bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactions between a lymphoma membrane-associated guanosine 5'-triphosphate-binding protein and the cytoskeleton during receptor patching and capping.
Authors: Bourguignon L Y , Walker G , Huang H S
Published: December 2022

In this study we have used several complementary techniques to isolate and characterize a lymphoma membrane-associated 41-kDa protein that shares a number of structural and functional similarities with the alpha i subunit of the guanosine 5'-triphosphate (GTP)-binding protein (e.g., Gi alpha-like protein). In addition, using permeabilized lymphoma cells, we have found that: 1) GTP or GTP-tau-S augments, and pertussis toxin inhibits, phospholipase C (PLC) activity and receptor capping; and 2) the addition of lymphoma 41-kDa Gi alpha-like protein stimulates PLC activity and receptor patching/capping, and reverses the inhibitory effect of pertussis toxin on both activity and receptor patching/capping. Additional cytochemical and biochemical data indicate that the lymphoma 41-kDa protein is closely associated with several cytoskeletal proteins (e.g., actin, myosin, and fodrin) all of which colocalize under receptor cap structures. Furthermore, both the 41-kDa-mediated phospholipase C activity and receptor patching/capping are inhibited by cytochalasin D (a microfilament disrupting drug) and W-7 drug (a calmodulin inhibitor). Together, these data provide strong evidence for a functional association between the lymphoma membrane cytoskeleton and the 41-kDa (Gi alpha-like) protein. Specifically, this association appears to be required for the activation of phospholipase C that results in inositol triphosphate production, subsequent internal Ca2+ release, and finally surface receptor patching and capping.
CD18-dependent and -independent mechanisms of neutrophil emigration in the pulmonary and systemic microcirculation of rabbits.
Authors: Doerschuk C M , Winn R K , Coxson H O , Harlan J M
Published: December 2022

Neutrophil (PMN) migration in the systemic and pulmonary circulation of rabbits was compared by using different inflammatory stimuli to determine the role of the leukocyte adhesion complex, CD11/CD18, in each of these vascular beds. The adhesion complex was blocked by administering the anti-CD18 mAb 60.3. The data show that mAb 60.3 blocks PMN emigration into inflammatory foci in the abdominal wall produced by implanting sponges containing either hydrochloric acid, Streptococcus pneumoniae, Escherichia coli endotoxin, or PMA. mAb 60.3 also inhibited PMN emigration in response to peritoneal instillation of S. pneumoniae. The effect of mAb 60.3 on PMN emigration in the lungs varied depending upon the stimulus. PMN failed to migrate into the PMA-induced pneumonia; however, mAb 60.3 pretreatment only partially inhibited endotoxin-induced pneumonia and did not inhibit S. pneumoniae or hydrochloric acid-induced pneumonias. PMN lavaged from the alveolar spaces in the Streptococcal pneumonia had similar quantities of mAb 60.3 bound to their surfaces as the circulating PMN. We conclude that the CD11/CD18 complex mediates PMN adherence in the systemic circulation. However, PMN adherence in the pulmonary circulation may occur by either CD18-dependent or -independent mechanisms that are specific to the inciting stimulus.
CD18-deficient cells respond to lipopolysaccharide in vitro.
Authors: Wright S D , Detmers P A , Aida Y , Adamowski R , Anderson D C , Chad Z , Kabbash L G , Pabst M J
Published: December 2022

The CD11/CD18 complex of leukocyte adhesion molecules has been shown to bind LPS on the surface of gram negative bacteria and LPS-coated erythrocytes (J. Exp. Med. 164:1876, 1986). LPS elicits several responses in leukocytes including secretion of TNF-alpha and IL-1 beta, and priming for enhanced release of oxygen radicals such as superoxide anion. To determine if expression of CD18 molecules is necessary for these effects of LPS, we have examined the responses of leukocytes from CD18-deficient patients. Three of the patients in this study are characterized for the first time here, and three were described elsewhere. Monocytes and macrophages from CD18-deficient patients synthesized normal amounts of IL-1 beta and TNF-alpha in response to LPS. Further, PMN and monocytes from CD18-deficient patients showed normal priming for enhanced release of superoxide anion in response to LPS. Although a small contribution of CD18 molecules to some responses cannot be ruled out by our data, we may conclude that CD18 molecules are not essential for cellular responses to LPS.
Characterization of the receptor for tumor necrosis factor (TNF) and lymphotoxin (LT) on human T lymphocytes. TNF and LT differ in their receptor binding properties and the induction of MHC class I proteins on a human CD4+ T cell hybridoma.
Authors: Andrews J S , Berger A E , Ware C F
Published: December 2022

TNF-alpha and lymphotoxin (LT or TNF-beta) are structurally related cytokines that share several proinflammatory and immunomodulatory activities. The shared biologic activities of TNF and LT have been attributed to their binding to a common cell surface receptor(s). We observed that rTNF enhanced the expression of MHC class I proteins on the human T cell hybridoma, II-23.D7, however LT was largely unable to regulate MHC expression. To determine the molecular basis of this disparity between LT and TNF the receptor binding characteristics of rTNF and rLT were investigated by direct and competitive radioligand assays on the II-23.D7 T hybridoma, and for comparison, anti-CD3 activated human T lymphocytes. Specific 125I-rTNF binding to the II-23.D7 line revealed a single class of sites with a Kd = 175 pM and 3000 sites/cell; anti-CD3 activated T cells exhibited specific TNF binding with similar properties. The relationship of receptor occupancy to the induction of MHC class I Ag yielded a hyperbolic curve indicating a complex relationship between rTNF binding and biologic response. LT appeared to function like a partial agonist in that rLT was 10- to 20-fold less effective than rTNF in competitively inhibiting 125I-rTNF binding on the II-23.D7 line. Scatchard type analysis revealed a single class of low affinity binding sites for 125I-rLT. No differences in the competitive binding activity of rTNF and rLT were observed on the anti-CD3-activated T cells. Receptors for rTNF and rLT were immunoprecipitated from the II-23.D7 and activated T cells with anticytokine antibodies after cross-linking of radioiodinated rTNF or rLT to intact cells by using chemical cross-linking reagents. Analysis of the cross-linked adducts by SDS-PAGE and autoradiography indicated a major adduct of 92 kDa for rTNF and 104 kDa for rLT. Enzymatic digestion with neuraminidase or V8 protease revealed a unique structure to these adducts consistent with the cross-linking of a single chain of cytokine to a cell surface glycoprotein. rTNF inhibited the formation of the 104-kDa adduct formed with 125I-rLT on the II-23.D7 line, indicating these two cytokines bind to the same receptor of approximately 80 kDa. These results suggest that the disparate activities of LT and TNF to induce MHC class I proteins on the II-23.D7 cells are, in part, associated with a modified state of a common receptor.
Constitutive production of human interleukin for DA cells/leukemia inhibitory factor by human tumor cell lines derived from various tissues.
Authors: Gascan H , Anegón I , Praloran V , Naulet J , Godard A , Soulillou J P , Jacques Y
Published: December 2022

The cytokine HILDA (human IL for DA cells)/LIF (leukemia inhibitory factor), previously reported to trigger the proliferation of the DA1.a cell line was purified to homogeneity from the culture supernatant of the 5637 bladder carcinoma cell line by using a four step procedure, including cationic exchange chromatography at pH 6, Con A affinity chromatography, reverse phase HPLC, and gel filtration HPLC. The purified radiolabeled protein was analyzed in SDS-PAGE and a single band with a molecular mass of 43 kDa was revealed. The iodinated material was then tested in a radioreceptor assay using the M1 cell line as a second target cell line. The binding of the natural 125I hormone was abrogated in a dose dependent and specific fashion by either natural and rHILDA. The limit of signal detection of displacement for the radiolabeled ligand by unlabeled sample was found to be 0.05 ng/ml. The proliferative DA1.a assay and the M1 radioreceptor assay were used to analyze the HILDA content of 17 human tumor cell line culture supernatants, and 12 among them were spontaneously secreting a detectable level of LIF. The secreted amount of HILDA was largely enhanced by treating the cell line with 30 to 80 nM PMA; in these conditions a 10 to 20x increase in the detected amount was observed. The specificity of the detected signal was reinforced by analyzing the mRNA expression of HILDA in the tumor cell lines. The secreting cell lines were found to express various forms of LIF transcripts with a major specie at 3.8 kb.
Mac-1 (CD11b/CD18) mediates adherence-dependent hydrogen peroxide production by human and canine neutrophils.
Authors: Shappell S B , Toman C , Anderson D C , Taylor A A , Entman M L , Smith C W
Published: December 2022

Human neutrophils exposed to protein-coated polystyrene or cultured endothelial monolayers produce large quantities of H2O2 in response to soluble stimuli that elicit little or no secretion of reactive oxygen species from cells in suspension. To characterize the mechanisms involved in this adherence-dependent respiratory burst, we have investigated the possible role of one integrin known to participate in the adhesion of neutrophils to endothelial cells, CD11b/CD18 (Mac-1). H2O2 production was examined with chemotactic factor-stimulated human and canine neutrophils exposed to protein-coated surfaces and cultured human and canine endothelial cells. The two protein-coated surfaces used were type I collagen-coated glass or plastic, a surface to which neither human nor canine neutrophils adhered, and keyhole limpet hemocyanin (KLH)-coated glass or plastic, a surface to which human and canine neutrophils adhered only after chemotactic stimulation. FMLP-stimulated human neutrophils and platelet activating factor-stimulated canine neutrophils failed to produce detectable H2O2 when in contact with type I collagen, but secreted large amounts of H2O2 when adherent to KLH or endothelial cell monolayers. FMLP-stimulated neutrophils from patients with CD18-deficiency failed to adhere to any of these surfaces and failed to produce H2O2 under these conditions. mAb reactive with CD18 and CD11b were equally effective in markedly inhibiting the adhesion of normal human neutrophils to these surfaces and markedly inhibited the production of H2O2. A mAb reactive with CD18 blocked adhesion of stimulated canine neutrophils, and mAb directed against both CD18 and CD11b blocked H2O2 production by canine neutrophils on KLH and endothelium. A nonbinding mAb and a mAb reactive with CD11a did not inhibit H2O2 production of human cells on KLH or endothelial monolayers, and nonbinding and binding control mAb did not inhibit H2O2 production by canine neutrophils. These results indicate that Mac-1 (CD11b/CD18) can mediate adhesion-dependent H2O2 production by human and canine neutrophils exposed to chemotactic factors.
Requirement of lipophosphoglycan for intracellular survival of Leishmania donovani within human monocytes.
Authors: McNeely T B , Turco S J
Published: December 2022

The function of the lipophosphoglycan of Leishmania donovani parasites was investigated in human peripheral monocytes. In contrast to wild-type L. donovani which grow in monocytes, incubation of monocytes with two variant lines of L. donovani, defective in lipophosphoglycan expression, resulted in the entry of the variant cells into the monocytes and their subsequent destruction. Passive transfer of lipophosphoglycan to the variant cells led to prolonged survival in monocytes. These results indicate that lipophosphoglycan is required by the parasite for intracellular survival. To investigate one possible protective role of the glycoconjugate, preincubation of monocytes with a suspension of lipophosphoglycan and subsequent treatment of the cells with PMA or opsonized zymosan resulted in an attenuation of the oxidative burst; the attenuation effect was concentration dependent on the glycoconjugate and independent of preincubation time. Moreover, hydrophobic beads, coated with lipophosphoglycan, were phagocytized by monocytes and found to inhibit oxygen consumption in monocytes activated with PMA. These results suggest a possible relationship between the absence of lipophosphoglycan in the variant parasites and their inability to survive within monocytes. Although the precise molecular basis remains to be elucidated, the ability of lipophosphoglycan to impair the microbial oxidative response may be a contributing factor in its requirement for intracellular survival.
Host-specific evasion of the alternative complement pathway by schistosomes correlates with the presence of a phospholipase C-sensitive surface molecule resembling human decay accelerating factor.
Authors: Pearce E J , Hall B F , Sher A
Published: December 2022

Schistosoma mansoni parasites recovered from the blood stream were found to be nonactivators of the alternative complement pathway (ACP) when exposed to sera of homologous but not heterologous host species. Schistosomes could be converted into activators of the homologous ACP by treatment with phospholipase C. Antibodies to either human or guinea pig decay accelerating factor (DAF), a 70-kDa glycosylphosphatidylinositol anchored membrane glycoprotein which controls ACP activation on the mammalian cell plasma membrane, bound to the surface of immature schistosomes and immunoprecipitated a molecule of similar molecular mass from detergent extracts of surface iodinated parasites. Phospholipase C treatment drastically reduced the reactivity of the worms with the anti-DAF antibodies. These data suggest that schistosomes evade the ACP by inserting functional host DAF into their surfaces, possibly through adsorption of the molecule's lipophilic diacyglycerol membrane anchor moiety into the outer lipid bilayer of the parasite.
Mouse V lambda x gene sequence generates no junctional diversity and is conserved in mammalian species.
Authors: Sanchez P , Marche P N , Rueff-Juy D , Cazenave P A
Published: December 2022

The lambda x, a new mouse Ig lambda L chain, is produced by rearrangement of the V lambda x, J lambda 2, and C lambda 2 gene segments. The V lambda x amino acid sequence is as divergent to other V lambda as to Vk gene sequences. Additionally, its third hypervariable region (CDR3) is four amino acids longer than those of all other variable gene segments of murine L chain. We have cloned and sequenced the germ-line V lambda x gene and found that the unexpected CDR3 length is encoded by the V lambda x gene. Junctional diversity is prevented by a TAA termination codon localized at the V lambda x 3' extremity. Moreover, we show a striking conservation of the V lambda x sequence in various mammalian species. Portions of the V lambda x sequence display more than 70% of nucleotide sequence identity with rabbit and human variable regions. These results suggest that V lambda x predated the divergence of mammalian species.
Differential regulation of Ca2+ mobilization in human thymocytes by coaggregation of surface molecules.
Authors: Deusch K , Daley J F , Levine H , Languet A J , Anderson P , Schlossman S F , Blue M L
Published: December 2022

Variations in intracellular Ca2+ levels in developing thymocytes are likely to play a major role in both the activation-associated differentiation of thymocytes and in the selection or clonal deletion of cells. Here we examine the role of CD4, CD8, CD2, and CD45 in the regulation of intracellular Ca2+ levels in mature and immature thymocytes. Mature and immature thymocytes, distinguished on the basis of their CD5 expression, were analyzed simultaneously for their ability to mobilize Ca2+ after coaggregation of their CD3/TCR with other thymic surface Ag. Flow cytometric analysis by using Indo-1 showed that coaggregation of CD4, CD8, and CD2 with CD3/TCR clearly enhances a minimal signal delivered via CD3/TCR on immature thymocytes. Coaggregation with class I MHC had no discernible effect. The responsiveness of immature thymocytes correlated strictly with CD3 surface expression, such that loss of responsiveness occurred with reduced CD3 cell-surface density. However, even thymocytes with very low CD3 expression were able to respond to triggering via CD3 under optimal conditions, indicating that the CD3 signal-transducing mechanism is functional on early thymic cells. Intracellular increases in Ca2+ concentrations induced via CD3, could effectively be inhibited by cross-linking of CD45 and CD3 on immature thymocytes. Although triggering via CD2 alone induced a strong Ca2+ flux, prolonged incubation with activating anti-CD2 antibodies made thymocytes refractory to subsequent triggering. Refractoriness was associated with partial loss of surface CD3 and CD3 zeta. Our results indicate that thymic surface Ag are differentially involved in the regulation of intracellular Ca2+ levels in immature as well as mature thymocytes.
Metabolic activity is necessary for activation of T suppressor cells by B cells.
Authors: Elkins K L , Stashak P W , Baker P J
Published: December 2022

Ag-primed B cells must express cell-surface IgM, but not IgD or Ia Ag, and must remain metabolically active, in order to activate suppressor T cells (Ts) specific for type III pneumococcal polysaccharide. Ag-primed B cells that were gamma-irradiated with 1000r, or less, retained the ability to activate Ts; however, Ag-primed B cells exposed to UV light were not able to do so. gamma-Irradiated and UV-treated Ag-primed B cells both expressed comparable levels of cell-surface IgM, and both localized to the spleen after in vivo transfer; neither could proliferate in vitro in response to mitogens. By contrast, gamma-irradiated primed B cells were still able to synthesize proteins, whereas UV-treated primed B cells could not. These findings suggest that in order for Ag-primed B cells to activate Ts, they must a) express cell-associated IgM (sIgM) antibody bearing the idiotypic determinants of antibody specific for type III pneumococcal polysaccharide, and b) be able to synthesize protein for either the continued expression of sIgM after cell transfer, or for the elaboration of another protein molecule that is also required for the activation of Ts; this molecule does not appear to be Ia Ag.
Characterization of the neutral aminopeptidase activity associated to the mouse thymocyte-activating molecule.
Authors: Gorvel J P , Vivier I , Naquet P , Brekelmans P , Rigal A , Pierres M
Published: December 2022

We previously characterized a dimeric, Mr = 115,000, developmentally regulated mouse T cell-activating molecule (THAM). We show in this report that the THAM-specific mAb H194-112 exhibits strong reactivity with several nonlymphoid tissues including polarized enterocytes from small intestine, kidney cortical tubuli, and liver bile ductuli, as well as kidney glomeruli and lung alveoloar pneumocytes. Both the tissue distribution and the structural features of THAM made it likely that this molecule belongs to the ectoenzyme family. This was confirmed by the following experimental evidence: 1) the H194-112+ molecules from enterocyte brush borders (BB) or M14.T thymoma cells were recognized by several antisera specific for intestinal aminopeptidase N (AP-N); 2) mAb H194-112 was found to immunodeplete the AP-N activity from both thymic or enterocyte BB detergent extracts; 3) the hydrophilic, Mr = 115,000 form obtained by papain treatment of thymoma or enterocyte BB could be immunopurified on H194-112 column and exhibited, after hypotonic elution, strong enzymatic activity on the AP-N substrates (i.e., leucyl or alanyl beta-derivatives); 4) mAb H194-112 was found to inhibit the AP-N activity when assayed on alanyl but not leucyl beta-naphthylamide substrate; and 5) preincubation of AP-N with mAb H194-112 prevented the inhibiting effects of bestatin and D,L-methionyl hydroxamate on AP-N activity. These data add a new member to the list of functional ectoenzymatic markers of lymphoid cells (i.e., CD10, CD13, CD26, CD55, and CD73). In view of the known immunomodulating properties of bestatin, one may speculate that the T cell-activating effects of mAb H194-112 is related to an impairment of a surface enzymatic function regulating lymphoid cell activation.
Increased lymphocyte adherence to human arterial endothelial cell monolayers in the context of allorecognition.
Authors: Colson Y L , Markus B H , Zeevi A , Duquesnoy R J
Published: December 2022

The interactions of alloreactive T lymphocytes with the vascular endothelium were studied in an in vitro model of lymphocyte adherence to cultured human arterial endothelial cell (HAEC) monolayers. Donor-primed lymphocytes (DPL) were shown to have significantly greater adherence to donor HAEC than were third-party primed lymphocytes. Limiting dilution analysis of adherent DPL showed an enrichment of donor-reactive lymphocytes compared with nonadherent DPL. This study examines the allospecific nature of this increased lymphocyte adherence. HAEC constitutively express class I HLA Ag and can be induced by IFN-gamma to express class II Ag. DPL adherence to class I+ HAEC was inhibited only in the presence of mAb directed against class I Ag. DPL adherence to class I+ and class II+ HAEC was inhibited in the presence of mAb directed against class I and class II Ag. Class I- and class II-specific adherence was also shown to involve CD8 and CD4 molecules, respectively, whereas lymphocyte function-associated Ag do not appear to play a major role in long term alloreactive lymphocyte adherence to HAEC. These findings suggest that alloreactive lymphocyte adherence to HAEC is mediated by two mechanisms. One is based on allorecognition, primarily of HLA Ag, and the other is related to presumably non-Ag-specific interactions between activated lymphocytes and the vascular endothelium. The studies presented provide evidence to suggest that HLA-specific lymphocyte adherence to endothelium may significantly contribute to the development of alloreactive lymphocyte infiltrates within the allograft.
Aqueous humor contains transforming growth factor-beta and a small (less than 3500 daltons) inhibitor of thymocyte proliferation.
Authors: Granstein R D , Staszewski R , Knisely T L , Zeira E , Nazareno R , Latina M , Albert D M
Published: December 2022

The anterior chamber of the eye is an immunologically privileged site in which allografts survive longer than at other body sites. In this regard, it is relevant that aqueous humor (AH) inhibits lymphocyte proliferation. In order to analyze AH for specific substances that inhibit thymocyte proliferation, samples of human AH, murine AH, and rhesus monkey AH were added to cultures of thymocytes stimulated by IL-1 or IL-2 in the presence of PHA. All samples of AH tested had potent inhibitory activity on thymocyte proliferation in this system. Inhibitory activity was lost by heating AH to 80 degrees C for 1 h. Dialysis of murine AH indicated that species smaller than 3500 Da were capable of mediating this activity; we have termed the factor(s) responsible for this "small inhibitory factor(s) of AH." Retentate, containing species larger than 3500 Da, retained inhibitory activity, but less than nondialyzed AH. Assays for PGE2 demonstrated that murine and human AH contained small quantities of PGE2. These quantities were insufficient to inhibit thymocyte proliferation in our assay system. Furthermore, AH from mice treatend with indomethacin had full inhibitory activity. Assays for transforming growth factor beta (TGF-beta) after acid activation demonstrated significant quantities of latent TGF-beta within human and murine AH which could be largely neutralized by antisera to TGF-beta. Active TGF-beta "activity" was also present without acid activation in samples of AH at a level approximately 20% that of latent TGF-beta. However, most of this "activity" could not be neutralized by antisera to TGF-beta. AH contains factors capable of limiting thymocyte proliferation.
Partial characterization of a C5a-inhibitor in peritoneal fluid.
Authors: Ayesh S K , Ferne M , Flechner I , Babior B M , Matzner Y
Published: December 2022

We have recently described a 40-kDa protein in peritoneal fluid that neutralized the chemotactic activity of the C fraction C5a. It was deficient in peritoneal fluids of patients suffering from familial Mediterranean fever. Further characterization of the inhibitor with the use of 125I-rC5a binding to dibutyryl cAMP-induced U937 cells revealed dependence on the peritoneal fluid concentration, on the time of incubation and on temperature and pH. Fractionation of 125I-C5a on Sephadex G-50 column, before and after incubation with peritoneal fluid, revealed similar fractionation patterns despite loss of biologic activity of the treated C5a (but not its binding to polyclonal anti-C5a antibody). Analysis of rC5a by SDS-PAGE before and after treatment with partially purified C5a inhibitor, revealed slight modification of the inhibitor-treated C5a. Using various protease inhibitors (i.e., PMSF) suggested that the C5a inhibitor is a serine protease. It neutralized C5a by means of limited proteolysis which did not change C5a immunologic properties and changed only slightly its m.w. but abolished its receptor binding and chemotactic functions. It is suggested that the C5a inhibitor plays a role in the regulation of inflammation in serosal tissues and that its deficiency in familial Mediterranean fever may explain the attacks of sterile inflammation characteristic of this disease.
IFN-gamma production in tissues of mice during acute infection with lymphocytic choriomeningitis virus.
Authors: Gessner A , Drjupin R , Löhler J , Lother H , Lehmann-Grube F
Published: December 2022

As shown by a single-cell solid-phase ELISA, splenocytes as well as liver lymphoid cells from unmanipulated specific-pathogen free mice synthesized and released IFN-gamma. Synthesis of this lymphokine could not be demonstrated either on the transcriptional level by Northern blotting or immunocytochemically. Thus, IFN-gamma is constitutively produced in mice, although in low quantities. During acute infection with lymphocytic choriomeningitis virus, IFN-gamma mRNA became detectable in spleen and brain but not in the liver. In spleens and livers of these mice, the numbers of cells synthesizing the lymphokine were increased and many were seen in foot tissue undergoing a delayed-type hypersensitivity reaction after intraplantar inoculation of the virus. In contrast, few IFN-gamma-producing cells were found in the inflammatory infiltrates of leptomeninges and choroid plexus after intracerebral infection.
Induction of virus-neutralizing antibodies by bacteria expressing the C3 poliovirus epitope in the periplasm. The route of immunization influences the isotypic distribution and the biologic activity of the antipoliovirus antibodies.
Authors: LeClerc C , Martineau P , Van der Werf S , Deriaud E , Duplay P , Hofnung M
Published: December 2022

Two viral epitopes (C3 neutralization epitope from poliovirus type 1 and the 132-145 peptide from the PreS2 region from hepatitis B virus) have been expressed in the Escherichia coli periplasm as protein fusion with the maltose binding protein (MalE protein). Immunization of mice with live bacteria expressing the foreign viral epitopes in their periplasm elicited high antibody titers against the viral peptide as well as against the corresponding virus. This demonstrates for the first time in the case of defined epitopes that, when live bacteria are used as immunogens, presentation at the cell surface is not a prerequisite to obtain an antibody response. On the other hand, the induction of antiviral antibody responses by these recombinant bacteria depended dramatically on the route of immunization: a response was induced by live bacteria through the i.v. route but not through the s.c. route. However, when bacteria were heat killed or when the MalE hybrid protein was released under a soluble form from the cell, a response was induced even upon s.c. immunization. From these results, we suggest that in order to induce high levels of antibodies by the s.c. route, a major parameter for bacterial Ag would be their capacity to be released into a soluble form before the interaction of the bacteria with the APC. This would permit the presentation by B cells rather than by phagocytic cells. Finally, we demonstrate that the route of immunization influences the isotypic distribution and the neutralizing activity of the antipoliovirus antibodies. Such results may have major implications for the development of bacterial vaccines based on fusion proteins.
Human IgG Fc receptor II mediates antibody-dependent enhancement of dengue virus infection.
Authors: Littaua R , Kurane I , Ennis F A
Published: December 2022

It is known that anti-dengue virus antibodies at subneutralizing concentrations augment dengue virus infection of IgG FcR (Fc gamma R)-positive cells, and this phenomenon is called antibody-dependent enhancement. This is caused by the uptake of dengue virus-antibody complexes by Fc gamma R. We previously reported that Fc gamma RI can mediate antibody-dependent enhancement. In this study we use an erythroleukemia cell line, K562, which has Fc gamma RII, but does not have Fc gamma RI or Fc gamma RIII, to determine if Fc gamma RII can mediate infection by dengue virus-antibody complexes. Polyclonal mouse anti-dengue virus antibody significantly augments dengue virus infection of K562 cells, whereas normal mouse serum does not. A mAb IV.3, which is specific for Fc gamma RII and is known to inhibit the binding of Ag-antibody complex to Fc gamma RII, inhibits dengue antibody-mediated augmentation of dengue virus infection. It has been reported that Fc gamma RII binds to mouse IgG1, but not to mouse IgG2a. A mouse IgG1 anti-dengue virus mAb (3H5) augments dengue virus infection of K562 cells, but a mouse IgG2a anti-dengue virus mAb (4G2) does not. 4G2 augments dengue virus infection of a human monocytic cell line, U937, which has Fc gamma RI. Based on these results we conclude that Fc gamma RII mediate antibody-dependent enhancement of dengue virus infection in addition to Fc gamma RI.
IL-1 and tumor necrosis factor synergistically stimulate fibroblast IL-6 production and stabilize IL-6 messenger RNA.
Authors: Elias J A , Lentz V
Published: December 2022

We characterized the effects of rIL-1 and rTNF on human lung fibroblast IL-6 production. rIL-1 was a potent stimulator, rTNF only marginally stimulated, and rIL-1 and rTNF in combination synergistically stimulated IL-6 protein production. These changes were associated with proportionate alterations in IL-6 mRNA accumulation. Nuclear run-on analysis demonstrated that the effects of rIL-1 and rTNF individually were associated with increased IL-6 gene transcription. In contrast, alterations in gene transcription could not fully explain the synergistic effects of rIL-1 and rTNF in combination. However, IL-6 mRNA was significantly more stable in cells stimulated with rIL-1 plus rTNF than in cells stimulated with rIL-1 alone. Thus the synergistic effects of rIL-1 and rTNF in combination were mediated, at least partially, by an alteration in the stability of IL-6 mRNA. Alterations in mRNA stability may be an important mechanism of cytokine-cytokine synergy.
IL-3 and granulocyte-macrophage colony-stimulating factor stimulate two distinct phases of adhesion in human monocytes.
Authors: Elliott M J , Vadas M A , Cleland L G , Gamble J R , Lopez A F
Published: December 2022

IL-3 and granulocyte-macrophage CSF are hemopoietic growth factors involved in monocytopoiesis and functional stimulation of circulating blood monocytes. We demonstrate that both cytokines enhance the adhesion of purified human monocytes to cultured human umbilical vein endothelial cells and to plastic surfaces. The stimulation seen was biphasic: an early phase detectable by 10 min, and a late phase seen after 9 h of in vitro culture. IL-3- and granulocyte-macrophage-CSF-stimulated adhesion was seen at concentrations as low as 6 pM, with maximal monocyte adhesion of up to 60% seen at concentrations of 60 pM and above. Both phases of stimulated adhesion were partially inhibited by a monoclonal antibody to CD18, the common beta-chain of the leukocyte functional Ag family of adhesion molecules, but not by an antibody to CD11b, the alpha-chain of MAC-1. However, a difference in the mechanism by which the early and late phases of stimulated adhesion arise could be shown by the use of cycloheximide as an inhibitor of protein synthesis. Although the late phase was totally dependent on de novo protein synthesis, early phase adhesion was not inhibited by cycloheximide, suggesting receptor redistribution or conformational change as the mechanism mediating enhanced adhesion at this time. These findings may be relevant to the pathogenesis of inflammatory disease and may have implications for the clinical use of these cytokines.
Alveolar and peritoneal macrophages bear three distinct classes of Fc receptors for IgG.
Authors: Anderson C L , Looney R J , Culp D J , Ryan D H , Fleit H B , Utell M J , Frampton M W , Manganiello P D , Guyre P M
Published: December 2022

The FcR for IgG on the plasma membrane of cells of the mononuclear phagocyte system mediate a number of different biologic responses such as phagocytosis, pinocytosis, superoxide generation, and antibody-dependent cytotoxicity. In the interest of understanding the pathophysiology of these processes we have begun to characterize the FcR for IgG on two readily available sources of macrophages--the lung and the peritoneum--using antireceptor mAb. We find that all three of the distinct classes of FcR for IgG which have been described in man are present on both pulmonary and peritoneal macrophages. Most monocytes, we suggest, bear low numbers of Fc gamma RIII whereas a small subpopulation of monocytes expresses substantial numbers of Fc gamma RIII. Furthermore, we find that two different forms of Fc gamma RIII differ in their capacity to bind anti-Fc gamma RIII mab 3G8 in the presence of human IgG. Human IgG does not block the binding of mAb 3G8 to neutrophils, but it does block 3G8 binding to macrophages and large granular lymphocytes; this finding correlates with the expression of the two Fc gamma RIII genes, I and II, in man. Studies aimed at illuminating the molecular mechanisms of Fc gamma R-mediated processes in macrophages will require consideration of the receptors of all three classes.
Activated human monocytes oxidize low-density lipoprotein by a lipoxygenase-dependent pathway.
Authors: McNally A K , Chisolm G M , Morel D W , Cathcart M K
Published: December 2022

Monocyte-mediated oxidation of low-density lipoprotein (LDL) converts the lipoprotein to a potent cytotoxin. The oxidation process requires monocyte activation and requires superoxide anion since it can be blocked by superoxide dismutase. In this study, the requirement for lipoxygenase activity is shown, in that 1) inhibitors of lipoxygenase prevent the alteration of LDL, 2) copper (II) (3,5-diisopropylsalicylic acid), an agent shown to enhance lipoxygenase activity in a cell-free system, similarly enhances monocyte-mediated LDL alteration, and 3) the (3,5-diisopropylsalicylic acid)-enhanced monocyte-mediated modification of LDL can be completely blocked by inhibitors of lipoxygenase or by superoxide dismutase. These data suggest an integral role for monocyte lipoxygenase in the generation by activated monocytes of the extracellular superoxide anion that participates in the oxidation of LDL and the conversion of LDL to a cytotoxin. Monocyte-modified LDL may be a mediator in tissue damage that accompanies atherosclerosis or occurs at sites of inflammation.
Rabbit Ig kappa 1b6 gene structure.
Authors: Dreher K L , Asundi V , Wolf B , Bruggeman L
Published: December 2022

Previous studies employing Southern blot analyses have detected multiple kappa-homologous sequences within EcoRI-digested DNA isolated from kappa 1b6 homozygous rabbits and kappa 1b6 L chain secreting RMH H158 cell line. These results are very unexpected because the published partial protein sequence for the kappa 1b6 C region is incompatible with an EcoRI restriction endonuclease recognition sequence at the nucleotide level for this allotype. To determine their identity, the kappa-homologous sequences were isolated from DNA extracted from a kappa 1b6 L chain secreting RMH H158 cell line by molecular cloning. Structural analyses demonstrated these sequences to contain genetic information encoding the majority of the kappa 1b6 L chain gene locus. The protein sequence deduced from the kappa 1b6 C region gene was shown to differ from the published partial kappa 1b6 C region protein sequence at five amino acid positions. One of these differences results in a glycine to serine interchange that introduces an EcoRI restriction endonuclease recognition site within the kappa 1b6 C region gene. Subsequent genomic Southern blot analyses confirmed this structural assignment. Based on these data, the EcoRI-sensitive kappa-homologous fragments present within the genomes of the RMH H158 cell line and kappa 1b6 homozygous rabbits represent the nominal kappa 1 gene and not an alternative kappa isotype or kappa pseudogene. Rabbit Ig kappa 1 allelic nucleotide sequence homology comparisons have shown the isolated kappa 1b6 J-C gene locus to display common structural features previously identified in other kappa 1 alleles.
The genomic organization of the CD28 gene. Implications for the regulation of CD28 mRNA expression and heterogeneity.
Authors: Lee K P , Taylor C , Petryniak B , Turka L A , June C H , Thompson C B
Published: December 2022

CD28 is a 90-kDa homodimeric glycoprotein present on the surface of a large subset of T cells that appears to play an important role in the modulation of T cell activation. Although a number of physiologic effects associated with CD28 stimulation have been defined, relatively less is known about the structure and expression of the CD28 gene itself. We now show that CD28 is expressed in both Th cells and plasma cells as a series of four distinct CD28 mRNA species: 1.3-, 1.5-, 3.5-, and 3.7-kb transcripts. The steady state expression of all four transcripts in CD28+ T cells was stimulated by PMA, suggesting that they might share a common phorbol-sensitive promoter. Consistent with this hypothesis, CD28 was found to be encoded by a single copy gene organized into four exons, each exon defining a functional domain of the predicted protein. All CD28 transcripts appear to initiate within a 61-bp palindrome. Generation of the four CD28 mRNA species from the CD28 gene involves two distinct posttranscriptional events. The longer pair of transcripts (3.5/3.7 kb) is generated by the use of an alternate nonconsensus polyadenylation signal. This results in the addition of 2167 bp beyond the first polyadenylation site utilized by the shorter (1.3/1.5 kb) pair of transcripts. The size difference between the 3.7- and 3.5-kb messages and between the 1.5- and 1.3-kb messages is generated by an internal splicing event that deletes 252 bp within exon 2, which encodes the extracellular domain. This deletion would result in the loss of 84 amino acids, including 4 of 5 extracellular cysteine residues. Although this deletion would result in significant disruption of CD28 secondary structure, it would not be expected to interfere with the ability of the resultant protein to be expressed on the cell surface. These findings suggest that variant isotypes of CD28 may be expressed on the cell surface with potentially different physiologic roles.
Human T cell receptor-gamma delta-expressing T-cell lines recognize MHC-controlled elements on autologous EBV-LCL that are not HLA-A, -B, -C, -DR, -DQ, or -DP.
Authors: Lam V , DeMars R , Chen B P , Hank J A , Kovats S , Fisch P , Sondel P M
Published: December 2022

HLA-loss variants of an EBV-transformed B lymphoblastoid cell line (EBV-LCL) 721 were used to investigate whether human MHC molecules other than known class I or class II were involved in autologous T cell responses. Bulk lymphocyte cultures of purified T cells primed to an autologous variant EBV-LCL that fails to express HLA-class II and has reduced cell surface HLA-class I expression, and oligoclonal TCR-gamma delta-bearing lines derived from them, could lyse both this EBV-LCL and an independently derived, class II expressing autologous variant EBV-LCL that bears no HLA-A, -B, or -C, suggesting the presence of additional HLA-like restriction elements. Cold target inhibition of cytolysis mediated by these lines indicated that a shared or cross-reactive MHC controlled restriction element other than the known MHC determinants was retained by the EBV-LCL variants. Single-cell derived clones from these T cell lines which expressed only the TCR-gamma delta showed this same target cell specificity pattern, proving recognition of MHC-controlled determinants by autologous gamma delta T cells. Anti-gamma delta antibody could inhibit cytolysis by the gamma delta-expressing lines, suggesting that the TCR-gamma delta was involved in recognition of the EBV-LCL targets. Flow cytometric analysis with separate HLA-reactive antibodies indicated that the restriction element for these cytolytic responses is a molecule serologically cross-reactive with HLA-B and -C Ag, yet is a determinant that cannot be HLA-A, -B, -C, -DR, -DQ or -DP.
Enhanced production of IL-6 in tumor-bearing mice and determination of cells responsible for its augmented production.
Authors: Utsumi K , Takai Y , Tada T , Ohzeki S , Fujiwara H , Hamaoka T
Published: December 2022

IL-6 is a cytokine secreted in normal individuals by monocytes, fibroblasts, and endothelial cells. We have found increased levels of IL-6 in the sera from MH134 hepatoma- and CSA1M fibrosarcoma-bearing mice. Concerning the capacity of these tumor cells themselves to produce IL-6 in vitro, they exhibited the distinct contrast, i.e., the MH134 tumor cells produced high levels of IL-6 whereas the CSA1M generated a marginal level of IL-6. It was, however, demonstrated that appreciably enhanced IL-6 production was observed in spleen cell culture supernatants from both types of tumor-bearing mice when compared to those obtained from normal mice. More importantly, in contrast to the production of IL-6 by non-T cell compartment of normal spleen cells, enhanced IL-6 production of spleen cells from tumor-bearing mice was ascribed to T cell compartment. Analysis of T cell phenotype has revealed that enhanced IL-6 production was mediated predominantly by Lyt-2+ but not by L3T4+ T cell subset. Thus, these results indicate that increased circulating IL-6 is elicited in the tumor-bearing state and that irrespective of the potential of tumor cells themselves to produce IL-6, T cells, especially Lyt-2+ T cells from tumor-bearing mice are responsible for such a high level of IL-6 production.
Regulatory role of CD4/L3T4 molecules in IL-2 production by affecting intracellular Ca2+ concentration of T cell clone stimulated with soluble anti-CD3.
Authors: Tamura T , Mizuguchi J , Nariuchi H
Published: December 2022

To elucidate the role of CD4 molecule in T cell activation, the effect of anti-CD4 on T cell IL-2 production was examined by using an alloreactive Th clone. The alloreactive T cell used in the present experiments produced IL-2 in response to soluble anti-CD3 epsilon-chain (anti-CD3) without accessory cell or insoluble antibody carrier. The IL-2 production was suppressed by the addition of anti-CD4 in cultures. An intracellular free Ca2+ concentration ([Ca2+]i) of the T cell clone was elevated by anti-CD3 stimulation, but the elevation was suppressed in the presence of anti-CD4. When the clone was stimulated in Ca2(+)-free medium, the elevation of [Ca2+]i was not observed. When Ca2+ influx was induced by calcium ionophore A23187 or ionomycin, the clone produced IL-2 in response to anti-CD3 in the presence of anti-CD4. When polyclonal T cell line or several other alloreactive T cell clones were examined for their anti-CD3 response, essentially the same results as mentioned above were obtained. Taken together, these results suggest that the slow and sustained elevation of [Ca2+]i is an essential signal for IL-2 production of T cells, and that anti-CD4 suppresses the IL-2 production by interfering the [Ca2+]i elevation. The significance of CD4 molecules in murine T cell activation was discussed.
In vivo and in vitro regulation of IgE production in murine hybridomas.
Authors: Mathur A , Van Ness B G , Lynch R G
Published: December 2022

Normal BALB/c mice injected i.p. with the IgE-secreting hybridomas B53 (epsilon, kappa anti-DNP), SE1.3 (epsilon, kappa, anti-arsonate) or A3B1 (epsilon, kappa, anti-TNP) were monitored for serum IgE concentrations and frequencies of splenic T lymphocytes with surface membrane receptors for the Fc portion of IgE (Fc epsilon R+ T lymphocytes). Mice with B53 or SE1.3 hybridomas initially developed high concentrations of IgE and CD8+ Fc epsilon R+ T lymphocytes, followed by a progressive decline in both serum IgE and expression of cytoplasmic epsilon-chains in the hybridoma cells. Serum IgE concentrations in mice with A3B1 hybridomas progressively increased without development of Fc epsilon R+ T lymphocytes nor a subsequent decline in IgE or change in cytoplasmic epsilon-chain expression in the A3B1 cells. An in vitro system in which the IgE-secreting hybridoma cells were cocultured with spleen cells harvested from mice with established B53 tumors was used to investigate the mechanisms involved in the inhibition of IgE production by the hybridoma cells. The results of these studies indicate that: 1) the induction/upregulation of Fc epsilon R on CD8+ T lymphocytes in vivo requires factors in addition to high serum IgE concentrations; 2) in addition to CD8+ Fc epsilon R+ T lymphocytes and monocytes, another, as yet unidentified, splenic cell component appears to contribute to the process by which epsilon-chain expression in IgE-secreting hybridoma cells is suppressed, and 3) a hybridoma (A3B1) that fails to induce CD8+, Fc epsilon R+ T lymphocytes in vivo and is not inhibited in IgE expression in vivo, nonetheless is inhibited in IgE expression in vitro when cocultured with spleen cells from mice with B53 tumors.
Egr-1 expression in surface Ig-mediated B cell activation. Kinetics and association with protein kinase C activation.
Authors: Seyfert V L , McMahon S , Glenn W , Cao X M , Sukhatme V P , Monroe J G
Published: December 2022

We have studied the expression of an immediate/early type gene, Egr-1, in murine B lymphocyte responses to Ag receptor-generated signals. The Egr-1 gene encodes a zinc finger protein with sequence-specific DNA binding activity and is believed to act as an intracellular "third messenger," to couple receptor-generated signals to activation-associated changes in gene expression. We show here that Egr-1 mRNA expression is rapidly and transiently (returning to basal levels by 6 h) induced after receptor crosslinking with anti-receptor antibodies. Egr-1 protein expression is more prolonged, maintaining detectable levels through 12 h. The induction of Egr-1 is a primary response to Ag receptor signaling, as it is independent of new protein synthesis and is inhibited by actinomycin D. We have also examined the linkage of Egr-1 to known signaling pathways associated with G0 to G1 transition by these cells in response to signals generated through the B cell Ag receptor. Egr-1 mRNA was not induced after elevation of intracellular free Ca2+. In contrast, the pharmacologic agents PMA and SC-9, which directly activate protein kinase C, both cause marked increases in Egr-1 mRNA levels with the same kinetics as observed after anti-receptor antibody stimulation. Further, the protein kinase C inhibitors H7, sangivamycin, and staurosporin block anti-receptor antibody-induced expression of Egr-1, thus, B cell Ag receptor-linked Egr-1 expression is likely coupled to the protein kinase C component of transmembrane signaling. Preliminary promoter mapping studies are consistent with this conclusion, because both PMA and anti-receptor antibody act through the same or overlapping cis-regulatory elements.