bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteolysis of C3 on U937 cell plasma membranes. Purification of cathepsin G.
Authors: Maison C M , Villiers C L , Colomb M G
Published: December 2022

Covalent binding of C3 fragments to U937 cell membranes involved a cell surface-associated proteolytic activity. Two proteases able to cleave C3 were purified from U937 plasma membranes. Purification involved solubilization of the membranes and ion exchange chromatography. One of the purified proteases was identified as elastase, based upon a substrate specificity for benzyloxycarbonylalanine-o-nitrophenyl ester and complete inhibition by elastatinal and methoxysuccinyl-alanyl-alanyl-prolyl-valyl-chloromethyl-ketone. The other protease (m.w. 28,000) is cathepsin G, as deduced from the amino acid composition, the amino-terminal sequence, and the substrate specificity for succinyl-alanyl-alanyl-phenylalanine-p-nitroanilide. These two lysosomal proteases are present on the U937 cell surface, as confirmed by immunofluorescence analysis. Plasma membrane elastase and cathepsin G from U937 cells cleave C3 into C3a- and C3b-like fragments; further incubation leads to C3c- and C3dg-like fragments, as judged from SDS-PAGE analysis of the digests. Sequencing of the C3b-like fragment purified by reverse phase chromatography indicates that initial cleavage of C3 by purified cathepsin G occurs at two positions in the amino-terminal part of the alpha-chain, at a Arg-Ser bond located between residues 748 and 749 and at a Leu-Asp bond between residues 751 and 752. These proteases are, thus, able to generate, on the U937 surface, active fragments of C3, which are likely to be involved in cell-protein and cell-cell interactions.
Human T cell recognition of influenza A nucleoprotein. Specificity and genetic restriction of immunodominant T helper cell epitopes.
Authors: Brett S J , Blau J , Hughes-Jenkins C M , Rhodes J , Liew F Y , Tite J P
Published: December 2022

The characterization of human T cell antigenic sites on influenza A nucleoprotein (NP) is important for subunit vaccine development for either antibody boosting during infection or to stimulate T cell-mediated immunity. To identify such sites, 31 synthetic peptides that cover more than 95% of the amino acid sequence from NP of influenza A/NT/60/68 virus were tested for their ability to stimulate PBL from 42 adult donors. The most frequently recognized region was covered by a peptide corresponding to residues 206-229 of NP, with 20/42 (48%) of responders. In many individuals this was also one of the peptides that stimulated the strongest T cell responses. Other regions that were also frequently recognized were 341-362 by 13/42 (30%), 297-318 by 10/42 (23%), and 182-205 by 9/42 (21%) of individuals. These peptides covered highly conserved regions in NP of influenza A viruses, suggesting that they could be useful in boosting cross-reactive immunity against the known type A virus strains. In order to define the class II restriction molecules used to present particular T cell epitopes, 22 persons from the donor panel were HLA-typed. The majority of persons who expressed DR2, and proliferated to NP also responded to the major immunodominant region 206-229. In addition, this peptide was also immunodominant in the one person expressing DRw13. The observation that recognition of this peptide is associated with DR2 was confirmed by using short term T cell lines and APC from a panel of typed donors. Further results with virus-specific T cell lines and clones and transfected L cells expressing DR molecules showed that DR1 could also present this peptide. Therefore the results suggest that recognition of 206-229 is associated with at least three different DR haplotypes and this may explain the high frequency with which this peptide is recognized in the population. The fine specificity of the response to peptide 206-229 was distinct when presented by DR1- or DR2-expressing APC. The DR1 response was dependent on the N terminus, and the DR2 response was directed to the C terminus of the peptide.
Engagement of CD14 on human monocytes terminates T cell proliferation by delivering a negative signal to T cells.
Authors: Lue K H , Lauener R P , Winchester R J , Geha R S , Vercelli D
Published: December 2022

We have recently shown that engagement of the human monocytic Ag CD14 by murine mAb induces lymphocyte function-associated antigen-1/intercellular adhesion molecule-1-dependent homotypic adhesion. To determine whether CD14 plays a role in monocyte-T cell interactions, we tested the effect of anti-CD14 mAb on the proliferation of human T cells. Our results show that anti-CD14 mAb strongly inhibited T cell proliferation induced by Ag, anti-CD3 mAb, and mitogenic lectins. Inhibition by anti-CD14 mAb was epitope-dependent and required physical contact between monocytes and T cells. CD14 engagement did not affect IL-2R expression or IL-2 synthesis but induced a state of unresponsiveness that was not IL-2 specific; proliferation of anti-CD3-activated T cell blasts in response to both IL-2 and IL-4 was abrogated by addition of monocytes preincubated with anti-CD14 mAb. Inhibition of T cell proliferation after engagement of CD14 on monocytes was likely to result from delivery of a negative signal to T cells, rather than from disruption of a costimulatory monocyte-derived signal, because incubation of monocytes with anti-CD14 mAb also inhibited monocyte-independent T cell proliferation induced by PMA and ionophore. These results, together, point to a role of CD14 in the monocyte-dependent regulation of T cell proliferation.
Cross-linkage of Ly-6A/E induces Ca2+ translocation in the absence of phosphatidylinositol turnover and mediates proliferation of normal murine B lymphocytes.
Authors: Snapper C M , Yamada H , Mond J J , June C H
Published: December 2022

Ly-6A/E is a phosphatidylinositol (PI)-linked membrane protein whose expression is induced or upregulated on normal murine T and B cells by IFN-gamma. Cross-linkage of Ly-6A/E expressed on normal murine T cells stimulates Ca2+ translocation, and in the presence of a protein kinase C (PKC) activator, lymphokine secretion, and cellular proliferation. Utilizing an anti-Ly-6A/E mAb, we studied the effect of cross-linking Ly-6A/E on IFN-gamma-treated resting B cells, for Ca2+ translocation, PI turnover, and cellular proliferation. Since these events are known to be stimulated by cross-linkage of B cell membrane (m)Ig, we compared the changes mediated through these respective membrane proteins. We show that cross-linkage of B cell Ly-6A/E stimulates a large, rapid, and sustained increase in the concentration of intracellular free calcium ([Ca2+]i) comparable in magnitude, though somewhat delayed, relative to that observed after cross-linking of mIg. Cross-linkage of B cell Ly-6A/E does not, however, stimulate detectable PI turnover, in contrast to PI turnover induced by ligation of mIg. Both the Ly-6A/E- and mIg-mediated increase in [Ca2+]i occur through mobilization of internal Ca2+ stores as well as entry of Ca2+ into the cell from the extracellular compartment. Ly-6A/E-mediated Ca2+ translocation appears to be under the regulation of PKC in that short term pretreatment of B cells with the PKC activator, PMA, inhibits the Ly-6A/E- as well as the mIg-mediated increase in [Ca2+]i, whereas prolonged exposure to PMA, under conditions that lead to depletion of PKC, results in an augmentation in Ca2+ translocation after ligation of either Ly-6A/E or mIg. Co-capping studies indicate that Ly-6A/E and mIg cap independently in the B cell membrane, thus suggesting that the Ly-6A/E-induced effects on Ca2+ translocation are not mediated through simultaneous modulation of mIg. Anti-Ly6A/E, by itself, does not stimulate an increase in [3H]thymidine incorporation by IFN-gamma-treated resting B cells, but induces a striking increase in the presence of PMA. By contrast, anti-Ig by itself stimulates significant increases in [3H]thymidine incorporation that is inhibited by PMA. Thus, Ly-6A/E is a potent mediator of B cell activation that may use a signal transduction system in quiescent B cells that is distinct from that of the Ag receptor.
Role of Mls-1 locus and clonal deletion of T cells in susceptibility to collagen-induced arthritis in mice.
Authors: Anderson G D , Banerjee S , Luthra H S , David C S
Published: December 2022

The role of T cell-mediated and humoral immunity to type II collagen has been well documented in collagen-induced arthritis (CIA). Previous work from our laboratory has indicated that genomic deletions of TCR V beta genes may play a role in CIA resistance in mice. This indicated a selectivity of TCR usage by autoreactive T cells in CIA in mice. Certain strains of mice, although having a normal genomic V beta TCR repertoire, can show clonal deletion of peripheral T cells that bear specific V beta gene products in their TCR. These clonally deleted T cells are reactive with self-Ag such as minor lymphocyte stimulation (Mls) Ag. An Mls-congenic strain, BALB.D2.Mlsa, which differs only at the Mls-1 a locus from BALB/c (Mls-1b), was used to examine the effect of clonal deletion of Mls-1a-reactive T cells in CIA. These two strains were crossed to three CIA-susceptible strains, B10.RIII (H-2r, Mls-1b), DBA/1 (H-2q, Mls-1a), and B10.Q (H-2q, Mls-1b), and the crosses were injected with type II collagen. A significantly decreased incidence of arthritis was observed in the (BALB.D2.Mlsa x B10.Q)F1 hybrids, compared with (BALB/c x B10.Q)F1 hybrids, upon immunization with chick type II collagen. The BALB.D2.Mlsa cross mice also had significantly lower levels of antimouse collagen antibodies. Flow cytometric analysis confirmed the clonal deletion of Mls-1a-reactive V beta 8.1, V beta 6, V beta 7, and V beta 9 subsets in the (BALB.D2.Mlsa x B10.Q)F1 hybrids. The study of H-2q/d mice in (BALB.D2.Mlsa x B10.Q) x B10.Q back-crosses demonstrated a significant correlation between CIA resistance and Mls-1a locus. On the other hand, B10.RIII crosses showed only a modest decrease in CIA incidence in the presence of Mls-1a. As expected, all the DBA/1 crosses had an equal incidence of CIA, which was somewhat less than that seen in DBA/1 mice themselves. These studies point out that the Mls-1a locus could play a role in decreasing CIA incidence by clonal deletion of T cells bearing specific V beta TCR, which may be involved in the pathogenesis of CIA. The influence of the clonal deletion of T cells on CIA, and hence the usage of specific V beta TCR by autoreactive anti-type II collagen T cells, however, depends not only on the source of the type II collagen and the MHC class II molecules involved but also on other background genes in mice.
Suppressive effects of monocytic cells and transforming growth factor-beta on natural killer cell differentiation in autoimmune viable motheaten mutant mice.
Authors: Koo G C , Manyak C L , Dasch J , Ellingsworth L , Shultz L D
Published: December 2022

Viable motheaten (mev) mice are homozygous for a recessive single gene mutation at chromosome 6. These mice develop numerous inflammatory and arthritic syndromes and exhibit abnormal B cell functions as well as lower T and NK cell activity. In this study, the differentiation of NK cells in mev mice was examined to elucidate the underlying basis for decreased NK activity. Although NK cells appear to be present in mev mice, their activity was demonstrable only when the spleen cells were enriched by nylon wool passage. Similarly bone marrow cells from these mice could be shown to contain precursors of NK cells when they were passed over nylon wool and transplanted into irradiated recipients. The adherent cells from both the spleen and bone marrow of mev mice suppressed the differentiation of NK cells from normal splenic populations. These suppressive adherent cells were F4/80(+), AsGm-1(+), Qa-5(+), and NK-1.1(+). They were not cytolytic when cultured in IL-2. Antibodies to a number of cytokines, such as IFN-alpha, -beta, and gamma, or TNF-alpha, could not reverse the suppressive effect of the adherent cells. Addition of anti-TGF-beta antibody could, however, overcome the suppression, suggesting that TGF-beta was partly responsible for the defective NK differentiation in the mev mice.
Unusual alpha beta-T cells expanded in autoimmune lpr mice are probably a counterpart of normal T cells in the liver.
Authors: Seki S , Abo T , Ohteki T , Sugiura K , Kumagai K
Published: December 2022

Autoimmune MRL-lpr/lpr (lpr) mice develop severe lymphadenopathy, characterized by the accumulation of alpha beta-T cells with CD4-8- double negative (DN) phenotype, at the onset of disease. We previously demonstrated that the liver is a major site for the proliferation of such DN alpha beta-T cells. Herein, we further demonstrate that a large proportion of alpha beta-T cells in the liver and other organs, except the thymus, of lpr mice have unique properties, such as DN phenotype, relatively dull TCR intensity, a preponderance of V beta 8+ cells, and Pgp-1 expression. Interestingly, alpha beta-T cells in the liver of normal mice were found to consist of T cells with intermediate intensity of TCR (i.e., brighter than thymic dull TCR and lower than thymic bright TCR) as well as with bright intensity of TCR in the immunofluorescence test. These hepatic alpha beta-T cells with intermediate TCR in normal mice were found to have properties similar to those of alpha beta-T cells in lpr mice. These results suggest that abnormal alpha beta-T cells in lpr mice are a counterpart of normal T cells in the liver. An abnormal expansion of such T cells in the liver might be fundamental to the pathogenesis involved in these autoimmune mice.
Granulocyte-macrophage colony-stimulating factor promotes the proliferation of human alveolar macrophages in vitro.
Authors: Nakata K , Akagawa K S , Fukayama M , Hayashi Y , Kadokura M , Tokunaga T
Published: December 2022

The effects of granulocyte-macrophage (GM-CSF) or macrophage-CSF on in vitro proliferation of human alveolar macrophages (AM) were evaluated. AM of healthy volunteers incubated with recombinant human GM-CSF revealed incorporation of [3H]thymidine in vitro. The maximum incorporation was observed at 20 U/ml of GM-CSF on day 3. The proportion of proliferating cells incubated with 20 U/ml of GM-CSF from day 3 to day 4 was 8 to 11% of the total, whereas 3 to 5% of cells proliferated without GM-CSF. The number of cell nuclei increased 1.30- to 1.68-fold in the initial 7 days during incubation with 20 U/ml of GM-CSF, whereas there was a 1.07- to 1.13-fold increase without GM-CSF. Conditioned media obtained by the incubation with human lung tissue exhibited similar effects as recombinant human GM-CSF on macrophages. The effects were completely abrogated by antibody against human GM-CSF. Immunohistochemically, GM-CSF was detected in lung cells including AM, alveolar epithelium, alveolar interstitial cells, and endothelial cells. In contrast, recombinant macrophage-CSF did not induce the proliferation of human AM, although it has been known to promote the proliferation of murine AM. These observations suggest that GM-CSF plays an important role among the regulatory factors that locally support the population of AM in human lungs.
Effect of natural polymorphism at residue 86 of the HLA-DR beta chain on peptide binding.
Authors: Busch R , Hill C M , Hayball J D , Lamb J R , Rothbard J B
Published: December 2022

Class I and class II MHC glycoproteins are highly polymorphic molecules that bind antigenic peptides and present them on cell surfaces for recognition by T lymphocytes. Even though MHC polymorphism has long been known to affect both peptide binding and recognition by the TCR, the role of individual amino acids of MHC proteins in these interactions is poorly understood. To examine the effect of a small number of amino acid residues on T cell stimulation, B lymphoblastoid cell lines homozygous for the closely related DR1 subtypes, Dw1 and Dw20, and the DR4 subtypes, Dw4 and Dw14, were compared for their ability to present an immunogenic influenza hemagglutinin peptide (HA307-319) to an Ag-specific, DR1,4-restricted T cell clone. B cell lines expressing DR1 Dw20 and DR4 Dw14 presented HA307-319 much less efficiently than DR1 Dw1 and DR4 Dw4 and bound a biotinylated analogue of the same peptide less well. Analysis of DRB1 gene sequences suggested that polymorphism at residue 86 had a major effect on peptide binding. Differences in binding of a set of HA307-319 analogues biotinylated at each residue to cells expressing DR1 Dw1 and DR1 Dw20 suggested that the polymorphism affected the interactions of many peptide residues with the class II molecule. In inhibition assays, DR1 Dw1 and DR4 Dw4 were shown to differ from DR1 Dw20 and DR4 Dw14 in their length requirements for peptide binding. Using a larger panel of homozygous B cell lines expressing many class II haplotypes, a Ser-309 substituted HA307-319 analogue was shown to bind to most B cell lines expressing Val-86 containing alleles (including DR1 Dw20 and DR4 Dw14) but failed to bind most B cell lines expressing Gly-86 alleles (including DR1 Dw1 and DR4 Dw4). The results indicated that polymorphism at residue 86 influenced the specificity and affinity of peptide binding and affected the conformation of peptide-DR protein complexes without completely eliminating T cell recognition.
A single amino acid in the second Ig-like domain of the human Fc gamma receptor II is critical for human IgG2 binding.
Authors: Warmerdam P A , van de Winkel J G , Vlug A , Westerdaal N A , Capel P J
Published: December 2022

The low-affinity human Fc gamma RIIa is encoded by a single gene with allelic variation, defined by low-responder and high-responder alleles (LR and HR). The HR Fc gamma RIIa transcript interacts strongly with murine (m) IgG1 complexes, in contrast to the LR Fc gamma RIIa. Furthermore, the transcripts can be discriminated by mAb 41H16, which recognizes an epitope expressed on the HR Fc gamma RIIa molecule. We report that this receptor is also polymorphic in its reactivity with human (h) IgG2. Binding studies using well-defined hIgG dimers revealed that LR Fc gamma RIIa molecules can efficiently bind hIgG2, in contrast to HR Fc gamma RIIa. Previous work of others showed one amino acid difference between the allelic forms of Fc gamma RII. We, however, found a second amino acid difference between both allelic forms. In this study, hybrid Fc gamma RIIa molecules were constructed to determine the epitope for mAb 41H16 and the binding domain for mIgG1 and hIgG2 complexes. Our data point to the importance of the amino acid at position 131, located in the second Ig-like domain of Fc gamma RIIa. When an arginine residue is present at amino acid position 131, the receptor is recognized by mAb 41H16. Furthermore, the receptor can bind mIgG1-sensitized indicator E, but binds hIgG2 dimers only weakly. When a histidine residue is present at this amino acid position, hIgG2 dimers do bind efficiently to Fc gamma RII, whereas mIgG1-sensitized E and mAb 41H16 exhibit a strongly diminished binding.
Aggregated bovine IgG inhibits mannose receptor expression of murine bone marrow-derived macrophages via activation.
Authors: Schreiber S , Stenson W F , MacDermott R P , Chappel J C , Teitelbaum S L , Perkins S L
Published: December 2022

We previously described the presence of an inhibitory protein contained in the 20 to 40% (NH4)2SO4 precipitable fraction of FCS that down-regulates expression of mannose receptors on bone marrow-derived macrophages. We now identify aggregated bovine IgG as the main inhibitory component. Heat-aggregated bovine IgG was capable of down-regulating expression of the macrophage mannose receptor in a dose-dependent manner without inducing changes in ligand affinity whereas neither F(ab')2 fragments nor nonaggregated IgG displayed any inhibitory effect. Depleting of IgG from heat inactivated FCS by protein G affinity chromatography completely removes the inhibitory activity. Moreover, readdition of the Ig eluate from the protein G chromatography column restored inhibition in a dose-dependent manner. Macrophages were able to clear exogenously added aggregated bovine IgG, thus leading to loss of inhibitory activity in macrophage-conditioned media as compared to sham-conditioned media containing aggregated IgG. These results indicate that aggregated IgG down-regulates mannose receptor expression by macrophage activation via interaction with Fc-gamma R.
IgG subclass distribution of primary and secondary immune responses concomitant with viral infection.
Authors: Coutelier J P , van der Logt J T , Heessen F W
Published: December 2022

Infection with mouse hepatitis virus was found to selectively increase the proportion of IgG2a in antibodies elicited by a concomitant administration of unrelated T cell-dependent protein Ag. In contrast, T cell-independent responses were only marginally affected. This isotypic bias, which occurred when the virus was inoculated shortly before or after a primary immunization, persisted in subsequent secondary responses. However, infection concomitant to secondary antibody responses did not affect their isotypic distribution. These observations suggest that the virus can durably modify unrelated T cell responses that are initiated at the time of infection, which could have implications in the pathogenesis of autoimmune reactions.
Control of T cell responses to staphylococcal enterotoxins by stimulator cell MHC class II polymorphism.
Authors: Yagi J J , Rath S , Janeway C A
Published: December 2022

The bacterial toxic mitogens or superantigens are a family of related proteins that elicit potent T cell proliferative responses. These responses require APC that express MHC class II proteins, but they are not MHC restricted and they do not depend on a processing step, presumably because these mitogens bind directly to MHC class II molecules. These mitogens stimulate T cells by interacting in an unknown way with the portion of the TCR encoded by certain V beta gene segments. In this paper, we explore the importance of MHC class II polymorphism in T cell responses to staphylococcal enterotoxins. We find that certain MHC molecules present SEB to V beta 8-bearing T cells far better than others. These data suggest that one route of host defence against bacterial toxic mitogens may be to alter MHC class II molecules so that stimulation is inhibited.
CD24, a signal transducer modulating B cell activation responses, is a very short peptide with a glycosyl phosphatidylinositol membrane anchor.
Authors: Kay R , Rosten P M , Humphries R K
Published: December 2022

CD24 is a signal-transducing molecule on the surfaces of most human B cells that can modulate their response to activation signals by antagonizing IL-induced differentiation into antibody-forming cells and inducing proliferation in combination with signals generated by Ag receptors. A cDNA that directs the expression of CD24 on the surfaces of transfected COS cells was cloned by its homology to a cDNA encoding the murine M1/69-J11d heat stable Ag. The CD24 cDNA encodes a mature peptide of only 31 to 35 amino acids that is extensively glycosylated and is attached to the outer surface of the plasma membrane by a glycosyl phosphatidylinositol lipid anchor. Although CD24 is structurally similar to M1/69-J11d, and the two Ag appear to have a common genetic ancestry, the homology of CD24 to the M1/69-J11d Ag is confined to a small cluster of amino acids comprising potential N-linked glycosylation sites. Combined with the differences in expression patterns of the human and murine Ag, this indicates that CD24 and M1/69-J11d may not have equivalent functional roles in lymphoid development. The novel structure of CD24 suggests that signaling could be triggered by the binding of a lectin-like ligand to the carbohydrates projecting from the CD24 peptide, and transduced through the release of second messengers derived from the glycosyl phosphatidylinositol membrane anchor of CD24.
Ly-49 multigene family. New members of a superfamily of type II membrane proteins with lectin-like domains.
Authors: Wong S , Freeman J D , Kelleher C , Mager D , Takei F
Published: December 2022

Ly-49 (YE1/48, A1) is a dimer protein expressed on subpopulations of murine NK cells. It is a member of a superfamily of type II transmembrane proteins containing carbohydrate recognition domains (CRD). In the mouse genome, the detection of multiple restriction fragments that cross-hybridize with Ly-49 cDNA probes suggests the presence of related genes. In this study, we have isolated several genomic clones encoding portions of CRD sequences highly homologous to the CRD of Ly-49. By using primers based on the consensus sequences of the genomic clones, expression of Ly-49-related genes was detected by the polymerase chain reaction in various organs, including lung, kidney, liver, spleen, and thymus. Two full-length cDNA clones that are highly homologous to the Ly-49 gene were subsequently isolated from a lung cDNA library. At the nucleotide level, the two clones are 72% and 80% identical to Ly-49 in their translated regions, but their sequences are different from those of the genomic clones characterized to date. The two cDNA clones potentially encode type II transmembrane proteins containing CRD that are very similar to Ly-49. These amino acid sequences are also homologous to other members of the superfamily of CRD-containing type II transmembrane proteins, including hepatic lectins and the low affinity IgER (CD23). The homology is most evident in the CRD but is also significant in other domains. These results demonstrate the existence of several functional genes that are highly related to Ly-49. These genes comprise a subfamily within the superfamily of type II transmembrane proteins containing CRD.
B cell triggering by bacterial lipopeptide involves both translocation and activation of the membrane-bound form of protein kinase C.
Authors: Boscá L , Márquez C , Martínez C
Published: December 2022

B cell activation by the lipopeptide trispalmitoyl-cysteinyl-alanyl-glycine (TPP), the biologically active moiety of bacterial lipoprotein, results in protein kinase C (PKC) translocation from the cytosol to the plasma membrane, as well as a significant increase in the activity of membrane-associated PKC that can be observed by in vitro incubation with TPP of partially purified PKC at calcium concentrations in the range of those prevailing in unstimulated B cells. TPP does not affect either the phosphoinositide turnover or the cytosolic-free calcium concentration, but promotes an increase in the intracellular pH that can be blocked by the PKC-inhibitors staurosporine or H-7. Moreover, incubation of B cells with staurosporine suppressed the proliferative response promoted by TPP at a half-maximum effective dose of 16 nM. Activation by TPP of PKC isoenzymes resolved after hydroxylapatite chromatography revealed that the resulting beta I/beta II isoenzyme was more sensitive than the alpha isoenzyme. These results suggest that TPP might mediate B cell activation via interaction with the membrane-associated fraction of PKC.
Immunization with soluble murine CD4 induces an anti-self antibody response without causing impairment of immune function.
Authors: Cassatt D R , Sweet R W , Arthos J A , Truneh A
Published: December 2022

The T cell surface molecule CD4 (L3T4 in mouse) is important in the T lymphocyte response to Ag presented in association with MHC class II molecules. To examine the role of CD4 in immune function, we expressed a soluble form of murine CD4 by deleting the transmembrane and cytoplasmic regions of the L3T4 gene and transfecting the altered gene into Chinese hamster ovary cells. The recombinant soluble mouse CD4 (smCD4) retained the native conformation of the external portion, as indicated by the binding of L3T4 mAb. In vitro, smCD4 did not inhibit class II-dependent, Ag-specific, T cell proliferation or MLR, even at concentrations 300-fold greater, on a molar basis, than that of anti-CD4 mAb. Immunization of mice with smCD4 induced a strong anti-CD4 response. These antibodies showed some binding to native cell surface CD4, indicating that immunization with smCD4 generated an anti-self response. Analysis of lymphoid cells from spleen, lymph node, and thymus of smCD4-treated mice revealed no alteration in subset phenotypes. Also, Th cell function, as measured by response to soluble Ag, was not compromised. Thus, smCD4 did not inhibit T cell activity in vitro, and the autoimmune response arising from immunization with smCD4 had no apparent consequences for normal immune function.
Hydrocortisone and IL-4 induce IgE isotype switching in human B cells.
Authors: Jabara H H , Ahern D J , Vercelli D , Geha R S
Published: December 2022

Induction of IgE synthesis in human B cells requires two signals. The first signal is delivered by the cytokine IL-4. The second signal activates B cells and is delivered by T cells, EBV infection, or engagement of the B cell-specific Ag CD40. Hydrocortisone (HC) has recently been shown to synergize with IL-4 to induce IgE synthesis in CD5+ chronic lymphocytic leukemia B cells. We show herein that a combination of HC and rIL-4 induces IgE synthesis in highly purified normal peripheral blood B cells. HC and IL-4 acted directly on B cells, because T cells and monocytes were not required for IgE synthesis. IgE induction was shown to occur in surface IgE- B cells isolated by cell sorting. These results suggest that IgE synthesis results from isotype switching, rather than from expansion of a precommitted B cell population. Furthermore, IgE synthesis was induced in sorted CD5- B cells, indicating that the ability to produce IgE in response to HC and IL-4 is not constrained by CD5 expression. Endogenous IL-6 was critical for induction of IgE synthesis by HC and IL-4, because an anti-IL-6 antibody strongly inhibited IgE production. These data suggest that hormones may play an important role in the regulation of IgE synthesis.
Mechanisms of IgE homeostasis. Sequestration of IgE by murine type II IgE Fc receptor-bearing B cell hybridomas.
Authors: Chen S S
Published: December 2022

Among all classes of Ig, IgE exhibits the highest rate of fractional catabolism of which the site and mechanisms is not understood. We construct a panel of murine B cell hybridomas to investigate the catabolism of IgE; one of these hybridomas, 17A11, constitutively expresses high levels of type II IgE FcR (Fc epsilon RII, CD23) (Kd:1.77 nM; B max: 1.65 x 10(5], and is capable of clearing receptor-bound IgE. Receptor-mediated endocytosis of IgE ligand ensues after binding monomeric and DNP-BSA:IgE immune complexes, and the binding is inhibited by treating 17A11 with anti-CD23. IgE ligands are sequestered and are not susceptible to acid stripping from the cell surface. The internalized IgE ligands redistributed into acid hydrolase containing high density lysosomal vesicles and were degraded; metabolic inhibitors such as chloroquine and monensin that elevate intracellular pH of 17A11 also prevent entry of IgE ligand into lysosomes. These observations raise the possibility that normal Fc epsilon RII-bearing mature B cells in the circulation and lymphoid tissues may function in sequestration and catabolic turnover of IgE molecules through IgE or IL-4 up-regulated Fc epsilon RII uptake; B cell Fc epsilon RII may perform an important role in determining the short biological half-life of IgE molecules, and contributes to IgE homeostasis.
Type I transforming growth factor-beta receptors on neutrophils mediate chemotaxis to transforming growth factor-beta.
Authors: Brandes M E , Mai U E , Ohura K , Wahl S M
Published: December 2022

Participation of human polymorphonuclear neutrophils in the inflammatory response is mediated, in part, by soluble factors such as chemotactic peptides and cytokines. Although the cytokine, transforming growth factor beta (TGF-beta), has been shown to recruit monocytes and promote the inflammatory process, its effects on neutrophils are unknown. In this investigation, [125I]TGF-beta 1 affinity binding studies were employed to show that neutrophils express TGF-beta receptors (350 +/- 20 receptors/cell), which exhibit high affinity for the ligand (dissociation constant, 50 pM). Affinity cross-linking studies identified the receptors to be primarily of the type I class. In contrast to the receptors on monocytes, neutrophil TGF-beta receptors were not down-regulated by exposure to specific inflammatory mediators. Additional studies examined whether exposure of neutrophils to TGF-beta could enhance specific functions, as occurs with monocytes. TGF-beta was shown to cause directed migration of neutrophils at femtomolar concentrations, thus it is the most potent neutrophil chemotactic factor yet identified. Neutrophil production of reactive oxygen intermediates was not stimulated by TGF-beta, nor did TGF-beta enhance or depress subsequent PMA- or FMLP-stimulated superoxide production. However, the stable expression of neutrophil TGF-beta receptors, and the capacity of this cytokine to stimulate neutrophil chemotaxis, suggest that the pro-inflammatory effects of TGF-beta are mediated by neutrophils in addition to monocytes.
Effect of dietary alpha-linolenate on platelet-activating factor production in rat peritoneal polymorphonuclear leukocytes.
Authors: Horii T , Satouchi K , Kobayashi Y , Saito K , Watanabe S , Yoshida Y , Okuyama H
Published: December 2022

The effects of dietary alpha-linolenate (18:3, n-3) and linoleate (18:2, n-6) on platelet-activating factor (PAF) production were examined. Rats were fed an alpha-linolenic acid-rich (perilla oil) diet or a linoleic acid-rich (safflower oil) diet for 6 wk, and polymorphonuclear leukocytes (PMN) were elicited by peritoneal injection of casein. The overall phospholipid content and composition as well as the subclass distribution of choline and ethanolamine glycerophospholipids in PMN were not altered by these diets. However, with the perilla oil diet their content of a putative precursor of PAF, 1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine was approximately 50% of that with safflower oil diet. On exposure to various concentrations of FMLP, PAF formation by PMN in the perilla oil group was less than 50% of that by PMN in the safflower oil group. A larger difference in PAF productions by PMN in the two dietary groups was observed on their stimulation with calcium ionophore A23187. These results demonstrate that PAF production is modulated in some as yet unknown way by changing the alpha-linolenate/linoleate balance of the diet.
Selection of the same major T cell determinants of influenza nucleoprotein after vaccination or exposure to infectious virus.
Authors: Brett S J , Gao X M , Liew F Y , Tite J P
Published: December 2022

In the present study, we have compared the T cell antigenic determinants on nucleoprotein (NP) of influenza A/NT/60/68 virus recognized by BALB/c mice (H-2d) after vaccination using several different vehicles with the determinants recognized after exposure to infectious virus. Mice were immunized s.c. with: 1) purified recombinant NP with three different adjuvants--alum, saponin, or CFA; 2) whole inactivated A/Okuda virus in PBS or saponin; or 3) live attenuated Salmonella typhimurium AroA- vector expressing NP. A series of overlapping synthetic peptides that cover more than 90% of the amino acid sequence of NP were used to map the Th cell epitopes. The results showed that the same limited number of major epitopes were recognized after each of the different immunization regimes. Secondary in vivo boosting using the same vehicles as for the primary immunization did not increase the number of different T cell sites recognized. The T cell responses after intranasal infection with infectious A/NT/60/68 or A/PR/8/34 virus also showed a similar pattern of recognition of the major CD4-positive T cell epitopes. The only exception was that the region corresponding to residues 401-419 was only recognized after exposure to NP from A/NT/60/68 but not A/PR/8/34. This is probably because the two viruses differ in amino acid sequence at positions 408 and 411 within this part of the NP molecule. In contrast to the results observed with CD4-positive T cell epitopes, the major determinant recognized by CD8-positive T cells was only presented after live viral infection. The results in this study have important implications for vaccine design, inasmuch as they indicate that the same dominant CD4 T cell determinants on NP presented by vaccination with NP are also recognized by T cells from mice exposed to infectious virus.
Phenotypic complexity of intraepithelial lymphocytes of the small intestine.
Authors: Lefrancois L
Published: December 2022

A detailed phenotypic analysis of intraepithelial lymphocytes (IEL) of the small intestine was performed using multicolor fluorescence flow cytometry. CD4+8+ IEL (double positives; DP) could be detected in significant numbers in preparations from several mouse strains. DP IEL expressed Tcr alpha, beta and Thy1. Comparison of Tcr alpha, beta levels of thymocytes and IEL revealed that whereas the majority of DP thymocytes expressed low Tcr levels, DP IEL expressed high, mature T cell levels of Tcr. In addition, DP IEL were generally Ly3- (CD8 beta), unlike their thymic counterparts, which are Ly3+. Ly3 was not present on Tcr gamma, delta IEL, whereas CD4-8+ Tcr alpha, beta IEL contained Ly3- and Ly3+ subsets. The Ly3- population in either Tcr-bearing subset could be further subdivided by Thy1 expression. Ly1 (CD5) expression was also examined, and none of the Tcr gamma, delta IEL were Ly1+. Based on Thy1, Ly3, and Ly1 expression, four CD4-8+ Tcr alpha, beta IEL subsets were detected. The results indicate the cellular complexity of the IEL compartment rivals that found in the thymus. These findings are discussed in light of recent data suggesting an extrathymic origin of some IEL.
Successful treatment of experimental allergic encephalomyelitis with transforming growth factor-beta 1.
Authors: Johns L D , Flanders K C , Ranges G E , Sriram S
Published: December 2022

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional cytokine with immunosuppressive effects on T cells in vitro. Experimental allergic encephalomyelitis is an archetypal T cell-mediated autoimmune demyelinating disease of the central nervous system that often serves as a model for multiple sclerosis. In vivo administration of TGF-beta 1 into SJL mice was successful in reducing the incidence of clinical disease and the histologic severity of inflammation and demyelination in the brain and spinal cord. Immunohistochemical studies performed on control animals showed that TGF-beta-1, -2, and -3 were present in inflammatory perivascular lesions in the brain. The use of a naturally occurring cytokine with immunoregulatory functions in the treatment of an autoimmune disease is novel. However, potential long term complications of such therapy must be addressed before its use in human autoimmune disease such as multiple sclerosis.
IFN-gamma differentially modulates the susceptibility of L1210 and P815 tumor targets for macrophage-mediated cytotoxicity. Role of macrophage-target interaction coupled to nitric oxide generation, but independent of tumor necrosis factor production.
Authors: Leu R W , Leu N R , Shannon B J , Fast D J
Published: December 2022

IFN-gamma primes murine macrophages to render them responsive for triggering by subactivating concentrations of bacterial LPS to mediate nonspecific tumor cytotoxicity. However, IFN-gamma also has direct anti-proliferative effects on transformed cells that serve as sensitive tumor targets for cytotoxic macrophages. We investigated the effects of preexposure of L1210 mouse leukemia and P815 mouse mastocytoma targets to rIFN-gamma on changes in their susceptibility to cytotoxicity by LPS-activated mouse peritoneal macrophages (PM). Co-incubation of inflammatory PM and either L1210 or P815 targets with IFN-gamma and LPS produced a classical synergistic cytotoxicity for both targets over that of IFN-gamma or LPS alone. Similar synergistic augmentation of cytotoxicity occurred when effector PM were preprimed for 24 h with IFN-gamma before testing for cytotoxicity of untreated targets. However, pretreatment of L1210 and P815 targets for 24 h with IFN-gamma (50 U) before assay produced divergent results in that L1210 was more susceptible, whereas P815 was less susceptible to cytotoxicity by LPS-activated macrophages. Similar results were obtained when both macrophages and targets were pretreated separately with IFN-gamma for 24 h before their combined assay for tumor cytotoxicity. Pretreatment of L1210 targets for 1, 4, or 24 h with IFN-gamma produced similar effects on their increased susceptibility to macrophage cytotoxicity. In contrast, P815 pretreated for 1 and 4 h with IFN-gamma showed an early increased susceptibility to macrophage cytotoxicity followed by a decrease after 24 h pretreatment. The pretreatment of L1210 or P815 targets with IFN-gamma before their exposure to LPS-activated macrophages had no effect on the production of TNF. However, there was a corresponding increase in nitric oxide generation by LPS-activated macrophages after their exposure to IFN-gamma pretreated L1210 targets and a decrease in the presence of IFN-gamma-pretreated P815 targets that correlated with their changes in susceptibility to macrophage killing. Nitric oxide generation by macrophages alone in response to LPS was found to be greater than when effector macrophages were exposed to the tumor targets and this was either increased by L1210 or decreased by P815 that had been pretreated with IFN-gamma. Our results indicate that IFN-gamma may act directly and differentially on tumor targets to alter their susceptibility for macrophage cytotoxicity, which was coupled to changes in the generation of cytotoxic nitric oxide, rather than TNF production by the macrophage.(ABSTRACT TRUNCATED AT 400 WORDS)
Binding of human rhinovirus and T cells to intercellular adhesion molecule-1 on human fibroblasts. Discordance between effects of IL-1 and IFN-gamma.
Authors: Piela-Smith T H , Aneiro L , Korn J H
Published: December 2022

Intercellular adhesion molecule-1 (ICAM-1) is found on the surface of many hemopoietic and non-hemopoietic cells and can function as an adhesive ligand for the integrin, leukocyte function associated molecule-1 (LFA-1, CD11a/CD18). ICAM-1/LFA-1 interaction is thought to be of importance in many immune mediated cell-cell adhesion reactions. Recently, the major human rhinovirus (HRV) receptor has been identified as ICAM-1. HRV has been shown to bind specifically to ICAM-1 on transfected COS cells and to purified ICAM-1, which has been adsorbed to plastic microtiter wells. We have compared the ability of ICAM-1 expressed on the surface of human fibroblasts (FB) to function as a receptor for HRV as well as a receptor for LFA-1-bearing human T lymphocytes. We show that FB stimulation by the cytokines IFN-gamma or IL-1, both known inducers of ICAM-1 synthesis and expression in FB, induced an increase in HRV binding to treated cells, which could be inhibited by antibody to ICAM-1. In contrast, only IFN-gamma and not IL-1 treatment of FB resulted in an increased adhesion of T lymphocytes. Binding of HRV to IFN-gamma-treated FB inhibited the subsequent adhesion of T cells. We also show that prior stimulation of FB with IL-1 enhanced the adhesion of HRV to IFN-gamma-stimulated cells, although IL-1 pretreatment was inhibitory for T cell adhesion. As these two cytokines both up-regulate ICAM-1 on the surface of human FB, the contrasting effects of IFN-gamma and IL-1 on human FB ICAM-1 adhesion to HRV and to LFA-1 suggest that qualitative as well as quantitative alterations of the ICAM-1 molecule may contribute to its specificity of ligand recognition.
IL-6 augments Fc IgE receptor (Fc epsilon RII/CD23) expression on human monoblastic/monocytic cell lines U937, THP-1, and Mono-Mac-6 but not on blood monocytes. Regulatory effects of IL-4 and IFN-gamma.
Authors: Willheim M , Gessl A , Berger R , Schedle A , Luger T , Föster O , Boltz-Nitulescu G
Published: December 2022

Human monoblastic/monocytic leukemia cell lines U937, THP-1, Mono-Mac-6, and blood monocytes were incubated with various concentrations of human rIL-6 and other cytokines and analyzed for their capacity to bind several anti-Fc epsilon RII/CD23 mAb. A marked and dose-dependent increase in the percentage of CD23+ cells, as well as in the mean channel fluorescence intensity, as demonstrated by FACS analysis, was noted after 8- to 72-h incubation of U937 cells with 1 to 1000 U/ml of human rIL-6. Furthermore, rIL-4 synergized with rIL-6 and rIFN-tau in augmenting the Fc epsilon RII expression on U937 cells, whereas rIFN-tau and rIL-6 showed rather additive effects. The enhancement of CD23 expression on IL-6-treated U937 cells was blocked by anti-IL-6 antibodies. Northern blot analysis, employing cDNA probes for Fc epsilon RII, showed that U937 cells contain Fc epsilon RII-specific mRNA. The level of Fc epsilon RII-encoding mRNA was evidently increased by treatment of U937 cells with human rIL-6, rIL-4, or with rIL-6 + rIL-4. The expression of CD23 on THP-1 and Mono-Mac-6 cells was increased slightly by rIL-6 and markedly by rIL-4, rIFN-tau, or a mixture of them. Approximately 14% of blood monocytes, isolated from apparently healthy donors, constitutively possess Fc epsilon RII. In contrast to the cell lines, the Fc epsilon RII density and the percentage of blood monocytes bearing Fc epsilon RII was not augmented by IL-6. Furthermore, rIL-6, and more evidently rIFN-tau, down-regulate rIL-4-driven Fc epsilon RII expression on monocytes but not on monocytic cell lines. Our findings point to differences in the capability of mononuclear phagocytes to respond to cytokine treatment, which may be differentiation dependent, and suggest separate regulatory pathways.
Complement deposition by antibodies to Pseudomonas aeruginosa mucoid exopolysaccharide (MEP) and by non-MEP specific opsonins.
Authors: Pier G B , Grout M , Desjardins D
Published: December 2022

The failure of cystic fibrosis patients to limit chronic infection due to mucoid Pseudomonas aeruginosa might be due to ineffective opsonins produced against this bacterium. Nonopsonizing antibody to the bacterial capsule, mucoid exopolysaccharide (MEP), appears at elevated titers during chronic colonization of cystic fibrosis patients, as do opsonins not specific for MEP. Nonopsonic antibodies to MEP occur naturally in most adults and can be induced in animals by immunization. A limited number of humans produce MEP-specific opsonic antibodies after immunization. The purpose of this study was to compare the activation and deposition of C components onto the bacterial surface in the presence of these different antibodies. Opsonic killing uses the classical C pathway. MEP-specific opsonic and nonopsonic antibodies bound to whole bacteria and activated C to a comparable degree, but opsonic antibody deposited 3 to 40 times more C3 onto bacteria, mostly as C3bi, compared to nonopsonic antibody. In addition, two to three times as much nonopsonic mAb as opsonic mAb (both IgG2b) bound to the bacteria at comparable input concentrations, indicating the difference in C deposition was not due to differences in antibody binding. Non-MEP-specific opsonins also bound C3 to the bacteria, but only a mean of 27 +/- 14% was ester linked, compared with 81 +/- 11% of C3 deposited by MEP-specific opsonins. Immunoprecipitation experiments indicated that two-thirds of the C3 bound in the presence of MEP-specific opsonins was linked to MEP, whereas non-MEP-specific opsonins obtained from infected patients deposited the C3 onto LPS and other unidentified Ag. These data show that MEP-specific opsonins function by depositing C3 onto the outer bacterial surface that differentiates them from non-MEP-specific opsonins produced in response to chronic infection.