were released into the supernatant by 48 h of culture. In washed cell pellets, the location of the 10-kDa species was found to be in the plasma membrane, externally oriented, as determined by FACS analysis, iodination with the membrane impermeable reagent 125I-sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate, and by its removal with brief trypsin treatment. Partial amino acid sequence data suggested that the 14-, 13-, and 10-kDa species all share the same N-terminal. The 14- and 10-kDa ECEF species were recovered by electroelution from SDS-PAGE gels and tested for activity in the assay of eosinophil cytotoxic function. Because of the amino acid sequence similarities between the ECEF species and thioredoxin (TRX), rTRX (synthesized in Escherichia coli and purified) was also tested for activity. The 14-kDa ECEF and rTRX induced a slight, but consistent and statistically significant enhancement of eosinophil cytotoxic function. By comparison, lower doses of the 10-kDa ECEF induced a major increase in cytotoxic function. Thus the forms of ECEF differ in size, conditions required for synthesis, trafficking by the U937 cell after synthesis, and biologic activity. It is likely that these considerations bear on the involvement of ECEF in the pathophysiology of eosinophilia in vivo.
Monoclonal antibodies to the calcium-binding region peptide of human C-reactive protein alter its conformation.
Authors: Swanson S J , Mullenix M C , Mortensen R F
Published: December 2022

Five mouse mAb were generated against a synthetic peptide corresponding to the proposed Ca(2+)-binding region of human C-reactive protein (CRP). The peptide consists of amino acids 134 to 148 and possesses a calmodulin Ca(2+)-binding sequence. The mAb reacted with a surface epitope(s) on native, intact CRP as well as the closely related pentraxin protein, serum amyloid P-component. Three of the 5 mAb inhibited the Ca(2+)-dependent phosphorylcholine-(PC) binding activity of CRP, but did not bind to the PC-binding region itself. Four of the five mAb also inhibited the recognition of an epitope in the PC-binding site of CRP. Four of the mAb partially, or completely, protected CRP from selective cleavage by pronase between residues 146 and 147. The findings suggest that the Ca(2+)-binding region is on the surface of CRP, has substantial flexibility, and is probably responsible for the allosteric effects of Ca2+ ions on CRP.
Oscillations in free cytosolic calcium during IgE-mediated stimulation distinguish human basophils from human mast cells.
Authors: MacGlashan D , Guo C B
Published: December 2022

Free cytosolic calcium ([Ca2+]i) levels increase after the stimulation of either human basophils or mast cells with anti-IgE antibody. Previous studies found that the mast cell [Ca2+]i response was graded in magnitude, according to stimulus strength, and that the activated level was free of oscillations. The current studies demonstrate several new features of the mast cell and basophil response. First, in mast cells, the transition to activated [Ca2+]i levels was abrupt (width = 6.5 +/- 2 s), after a period of quiescence whose duration (5 to 300 s) was a function of the strength of the stimulus. At optimal concentrations of anti-IgE, 97% of mast cells showed only abrupt transitions and oscillation-free activated calcium levels. In contrast, basophils showed marked oscillations whose magnitudes were partially dependent on the strength of stimulus. Like the mast cell, there was a quiescent period before the first transition and this period was also dependent on the strength of the stimulus. Oscillations were generally superimposed on an elevated [Ca2+]i level at a frequency of 0.5 to 3/min, had half-widths of 5 to 20 s, and were markedly chaotic in the frequency domain. In general, oscillations were more apparent at suboptimal concentrations of anti-IgE. Despite the apparent contrast in basophil and mast cell responses, oscillations (1 or 2 in a 10-min period) could be observed in a small percentage of mast cells and some basophils showed characteristics of mast cells. We tentatively conclude that mast cells and basophils utilize a similar mechanism of calcium mobilization but that the nonlinear characteristics of the calcium response may account for the mast cell/basophil differences. These studies indicated that the calcium kinetics, as measured by population averages, did not reflect the kinetics observed at the single cell level. Both mast cells and basophils had characteristics which could be described as graded and characteristics resembling all-or-nothing processes; the magnitude of a response was graded according the strength of stimulus while the kinetics profile appeared as an all-or-nothing event.
Nucleocapsid or spike protein-specific CD4+ T lymphocytes protect against coronavirus-induced encephalomyelitis in the absence of CD8+ T cells.
Authors: Körner H , Schliephake A , Winter J , Zimprich F , Lassmann H , Sedgwick J , Siddell S , Wege H
Published: December 2022

To investigate the antiviral CD4+ T cell response in coronavirus MHV-JHM-induced encephalomyelitis, spleen and thymic lymphocytes from diseased rats were stimulated in culture with virus Ag, expanded and tested for their specificity to viral proteins and nucleocapsid (N) and spike (S) proteins that had been expressed in bacteria. A strong T cell response specific for N was measurable during acute disease, whereas S-specific T cells were only detectable in rats with a later onset of disease. CD4+ T cell lines with specificity for virus and either N or S protein were established and their influence on the course of a mouse hepatitis virus-JHM infection was investigated. All lines were of the CD4+ phenotype. Both N and S protein-specific CD4+ T cells conferred protection to infected Lewis rats and reduced the amount of infectious virus in the central nervous system. After transfer of CD4+ T cells and challenge with virus, an increase in the antiviral IgM response occurred, but neutralizing antibodies were not detectable during the period of virus clearance. Previous CD8+ cell depletion did not abrogate protection mediated by CD4+ T cell line transfer.
Natural auto- and polyreactive antibodies differing from antigen-induced antibodies in the H chain CDR3.
Authors: Chen C , Stenzel-Poore M P , Rittenberg M B
Published: December 2022

We describe three sets of natural (preimmune) polyreactive antibodies and Ag-induced antibodies that share the same VH-VL combinations. The amino acid homology in the VH and VL segments averaged 92%. These sets were found among 49 neonatal and adult natural mAb that were compared with 35 Ag-induced monoclonals produced during the memory response to phosphocholine (PC)-keyhole limpet hemocyanin. Both groups of monoclonals had been selected on the basis of a restricted fine specificity pattern, namely the ability to recognize PC-protein and p-nitrophenyl phosphocholine but not PC. All of the antibodies were tested for reactivity against a panel of 15 self and foreign Ag. Despite their common fine specificity as the basis for selection, 33/49 natural antibodies were poly/auto reactive whereas 0/35 Ag-induced antibodies had such poly/auto reactive properties. The natural antibodies were encoded by genes representing nine different VH families and several V kappa and V lambda families. There were a few replacement substitutions distinguishing the Ag-induced antibodies from the natural antibodies in each set; however, the most noteworthy difference was the extreme variability of CDR3 in the natural antibodies that differed in both length and amino acid sequence from each other and from Ag-induced antibodies. The results suggest that CDR3 of the H chain may play a critical role in distinguishing poly- from monospecific combining sites in natural and Ag-induced antibodies.
Cytokine gene expression after in vivo primary immunization with goat antibody to mouse IgD antibody.
Authors: Svetić A , Finkelman F D , Jian Y C , Dieffenbach C W , Scott D E , McCarthy K F , Steinberg A D , Gause W C
Published: December 2022

Cytokines are important mediators of effector lymphoid cell function during an immune response, but their expression during an in vivo immune response has not been well documented. We analyzed the kinetics of cytokine gene expression during the course of an in vivo primary immune response to goat antibody to mouse IgD antibody. Total RNA was purified from spleens taken from freshly killed BALB/c mice 1 to 7 days after immunization. The reverse transcriptase polymerase chain reaction was used to evaluate the expression of seven cytokine genes, all of which encode cytokines that are secreted by T cells and are important in T and/or B cell activation and differentiation. These were IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-9, and IL-10. IL-2 and IL-9 exhibited an early elevated expression at days 2 to 3, and declined as the expression of IL-4, IL-6, IL-10, and IFN-gamma increased. In contrast, IL-5 gene expression showed little change, exhibiting a similar pattern to the housekeeping gene, hypoxanthine-guanine phosphoribosyl transferase. Cell sorting of CD4+ and CD4- cells at day 3 and day 5 after immunization revealed that CD4+ cells were the predominant source of the elevated cytokines (with the exception of IL-6). Our results demonstrate a specific and highly reproducible cytokine gene expression pattern during the course of a primary in vivo immune response that is marked by an absence of a clear-cut Th1/Th2 dichotomy.
Anti-IgM-mediated growth inhibition of a human B lymphoma cell line is independent of phosphatidylinositol turnover and protein kinase C activation and involves tyrosine phosphorylation.
Authors: Beckwith M , Urba W J , Ferris D K , Freter C E , Kuhns D B , Moratz C M , Longo D L
Published: December 2022

The RL cell line is an EBV-negative, surface IgM, IgD-positive B lymphoma line, which is significantly growth arrested in the presence of acrylamide-linked antibodies to the surface IgM receptor. We demonstrate here that activation of protein kinase C (PKC) with PMA abrogates anti-IgM-induced phosphoinositide turnover and Ca2+ mobilization; however, growth inhibition is not affected. In addition, inhibitors of PKC are unable to reverse the anti-IgM-mediated growth inhibition. Two-dimensional gel electrophoresis reveals a different pattern of protein phosphorylation after treatment of RL with PMA or anti-IgM. These data strongly suggest that anti-IgM-induced growth inhibition does not rely on phospholipase C-mediated phosphoinositide turnover, Ca2+ mobilization, or PKC activation. On the other hand, the phosphatase inhibitor orthovanadate results in an augmentation of proteins phosphorylated on tyrosine and the growth inhibition which follows anti-IgM treatment. Furthermore, protein tyrosine kinase inhibitors, genistein and herbimycin A, are able to reverse the anti-IgM-induced inhibition of growth. These data demonstrate that multiple signaling pathways are activated by the interaction of anti-IgM with its ligand, and suggest that tyrosine kinase activation is a critical component of the inhibitory response.
Predominant T cell receptor gene elements in TNP-specific cytotoxic T cells.
Authors: Kempkes B , Palmer E , Martin S , von Bonin A , Eichmann K , Ortmann B , Weltzien H U
Published: December 2022

H-2b class I-restricted, TNP-specific CTL clones were obtained by limiting dilution cloning of either short term polyclonal CTL lines or spleen cells of TNP-immunized mice directly ex vivo. Sequence analyses of mRNA coding for TCR alpha- and beta-chains of 11 clones derived from CTL lines from individual C57BL/6 mice revealed that all of them expressed unique but clearly nonrandom receptor structures. Five alpha-chains (45%) employed V alpha 10 gene elements, and four of those (36%) were associated with J beta 2.6-expressing beta-chains. The alpha-chains from these four TCR, moreover, contained an acidic amino acid in position 93 of their N or J region-determined sequences. Clones isolated directly from spleen cells carried these types of receptors at lower frequency, 27% V alpha 10 and 19% J beta 2.6, indicating that bulk in vitro cultivation on Ag leads to selection for these particular receptors. However, even in TNP-specific CTL cloned directly ex vivo, V alpha 10 usage was increased about fivefold over that in Ag-independently activated T cells in H-2b mice (4 to 5%). The selection for V alpha 10/J beta 2.6-expressing cells was obtained repeatedly in other TNP-specific CTL lines from C57BL/6 mice but not in FITC-specific CTL from the same strain or in TNP-specific CTL lines from B10.BR (H-2k) or B10.D2 (H-2d) mice. We conclude from this (a) that the selection for V alpha 10/J beta 2.6+ T cells is driven by the complementarity of these receptors to a combination of TNP and MHC epitopes and (b) that predominant receptor structures reflect the existence of a surprisingly limited number of "T cell-relevant" hapten determinants on the surface of covalently TNP-modified cells.
Differential regulation of surface Ig- and Lyb2-mediated B cell activation by cyclic AMP. I. Evidence for alternative regulation of signaling through two different receptors linked to phosphatidylinositol hydrolysis in murine B cells.
Authors: Muthusamy N , Baluyut A R , Subbarao B
Published: December 2022

The differential effect of cAMP on the regulation of early biochemical and cellular functions mediated through two different receptors on murine B cells are reported here. Surface IgM, the Ag receptor, and Lyb2, a 45-kDa differentiation Ag are concomitantly expressed on mature murine B lymphocytes. Triggering of B cells through these molecules, independently, resulted in inositol 1,4,5-triphosphate (IP3) generation, increase in intracellular Ca2+ levels, and cell enlargement associated with progression of cells from G0 to G1 ultimately resulting in DNA synthesis. Pretreatment of resting B cells with cholera toxin as well as other agents that raise the intracellular cAMP [(cAMP)i] such as forskolin, N6,2'-O-dibutyryl cyclic AMP, and 3-isobutyl-1 methyl xanthine inhibited the Ag receptor but not Lyb2-mediated DNA synthesis. The elevation of (cAMP)i inhibited the surface IgM but not Lyb2-mediated IP3 generation, Ca2+ response, and progression from G0 to G1 phase of the cell cycle. Failure of forskolin or N6,2'-O-dibutyryl cyclic AMP to inhibit Lyb2-mediated responses did not appear to be due to induction of cAMP-specific phosphodiesterase activity. Concentrations of H8 [N-(2-(methylamino)-ethyl)-5-isoquinoline sulfonamide, diHCl] inhibitory to cAMP dependent PKA prevented the inhibitory effect of forskolin on surface IgM-mediated Ca2+ response, suggesting that cAMP exerted its effects through PKA. These findings suggest that distinct PLC-coupled receptors, such as sIgM and Lyb2 molecules in B cells, may use either alternative mechanisms for phosphatidylinositol 4,5-bisphosphate hydrolysis or may use different intermediary transducer molecules that differ in their sensitivity to increased (cAMP)i levels. Thus "cross-talk" among cAMP and phosphatidylinositol signaling pathways was demonstrated for IgM but not Lyb2-mediated B cell activation.
Antigen-specific IGG2A production in response to prostaglandin E2, immune complexes, and IFN-gamma.
Authors: Stein S H , Phipps R P
Published: December 2022

Immune complexes (IC) can inhibit the differentiation of B lymphocytes into IgM secreting plasma cells in both Ag-specific and polyclonal systems. This report describes the ability of IC and IFN-gamma, or IFN-gamma and PGE2 to regulate the class of Ig produced in an Ag-specific system. In an in vitro model system using fluorescein (FL)-Brucella abortus as Ag, we previously showed that IC composed of an anti-FL antibody and FL-Ag inhibited the ability of FL-specific B cells to develop into IgM plaque-forming cells. In addition, PGE2 sensitized resting B cells to IC, whereas pretreatment with IL-4 alleviated the IC-mediated decrease in the IgM anti-FL response. In this manuscript, we demonstrate that IFN-gamma has antagonistic effects compared to IL-4, and potentiates the IC-induced decrease in the IgM antibody response. Interestingly, we discovered that the virtual ablation of the anti-FL IgM response exhibited by B cells treated with IC and IFN-gamma was accompanied by a dramatic increase in the anti-FL IgG2a response. Furthermore, resting B lymphocytes pulsed with IFN-gamma and PGE2, but not PGF2 alpha, exhibited augmented IgG2a production. This effect was also observed when IFN-gamma-pulsed B cells were stimulated with other agents, such as dibutyryl cAMP and cholera toxin, that elevate intracellular levels of cAMP. In addition, RpcAMP, the R-isomer of a sulfur-modified cAMP and a cAMP antagonist, inhibited anti-FL IgG2a responses. The ability of cAMP elevators such as PGE2 to promote IgG2a production, as well as to increase the frequency of IgG2a secreting cells, was demonstrated in B lymphocytes treated with IFN-gamma and polyclonally activated by LPS. Overall, our results demonstrate that IFN-gamma and PGE2-treated B lymphocytes challenged with antigen or LPS generate elevated levels of IgG2a via a cAMP-dependent pathway. These observations suggest that in vivo, PGE2 secreted by cells such as macrophages potentiates the ability of IFN-gamma to promote an IgG2a response, the predominant murine antiviral Ig.
Catecholamines stimulate the IFN-gamma-induced class II MHC expression on bovine brain capillary endothelial cells.
Authors: Coutinho G C , Durieu-Trautmann O , Strosberg A D , Couraud P O
Published: December 2022

The brain has been considered for a long time as an immunologically privileged site because of the lack of a true lymphatic system and the existence of several barriers that isolate it from the periphery. In the last few years, it became evident that cells in the central nervous system (astrocytes, microglial cells, and brain capillary endothelial cells) can be induced to express class II MHC and present Ag to T lymphocytes. The brain capillary endothelial cells, which are strategically located at the interface between blood and brain, could be involved in the initiation of immune responses within the brain parenchyma. We have previously characterized bovine brain capillary endothelial cells in culture and shown that they maintain in vitro a fully differentiated phenotype associated with the blood-brain barrier endothelium. In order to assess the role of these cells in the development of immune responses in the brain, we initiated the present study on the regulation of their class II MHC surface expression. Our data indicate that this expression on bovine brain capillary endothelial cells is inducible by IFN-gamma and further stimulated by catecholamines through activation of beta-adrenergic receptors. However, this latter effect is not mimicked by forskolin, theophylline, or dibutyryl-cAMP, suggesting the involvement of a cAMP-independent mechanism.
Thymic microenvironment induces HIV expression. Physiologic secretion of IL-6 by thymic epithelial cells up-regulates virus expression in chronically infected cells.
Authors: Schnittman S M , Singer K H , Greenhouse J J , Stanley S K , Whichard L P , Le P T , Haynes B F , Fauci A S
Published: December 2022

The hallmark of infection with HIV-1 is progressive depletion and qualitative dysfunction of the CD4+ Th cell population in infected individuals. Clinical trials of antiretroviral agents have shown that, despite suppression of virus replication, regeneration of the T cell pool does not occur. One proposed explanation for the defective regenerative capacity of the CD4+ T cell pool is infection of early T lymphocyte progenitors or stem cells. An additional explanation could be failure of cells of the intrathymic microenvironment (thymic epithelial (TE) cells) to carry out critical nurturing functions for developing thymocytes, i.e., secretion of thymocyte-trophic cytokines and expression of adhesion molecules. This study examines the effect of HIV on cultured TE cells and determines the role of TE cells in the regulation of viral expression in chronically HIV-infected cells. We found no evidence of infection of TE cells after exposure to HIV-1. However, normal human serum induced secretion of IL-6 by TE cells; induction of TE IL-6 was partially blocked by anti-IFN-gamma antibodies. Moreover, supernatants from TE cells maintained in normal human serum up-regulated HIV replication in chronically HIV-1-infected cells. Because intrathymic T cell precursors can be infected with HIV and T cell precursors come into close contact with TE cells in the thymus, IL-6 secreted by TE cells during normal intrathymic development may induce HIV expression in infected thymocytes in vivo and promote the intrathymic spread of HIV.
IL-1 receptor antagonist regulation of acute phase protein synthesis in human hepatoma cells.
Authors: Bevan S , Raynes J G
Published: December 2022

The hepatoma cell line HuH-7 has recently been shown to synthesize serum amyloid A (SAA) in response to IL-1. IL-1 receptor antagonist (IL-1Ra) was able to completely inhibit the response of SAA to IL-1 but not the increase seen in response to IL-6. IL-1Ra was equally effective at inhibiting IL-1 alpha or IL-1 beta. At a 10-fold molar excess of IL-1Ra over IL-1 there was complete inhibition of the SAA response. Removal of IL-1 at 24 h rapidly reduced the SAA secreted over the next 24 h. Addition of IL-1Ra to the cells at this time was as effective as removal of IL-1 at inhibiting the subsequent secretion of SAA. IL-1Ra was less effective at inhibition of IL-1-induced haptoglobin secretion. We would conclude that IL-1Ra may play an important role in the regulation of acute phase protein synthesis.
Murine T cell-stimulatory peptides from the 19-kDa antigen of Mycobacterium tuberculosis. Epitope-restricted homology with the 28-kDa protein of Mycobacterium leprae.
Authors: Harris D P , Vordermeier H M , Roman E , Lathigra R , Brett S J , Moreno C , Ivanyi J
Published: December 2022

Fifteen overlapping synthetic peptides, spanning the entire amino acid sequence of the Mycobacterium tuberculosis 19-kDa protein, were used to identify epitopes recognized by murine T cells. Five of the 15 peptides tested were able to elicit in vitro lymph node T cell proliferative responses in C57BL/10 mice primed by footpad inoculation with homologous peptide. Analysis in congenic strains of mice revealed H-2 restriction in the response to four peptides. However, one peptide, 19.7 (residues 61 to 80), induced T cell responses in all four haplotypes tested. This peptide was also unique in being able to stimulate lymph node cells from C57BL/10 mice immunized with recombinant 19-kDa protein, killed M. tuberculosis, or live bacillus Calmette Guerin infection. T cell lines specific for peptide 19.7 were of the CD4 phenotype. Significantly, sequence analysis revealed that residues 61 to 80 of the 19-kDa protein exhibited considerable homology with a single 20-amino acid sequence (residues 120 to 140), but not with any other region of the 28-kDa protein expressed in Mycobacterium leprae. This finding is the first evidence of epitope-restricted homology between otherwise structurally unrelated microbial Ag.
Distinct patterns of expression of MHC class I and beta 2-microglobulin transcripts at early stages of mouse development.
Authors: Jaffe L , Robertson E J , Bikoff E K
Published: December 2022

The class I surface Ag recognized during tissue graft rejection are composed of a 40- to 45-kDa H chain and a 12-kDa L chain, beta 2-microglobulin (beta 2m). Regulation of MHC gene expression during early development is thought to play an essential role in maternal tolerance of the fetal allograft. Here we used in situ hybridization techniques to characterize the temporal and spatial pattern of expression of MHC class I mRNA in the developing mouse embryo. MHC class I transcripts were initially detected at day 9.5 postcoitus in the primary and secondary trophoblast giant cell populations. At this stage, none of the remaining fetal components of the developing placenta expresses MHC class I mRNA. By contrast, the outer zone of trophoblast giant cells is the only trophectoderm-derived tissue in the developing placenta that does not express detectable levels of beta 2m mRNA. These findings indicate that the onset of MHC class I and beta 2m gene expression in early postimplantation stage embryos is not coordinately regulated. In addition, we made use of pluripotent embryonic stem (ES) cells to study developmental regulation of MHC class I and beta 2m gene expression in vitro. ES cells can be induced to form endoderm-like cells by the technique of suspension culture. In contrast to retinoic acid-treated F9 cells, the endoderm derived from ES cells expressed high levels of beta 2m mRNA, but no detectable MHC class I transcripts. These findings are consistent with results of our in situ hybridization experiments showing high levels of expression of beta 2m mRNA, and extremely weak expression of MHC class I transcripts in derivatives of the visceral endoderm in vivo.
Repertoire diversity of antibody response to bacterial antigens in aged mice. II. Phosphorylcholine-antibody in young and aged mice differ in both VH/VL gene repertoire and in specificity.
Authors: Nicoletti C , Borghesi-Nicoletti C , Yang X H , Schulze D H , Cerny J
Published: December 2022

Aging of mice is accompanied by both quantitative and qualitative changes in antibody responses to phosphorylcholine (PC), an immunodominant epitope of Streptococcus pneumoniae R36a strain (Pn). In order to study these changes at the molecular level, we generated PC-specific hybridomas from young (3 to 4 mo) and aged (20 to 24 mo) mice of different strains after primary immunization with S. pneumoniae R36a strain. These mAb were tested for Ig VH and VL gene family utilization, idiotopic repertoire, and cross-reactivity with unrelated Ag. Hybridomas from young mice (BALB/c, C57BL/6, and D1.LP) uniformly expressed the VH-S107 and V kappa-22 genes as well as most idiotopes of the T15 family, which were identified with different anti-T15 mAb. In contrast, the PC-reactive mAb from aged mice were quite heterogeneous: only 2 out of 13 utilized VHS107, 1 of 13 used VH7183, and 3 of 13 used VHJ558 gene family. Moreover, none of these mAb used L chain encoded by V kappa 22(0/13), but surprisingly they frequently expressed some of the T15 idiotope. In addition, the PC-binding mAb from aged mice showed broad cross-reactivity with various mouse and foreign proteins, whereas the mAb from young mice did not. These results demonstrate the genetic shift in antibody response of aging mice to PC, which is accompanied by a change in the antibody specificity. Interestingly, the qualitative repertoire change appears to be unrelated to the magnitude of antibody response, for the aged BALB/c mice maintain a very high reactivity to PC.
The murine Slp gene. Additional evidence that sex-limited protein has no biologic function.
Authors: Ogata R T , Zepf N E
Published: December 2022

Sex-limited protein (Slp) is a mouse serum protein of unknown function that has approximately 95% amino acid sequence identity with murine complement component C4 but is inactive in the complement pathway. The gene for Slp lies in the S region of the murine H-2 complex adjacent to the gene Cyp21 that encodes the Cytochrome P-450 enzyme steroid 21-hydroxylase. We report the sequence of a 26,307 bp long segment of the mouse genome that includes both the Slp and Cyp21 genes. The sequence reported was assembled from the sequences of three overlapping lambda phage genomic clones from mouse strain B10.WR, which carries four tandem pairs of Slp and Cyp21 genes. We also report the sequence of a fourth lambda clone, 12,539 bp in length, carrying parts of a distinct pair of Slp and Cyp21 genes from B10.WR mice. The Slp gene at 14.3 kb in length is about 1 kb shorter than the C4 gene; this difference is due primarily to absences of a simple repetitive sequence and a middle repetitive MT element in the corresponding introns 14 and 15, respectively. The gene sequence reveals an intron/exon organization identical to that of the murine C4 gene, and also that the 9 nucleotide deletion in exon 18, which appears to be directly responsible for the absence of complement activity, is unrelated to differences in intron sequences. Detailed comparisons of C4 and Slp gene sequences indicate that nucleotide substitutions in the Slp gene are occurring at approximately the same rate in both exons and introns. This implies that the murine Slp gene resembles a pseudogene and supports previously reported evidence that the Slp protein has no biologic function.