bluuebunny/crossref_metadata_2025 huggingface dataset:
DMA and DMB are the only genes in the class II region of the human MHC needed for class II-associated antigen processing.
Authors: Ceman S , Rudersdorf R A , Petersen J M , DeMars R
Published: December 2022

Previous studies have shown that homozygous mutations between the LMP2 and DNA loci in the human MHC cause class II molecules to be abnormally conformed and unstable in the presence of SDS at low temperature, and impede class II-associated Ag processing and presentation. These abnormalities result from impaired ability to form intracellular class II/peptide complexes that predominate in normal cells. We show in this work that this defect results from deficient expression of either the DMA or the DMB gene. Human B-LCL.174 (DR3) cells, which have a deletion of all known expressible genes in the class II region, express transgene-encoded HLA-DR3, but have the abnormalities. Transfer of cosmid HA14, which contains the DMA and DMB genes, into .174 (DR3) cells restored normal DR3 conformation, stability in 0.4% SDS at 0 degree, and ability to process and present tetanus toxoid, but only when both DMA and DMB mRNAs were present. The requirement for both genetic expressions in engendering normal phenotypes was confirmed by transferring the cloned genes into .174 (DR3) cells separately or together. Because normal phenotypes were fully restored in transferent cells expressing DMA plus DMB, other genes in the approximately 1-mb homozygous class II region deletion in .174 (DR3) cells either do not participate in or are dispensable for apparently normal production of intracellular class II/peptide complexes. The properties of DM-deficient EBV-transformed B lymphoblastoid cell lines (LCLs) suggest ways of identifying humans in whom DM deficiency contributes to congenital immunodeficiency and malignancy.
Autologous rat myelin basic protein is a partial agonist that is converted into a full antagonist upon blockade of CD4. Evidence for the integration of efficacious and nonefficacious signals during T cell antigen recognition.
Authors: Mannie M D , Rosser J M , White G A
Published: December 2022

A central question of TCR function is based on the observation that some MHC ligands may bind TCR without stimulating biologic activity. To address the role of CD4 in this mechanism, we studied the interactions of the anti-CD4 mAb W3/25 with an encephalitogenic line of T helper cells. Proliferative responses to guinea pig (GP) myelin basic protein (GPMBP) were mediated by distinct W3/25-sensitive and W3/25-insensitive mechanisms whereas responses to autologous rat (R) MBP (RMBP) were exclusively mediated by W3/25-sensitive pathways. In assays of IL-2 production, RMBP was a partial agonist that stimulated an intermediate level of IL-2 production but antagonized high levels of GPMBP-stimulated IL-2 production to that intermediate level. In the presence of W3/25, RMBP lacked stimulatory activity but instead exhibited inhibitory activity that completely blocked GPMBP-stimulated proliferative responses. The inhibitory mechanism did not involve antigenic competition for MHC glycoproteins, a blockade of mitogenic signaling, or induction of high zone tolerance. Rather, the mechanism involved specific occupancy of TCR with antagonistic MHC ligands derived from the 72-86 region of RMBP. In proliferative assays, GPMBP was approximately 10-fold more active than RMBP. In the presence of W3/25 however, GPMBP-induced agonism and RMBP-induced antagonism exhibited overlapping dose-response curves. RMBP and W3/25 not only fully inhibited GPMBP-stimulated proliferation, this synergistic combination also elicited an extended phase of T cell energy. In conclusion, RMBP-derived MHC ligands occupy TCR to exhibit full efficacy for CD4-dependent signaling pathways while simultaneously lacking efficacy for W3/25-resistant signaling pathways. These data support an "integrative" model of T cell Ag recognition whereby MHC glycoproteins actively guide specific clustering of MHC-ligated TCR to enable quantitative comparisons of efficacious (nonself) and nonefficacious (self) signals by T cells.
Human heart mast cells. Isolation, purification, ultrastructure, and immunologic characterization.
Authors: Patella V , Marinò I , Lampärter B , Arbustini E , Adt M , Marone G
Published: December 2022

We have isolated, partially purified, and characterized the mast cells from human heart tissue. The histamine content of left and right ventricles and septum of hearts obtained from 25 patients undergoing heart transplantation was 5.4 +/- 0.6, 5.3 +/- 0.5, and 5.6 +/- 0.5 micrograms/g of wet tissue, respectively. Ultrastructural study of cardiac mast cells revealed scroll, crystal, and mixed granules, homogeneously dense granules, and lipid bodies in the cytoplasm. A mild collagenase digestion was used to disperse the heart mast cells; the average yield was 3.2 +/- 0.6% (range: 0.8 to 13.6%). The average histamine and tryptase content/heart mast cells was 3.3 +/- 0.2 pg (n = 25) and 24.2 +/- 4.3 micrograms/10(6) cells (n = 11), respectively. Survival of cardiac mast cells after overnight culture was 71.9 +/- 5.4% (n = 23). The purification of human heart mast cells can be brought from less than 0.1 to 12% by a combination of low-speed centrifugation over albumin (2%) solution and Percoll gradient. Viability as shown by trypan blue exclusion was greater than 90%. Heart mast cells released histamine in response to immunologic (anti-IgE, anti-Fc epsilon RI, and C5a) and nonimmunologic stimuli (recombinant human stem cell factor, A23187, and compound 48/80) but did not respond to substance P, FMLP, 12-O-tetradecanoylphorbol-13-acetate, or acetylcholine. There was a linear correlation between the percentage of release caused by anti-IgE and anti-Fc epsilon RI, whereas there was no correlation between the release caused by C5a and anti-IgE-mediated stimuli. Cross-linking with anti-IgE of IgE on heart mast cells induced the release of tryptase (10.1 +/- 2.1 micrograms/10(7) cells; n = 10) and the de novo synthesis of PGD2 (17.3 +/- 4.3 ng/10(6) cells; n = 10) and of leukotriene C4 (19.1 +/- 4.5 ng/10(6) cells; n = 10). There was a linear correlation between the percentage of histamine secretion and tryptase release (r = 0.67; p < 0.001) induced by cross-linking of Fc epsilon RI. similarly, there was a significant correlation between percentage of histamine secretion and PGD2 (r = 0.63; p < 0.001) and LTC4 (r = 0.64; p < 0.001) release. Immunoelectron microscopy demonstrated the presence of chymase in cardiac mast cells. Mast cells isolated from human heart can be a useful model with which to study the role of these cells and their mediators in cardiac anaphylaxis and cardiovascular diseases.
Derivatization with monomethoxypolyethylene glycol of Igs expressing viral epitopes obviates adjuvant requirements.
Authors: Brumeanu T D , Zaghouani H , Elahi E , Daian C , Bona C A
Published: December 2022

Ig molecules expressing within the CDR3 loop viral B or T cell epitopes were derivatized with mPEG 5,000. Pegylated Ig were used to investigate the in vitro and in vivo effect of pegylation on the immunogenicity of viral epitopes expressed in chimeric Ig. Two chimeras were used in this study: Ig-HA carrying a CD4 epitope corresponding to amino acid residues 110-120 of the hemagglutinin (HA) of PR8 influenza A virus and Ig-V3C, a murine-human chimera carrying a consensus B cell epitope from the V3 loop of HIV-1 gp120 protein. Pegylated Ig-HA (Ig-HA-mPEG) with 6 to 8% substituted lysine residues showed in vivo resistance to enzymatic degradation and persisted significantly in blood circulation and lymphoid organs. Moreover, Ig-HA-mPEG was able to activate in vitro HA110-120-specific hybridoma T cells and to prime T cell proliferative response in vivo without requirement for adjuvant. Also, mildly pegylated Ig-V3C (Ig-V3C-mPEG) administered into BALB/c mice in the absence of adjuvant induced specific Ab response to V3C peptide with insignificant response to xenogeneic human Ig determinants.
B cell antigen receptor-mediated apoptosis. Importance of accessory molecules CD19 and CD22, and of surface IgM cross-linking.
Authors: Chaouchi N , Vazquez A , Galanaud P , Leprince C
Published: December 2022

Engagement of the B cell Ag receptor can induce a suicide pathway in various B cell types. Earlier studies showed that anti-IgM mAb treatment triggers apoptotic death in the Burkitt lymphoma-derived cell line, Ramos. We show that two B cell surface molecules, CD19 and CD22, which have been reported to interact either functionally or structurally with the B cell Ag receptor, also stimulate cell suicide when sufficiently aggregated, both in the Ramos and EBV-infected Ramos AW cell lines. In conditions of lower cross-linking, both molecules enhance the apoptotic response induced by a suboptimal dose of anti-IgM mAb in Ramos cells, reinforcing the notion that CD19 and CD22 may be involved in the death pathway and modulate Ag-induced B cell apoptosis. Similar outcomes were obtained with human tonsillar B cells, which enter the death program upon treatment with cross-linked anti-IgM, -CD19, or -CD22 mAbs. These results indicate that Ag-induced B cell suicide may affect mature B cells in the periphery and may be regulated via the interaction of CD19 and/or CD22 with their respective ligand(s). Early tyrosine phosphorylations were analyzed by Western blotting. The biologic outcome of these various treatments--cell survival or death--could not be related to any detectable new tyrosine-phosphorylated substrate, further questioning the biochemical basis of apoptosis signaling.
Stimulation of protein tyrosine phosphorylation by interaction of NK cells with fibronectin via alpha 4 beta 1 and alpha 5 beta 1.
Authors: Gismondi A , Milella M , Palmieri G , Piccoli M , Frati L , Santoni A
Published: December 2022

Recent evidence indicates that integrins do transduce signals that are involved in the regulation of a number of cellular processes. We have shown previously that human NK cells express alpha 4 beta 1 and alpha 5 beta 1 integrins, which mediate their adhesion to fibronectin (FN). Here we investigate whether cross-linking of beta 1 FN receptors on human NK cells stimulates tyrosine kinase activation. Our results indicate that cross-linking of beta 1 integrins on NK cells, either freshly isolated from PBL or generated from 10-day coculture of nonadherent PBMC with an irradiated EVB+ lymphoblastoid B cell line (long-term activated NK cells), altered the tyrosine phosphorylation pattern. In particular, we found stimulation of tyrosine phosphorylation of two proteins migrating with an apparent mass of 105 and 115 kDa, which was not observed after CD16 engagement. Phosphorylation of pp105-115 was already observed at 1 min, raised maximal values at 3 to 5 min, and persisted until 20 min after stimulation. Tyrosine phosphorylation of pp105-115 was also observed upon engagement of alpha 4 beta 1 and alpha 5 beta 1 FN receptors either with mAb directed against the alpha subunits or after NK cell adhesion to FN or its 120- and 40-kDa fragments. Pretreatment of NK cells with the tyrosine kinase inhibitor herbimycin A resulted in marked decrease of phosphorylation stimulated through alpha 4 beta 1 and alpha 5 beta 1 integrins, indicating that ligation of FN receptors on NK cells activates tyrosine kinase(s). Overall our results suggest that beta 1 integrins on NK cells play a major role as signaling molecules in the regulation of NK cell functions.
Complement activation by cross-linked B cell-membrane IgM.
Authors: Ohishi K , Kanoh M , Shinomiya H , Hitsumoto Y , Utsumi S
Published: December 2022

The B cell membrane IgM (mIgM) occurs in a monomeric form incapable of activating C. However, when cross-linked by a polyvalent ligand, mIgM may activate C by assuming a polymeric structure like secreted IgM. This possibility was tested with CR2- lymphoma cells, which did not activate C spontaneously. When CR2-deficient mIgM(lambda)+ Ramos cells were treated with F(ab')2 goat anti-lambda, then exposed to human serum, a marked C3 deposition took place, as examined by the flow cytometry. Similarly, C3 deposition on mIgM(kappa)+, mIgD(kappa)+ P32 cells was induced by F(ab')2 of either anti-kappa or anti-mu. Anti-delta was without effect, but the C3 deposition resulting from anti-kappa was markedly enhanced after mIgD was modulated by anti-delta. The mIgM-cross-linked cells bound C1q, and C3 deposition on these cells was abrogated by depletion of C1q, but not Factor B nor D, from serum. The C1-binding step of the mIgM-mediated C activation was inhibited by monomeric Fab' of polyclonal anti-mu containing a blocking Ab to the hemolytic activity of human IgM Forssman Ab. A large proportion of C3 deposits on mIgM-cross-linked cells was found to be associated with mIgM in the form of C3dg or C3d. These results demonstrate that cross-linked mIgM indeed triggers the classical pathway of C.
src-family tyrosine kinase p55fgr is expressed in murine splenic B cells and is activated in response to antigen receptor cross-linking.
Authors: Wechsler R J , Monroe J G
Published: December 2022

Stimulation of the B cell Ag receptor (BCR) triggers a complex cascade of intracellular signals that lead to activation, proliferation, and differentiation. One of the earliest and most critical events following Ag receptor cross-linking is the activation of receptor-associated tyrosine kinases, which phosphorylate a variety of substrates involved in transducing signals generated through the receptor. Several tyrosine kinases have been shown to be associated with the BCR, including the src family members Lyn, Fyn, Blk, and Lck and the non-src kinase Syk. Here we describe another src family kinase expressed in B cells, p55fgr. Although previous studies have suggested that Fgr expression in the mouse is restricted to cells of the myeloid lineage, we demonstrate that highly purified murine splenic B cells express the kinase as well. We also show that Fgr, like other B cell kinases, is activated in response to cross-linking of the Ag receptor. Unlike these other kinases, however, Fgr does not appear to be physically associated with the receptor, and does not become activated until several minutes after stimulation. Moreover, we find that Fgr is associated with a distinct set of cellular proteins. On the basis of these studies, we hypothesize that Fgr may play a unique role in the BCR-associated signaling pathway.
Distinctive serologic, chemical, and molecular properties of human lambda IV light chains.
Authors: Eulitz M , Ch'ang L Y , Zirkel C , Schell M , Weiss D T , Solomon A
Published: December 2022

Through extensive serologic, chemical, and molecular studies involving monoclonal Ig proteins and B cell-related populations, we provide definitive evidence that the V lambda IV subgroup of human light (L) chains is separate and distinct from V lambda III and all other known V lambda gene families. lambda IV and lambda III L chains were differentiated immunochemically using well-characterized polyclonal and monoclonal anti-V lambda subgroup-specific Abs. Prototypic L chains, originally classified as lambda IV on the basis of distinctive framework region 1 residues, were distinguished serologically from lambda IIIa, lambda IIIb, and lambda IIIc proteins and also from lambda I, lambda II, lambda VI, and lambda VIII L chains. Furthermore, by using anti-lambda IV reagents, we identified eight additional monoclonal V lambda IV-related populations, including three IgM rheumatoid factor-producing cell lines. The percentage of homology among the lambda IV proteins ranged from 83 to 100, vs 53 to 72 when compared with lambda III components. Moreover, lambda IV proteins shared particular subgroup-associated FR and complementarity-determining region residues. At the molecular level, the nucleotide sequences encoding two of the IgM lambda IV rheumatoid factors were identical to that found for the genomic counterpart, as well as to the previously reported IGLV3S1 and Humlv418 germ-line genes. Two other lambda IV cDNAs contained additional non-germ-line-encoded nucleotides at the V-J joint. The single or pauci-gene nature of the V lambda IV family was evidenced from Southern blotting and from the extensive sequence homology among lambda IV components. Our studies have provided further evidence for the prevalence of lambda IV L chains among Ig lambda autoantibodies, thus implying a functional significance for the V lambda IV subgroup.
Methylation status and transcriptional expression of the MHC class I loci in human trophoblast cells from term placenta.
Authors: Guillaudeux T , Rodriguez A M , Girr M , Mallet V , Ellis S A , Sargent I L , Fauchet R , Alsat E , Le Bouteiller P
Published: December 2022

Of the various molecular regulatory mechanisms that may be used by human trophoblast cells to down-regulate expression of HLA class I genes, we chose to investigate the methylation of DNA, generally associated with inhibition of transcription. We analyzed the methylation status of different HLA class I loci in villous and extravillous cytotrophoblast cells and in vitro-differentiated syncytiotrophoblast, purified from human term placenta, as well as in the human trophoblast-derived JAR and JEG-3 cell lines. We then compared methylation status and transcriptional activity. An inverse relationship was established between JAR and JEG-3: HLA-A, -B, and -G are methylated and repressed in JAR, whereas in JEG-3, HLA-A is methylated and repressed but HLA-B and -G are partially methylated and transcribed. HLA-E is unmethylated and transcribed in both cell lines. Apart from HLA-E, which is always unmethylated and transcribed, no such relationship exists for the other class I loci in trophoblast cells. Whereas nonclassical HLA-G and classical HLA-A and -B class I genes are undermethylated in both cytotrophoblast and syncytiotrophoblast, they are clearly transcribed in the former but minimally transcribed in the latter subpopulation. Thus, the down-regulation of class I gene expression in the in vitro-differentiated syncytiotrophoblast is unlikely to be caused by DNA methylation. Furthermore, there is no detectable expression of any class I molecule at the cell surface of either trophoblast cell subpopulation, suggesting a negative control on translation and/or on the secretory pathway to the plasma membrane.
RP105, a novel B cell surface molecule implicated in B cell activation, is a member of the leucine-rich repeat protein family.
Authors: Miyake K , Yamashita Y , Ogata M , Sudo T , Kimoto M
Published: December 2022

The RP105 Ag is a murine B cell surface molecule that transmits an activation signal into B cells following ligation with anti-RP105 mAb. The activation leads to protection of B cells from irradiation- or dexamethasone-induced apoptosis, and to B cell proliferation. A cDNA encoding the RP105 Ag was isolated. An encoded protein is a type I transmembrane protein consisting of 641 amino acids in a mature form. Northern hybridization with a probe specific for the cDNA clone detected a transcript with a size of approximately 3 kb. The transcript was observed in spleen, but not in thymus, kidney, muscle, heart, brain, or liver. Stable transfection of the cDNA clone conferred the expression of the RP105 Ag on a pro-B cell line, which was confirmed by immunofluorescence staining and immunoprecipitation with anti-RP105 mAb. The RP105 molecule possesses 22 tandem repeats of a leucine-rich motif. These repeated motifs are observed in members of the leucine-rich repeat protein family, and have been implicated in protein-protein interactions, such as cell adhesion or receptor-ligand binding. Amino- and carboxyl-flanking regions that are characteristically conserved among members of the family are located on both sides of tandemly repeated leucine-rich motifs in RP105 molecule. These results demonstrate that RP105 is a novel member of the leucine-rich repeat protein family, and the first member that is specifically expressed on B cells.
Crry and CD59 regulate complement in rat glomerular epithelial cells and are inhibited by the nephritogenic antibody of passive Heymann nephritis.
Authors: Quigg R J , Holers V M , Morgan B P , Sneed A E
Published: December 2022

Human glomerular epithelial cells (GEC) contain CD59, decay-accelerating factor, and membrane cofactor protein. Crry is the rodent analogue to the latter two proteins. We have previously shown that the nephritogenic Ab of passive Heymann nephritis, anti-Fx1A, impairs C regulation in rat GEC. Here we examined rat GEC C regulation. 125I-labeled GEC membrane proteins were immunoprecipitated with anti-Crry, anti-CD59, or anti-Fx1A. Crry and CD59 were present in GEC. Anti-Fx1A reacted with both Crry and CD59 from GEC, as well as with purified rCrry and CD59. The alternative C pathway was studied by incubating GEC in rat serum in Mg(++)-EGTA buffer. To inhibit the function of the C regulators, anti-Crry or anti-CD59 Ab were added to GEC. Inhibition of CD59 function alone had no effect on C regulation, whereas inhibition of Crry led to significant cytotoxicity from alternative pathway activation. Under conditions in which Crry was inactive, inhibition of CD59 further enhanced cytotoxicity. When the classical pathway of C was activated by GEC-bound IgG Ab, inhibition of either Crry or CD59 enhanced cytotoxicity, whereas inhibition of both Crry and CD59 together was additive. Therefore, Crry and CD59 are present and functionally active in GEC. Crry restricts C activation via both alternative and classical pathways. When the classical pathway of C is activated, or when Crry function is inhibited, CD59 limits C5b-9-mediated cytotoxicity. Anti-Fx1A binds to both Crry and CD59, which may account for its ability to activate the alternative pathway in vitro, and for its superior nephritogenicity in vivo.
Induction of persistent T cell hyporesponsiveness in vivo by monoclonal antibody to ICAM-1 in patients with rheumatoid arthritis.
Authors: Davis L S , Kavanaugh A F , Nichols L A , Lipsky P E
Published: December 2022

Rheumatoid arthritis patients undergoing treatment with a murine mAb (BIRR1) to ICAM-1 were studied to determine the effects of the treatment on T cell responsiveness assayed in vitro. Previous studies had demonstrated that over the 5-day treatment period, there was a transient increase in circulating T cells that returned to base line 3 days after therapy. The transient lymphocytosis correlated with a loss in delayed-type hypersensitivity reactivity during the time of Ab administration. However, neither the increase in T cell numbers nor the inhibition of delayed-type hypersensitivity responses correlated with the immediate clinical benefit of therapy or the prolonged nature of the response to therapy in some patients. The current studies show that after the return of lymphocyte numbers to pretreatment levels, a decrease in T lymphocyte responses to suboptimal activation signals, including accessory cell-dependent (low dose PHA and soluble anti-CD3 mAb) and accessory cell-independent stimuli (immobilized anti-CD3 mAb), was observed. However, responses to recall Ags were preserved. Depressed T cell responses were not the result of diminished accessory cell function or production of suppressive factors by monocytes, but rather reflected decreased IL-2 production. The duration of T cell hyporesponsiveness was variable in length but lasted up to 5 mo after treatment with anti-ICAM-1 mAb. The induction and persistence of T cell hyporesponsiveness correlated with an improvement in disease activity in treated patients. These studies show that treatment with anti-ICAM-1 mAb can induce T cell hyporesponsiveness that correlates with and may explain sustained therapeutic benefit in patients with rheumatoid arthritis.
Effect of hydrocortisone on spontaneous IgE and IgG4 production in atopic patients.
Authors: Kimata H , Lindley I , Furusho K
Published: December 2022

The effect of hydrocortisone on spontaneous human IgE and IgG4 production in atopic patients was studied. In cultures of mononuclear cells, hydrocortisone (HC) enhanced IgE and IgG4 production without affecting the production of IgM, IgA1, IgA2, IgG1, IgG2, or IgG3. In contrast, testosterone, beta-estradiol, progesterone, aldosterone, gonadotropin, and prolactin were without effect. HC had to be added at the initiation of culture to achieve this enhancing effect. IL-4 and IL-13 also enhanced spontaneous IgE and IgG4 production in mononuclear cells. However, the enhancement of IgE and IgG4 production by HC was not mediated by IL-4 or IL-13, as the addition of anti-IL-4 or anti-IL-13 Ab did not block the HC-induced enhancement. HC also enhanced IgE and IgG4 production in purified B cells, whereas neither IL-4 nor IL-13 did so. IL-5, IL-6, and TNF-alpha also enhanced IgE and IgG4 production in purified B cells; however, neither anti-IL-5, anti-IL-6, nor anti-TNF-alpha mAbs blocked the HC-induced enhancement. Purified surface (s)IgE+ and sIgG4+ B cells spontaneously produced IgE and IgG, respectively, whereas sIgE- and sIgG4- B cells failed to do so. HC enhanced IgE and IgG4 production in sIgE+ and sIgG4+ B cells, respectively, without affecting proliferation, whereas HC failed to induce IgE and IgG4 production in sIgE- and sIgG4- B cells, respectively. These results indicate that HC may selectively stimulate sIgE+ and sIgG4+ B cells.
V7, a novel leukocyte surface protein that participates in T cell activation. II. Molecular cloning and characterization of the V7 gene.
Authors: Ruegg C L , Rivas A , Madani N D , Zeitung J , Laus R , Engleman E G
Published: December 2022

V7 is a cell surface glycoprotein expressed on Ag-activated T cells, monocytes, and granulocytes, as well as subpopulations of T cells and accessory cells present in thymic medulla and tonsil. A mAb directed against V7 inhibits the proliferative response of T cells to allogeneic cells or immobilized anti-CD3 Ab, but not lectin mitogens, suggesting that V7 plays a role in TCR/CD3-mediated T cell activation. We have used the anti-V7 Ab in eukaryotic expression cloning experiments to isolate a cDNA clone containing a 3,340-bp insert that encodes V7 when transiently expressed in simian and murine fibroblastoid cells. DNA sequence analysis revealed a novel 1,021-amino acid open reading frame the structure of which conforms to the category of type I integral membrane proteins. The protein sequence includes a 20-residue putative hydrophobic signal sequence followed by a putative extracellular domain of 934 amino acids, a prototypic hydrophobic transmembrane spanning a domain of 25 residues, and finally a short and highly charged putative cytoplasmic domain of 42 residues. The extracellular domain contains seven pairs of regularly spaced cysteine residues, suggestive of Ig-like domains. On the basis of statistical analysis of the sequences of the putative cysteine loops, all seven of the Ig-like domains belong to the variable, or V-type, category. By using fluorescence in situ hybridization, we have mapped the V7 gene to human chromosome Ip13. Thus, the V7 glycoprotein represents a novel member of the Ig superfamily that is involved in critical intracellular signals essential for immune function.
IL-4 treatment can cure established gastrointestinal nematode infections in immunocompetent and immunodeficient mice.
Authors: Urban J F , Maliszewski C R , Madden K B , Katona I M , Finkelman F D
Published: December 2022

We examined the ability of a long acting formulation of IL-4 (IL-4/anti-IL-4 mAb complexes) to limit established infections of normal and immune deficient mice with two nematode parasites: Heligmosomoides polygyrus and Nippostrongylus brasiliensis. IL-4, at a dose of 5 to 20 micrograms every 3 to 4 days, rapidly decreased egg production and, over a period of 6 to 9 days, terminated infection in H. polygyrus-inoculated BALB/c mice. IL-4 treatment also considerably decreased egg production in H. polygyrus-inoculated CB.17 severe combined immunodeficient (SCID) mice and terminated infection in N. brasiliensis-inoculated SCID mice and anti-CD4 mAb-treated BALB/c mice. IL-4 was less effective at limiting H. polygyrus infection if administrated when parasites were in larval stages than if administered after adult worms had developed. The effects of IL-4 were inhibited completely by an mAb that specifically blocks the mouse IL-4R. These observations demonstrate that IL-4 can limit the fecundity and survival of gastrointestinal nematode parasites through effects on the host that are independent of the specific immune system.
Induction of IL-10 gene expression in human keratinocytes by UVB exposure in vivo and in vitro.
Authors: Enk C D , Sredni D , Blauvelt A , Katz S I
Published: December 2022

Numerous studies have demonstrated that ultraviolet B (UVB) irradiation has profound effects on the skin and systemic immune systems. Because many of the effects of UVB result in suppression of contact sensitivity responses and because IL-10 induces a Th2 rather than a Th1 response, we sought to determine whether UVB irradiation induces IL-10 transcription and subsequent protein secretion by human epidermal cells. Skin of nine volunteers was exposed to UVB or sham irradiation, and epidermal cell suspensions were prepared from suction blister roofs 24 h thereafter. mRNA was extracted using oligo dT-coated magnetic beads, and IL-10 cDNA was amplified with a sensitive RT-PCR technique. We found that IL-10 was constitutively expressed by epidermal cells in 5 of 9 volunteers and that IL-10 message was up-regulated by UVB exposure in all experiments. Since epidermis consists of a heterogeneous cell population with distinct cytokine profiles, we determined whether UVB caused enhanced IL-10 transcription and protein secretion in human keratinocyte cultures. In these experiments, IL-10 was constitutively expressed by keratinocytes and UVB up-regulated IL-10 gene expression in a dose-dependent manner 24 h after in vitro irradiation, coinciding with IL-10 protein secretion into the culture supernatants. Taken together, the findings indicate that UVB irradiation induces IL-10 in human keratinocytes and suggest that keratinocyte-derived IL-10 may be an important component of the immunosuppression that results from UVB irradiation.
IFN-gamma gene expression in pancreatic islet-infiltrating mononuclear cells correlates with autoimmune diabetes in nonobese diabetic mice.
Authors: Rabinovitch A , Suarez-Pinzon W L , Sorensen O , Bleackley R C , Power R F
Published: December 2022

Insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice results from selective destruction of pancreatic islet beta-cells following islet infiltration by mononuclear leukocytes. Cytokines produced by islet-infiltrating mononuclear cells may be involved in beta-cell destruction. Therefore, we analyzed cytokine mRNA expression, by reverse-transcriptase PCR (RT-PCR) assay, in mononuclear leukocytes isolated from pancreatic islets of four groups of mice: diabetes-prone female NOD mice; female NOD mice protected from diabetes by injection of CFA at an early age; male NOD mice with a low diabetes incidence; and female BALB/c mice that do not develop diabetes. We found that mRNA levels of IL-1 beta, IL-2, IL-4, IL-10, and IFN-gamma in mononuclear cells from islets of diabetes-prone female NOD mice increased progressively as these cells infiltrated the islets from age 5 wk to diabetes onset (> 13 wk). However, only IFN-gamma mRNA levels were significantly higher in islet mononuclear cells from 12-wk-old diabetes-prone female NOD mice than from less diabetes-prone NOD mice (CFA-treated females, and males) and normal mice (BALB/c). In contrast, IL-4 mRNA levels were lower in islet mononuclear cells from diabetes-prone female NOD mice than from NOD mice with low diabetes incidence (CFA-treated females and males). Splenic cell mRNA levels of IFN-gamma and IL-4 were not different in the four groups of mice. These results suggest that islet beta-cell destruction and diabetes in female NOD mice are dependent upon intra-islet IFN-gamma production by mononuclear cells, and that CFA-treated female NOD mice and male NOD mice may be protected from diabetes development by down-regulation of IFN-gamma production in the islets.
Involvement of nitric oxide in bone marrow-derived natural suppressor activity. Its dependence on IFN-gamma.
Authors: Angulo I , Rodríguez R , García B , Medina M , Navarro J , Subiza J L
Published: December 2022

Bone marrow (BM)-derived natural suppressor (NS) cells are strong inhibitors of lymphoproliferative responses. In this study we have assessed the involvement of nitric oxide (NO) in BM-derived NS activity, as detected in cocultures of BM and spleen cells stimulated with B cell (LPS) or T cell (Con A) mitogens. The results indicate that NS activity is readily inhibited by NG-monomethyl-L-arginine, a competitive inhibitor of NO synthase, or N-acetylcysteine, a free radical-scavenging thiol compound. High amounts of nitrite, a stable end product of NO, are detected only in supernatants of Con A- or LPS-stimulated spleen cells cocultured with BM cells enriched in NS activity (Fr3 cells). These amounts (15 to 55 microM) are strongly antiproliferative for both Con A and LPS responses, as was established with a nitrite curve made with a NO donor (sodium nitroprusside). Fr3 cells cultured alone release large quantities of NO and express inducible NO synthase (iNOS) mRNA upon LPS stimulation, but require spleen cells in cultures stimulated with Con A. Anti-IFN-gamma-neutralizing Abs blocked both NO production and NS activity, irrespective of the mitogen used; yet, only exogenous IFN-gamma is unable to promote successful NO production by Fr3 cells, but does induce detectable iNOS mRNA expression in these cells. Taken together the results indicate that: 1) NO is the major mediator of BM-derived NS activity; 2) BM cells enriched in NS activity produce large amounts of NO through an IFN-gamma-dependent iNOS induction.
Preferential species-restricted heavy/light chain pairing in rat/mouse quadromas. Implications for a single-step purification of bispecific antibodies.
Authors: Lindhofer H , Mocikat R , Steipe B , Thierfelder S
Published: December 2022

Conventional mouse/mouse or rat/rat hybrid-hybridoma supernatants contain up to 10 different IgG molecules consisting of various combinations of heavy and light chains. Hence, the yield of functional bispecific Ab is low, and purification is often complicated, hampering a general preclinical evaluation of, e.g., bispecific Ab-mediated tumor immunotherapy in animal models. In experiments to overcome this drawback we found that fusion of rat with mouse hybridomas opens the possibility of large scale production of bispecific Ab due to the increased incidence of correctly paired Ab and facilitated purification. In essence, rat/mouse quadroma-derived bispecific Ab have the following advantages: 1) enrichment of functional bispecific Ab because of preferential species-restricted heavy/light chain pairing (observed in four of four rat-mouse quadromas) in contrast to the random pairing in conventional mouse/mouse or rat/rat quadromas, and 2) a possible one-step purification of the quadroma supernatant with protein A. This simple chromatography step does not bind unwanted variants with parental rat/rat heavy chain configuration, and the desired rat/mouse bispecific Ab are retained, which can then easily be separated from parental mouse Ab by sequential pH elution.
Transcriptional regulation of mouse granulocyte-macrophage colony-stimulating factor/IL-3 locus.
Authors: Osborne C S , Vadas M A , Cockerill P N
Published: December 2022

Granulocyte-macrophage (GM)-CSF and IL-3 are hemopoietic growth factors whose genes are closely linked in both humans and mice. In humans, the GM-CSF and IL-3 genes are regulated by a cyclosporin A-inhibitable enhancer located 3 kb upstream of the GM-CSF gene that is inducible by signals that mimic TCR activation. To search for a murine homologue of this enhancer we probed mouse genomic DNA and located a 400-bp element 2 kb upstream of the mouse GM-CSF gene that was 76% homologous with the human GM-CSF enhancer. Like the human GM-CSF enhancer, this element formed a cyclosporin A-inhibitable DNase I-hypersensitive site in the murine T cell line EL4 upon activation with phorbol ester and calcium ionophore. Transient transfection assays showed that this homologue of the human enhancer acted as an inducible enhancer of the thymidine kinase promoter, the mouse IL-3 promoter, and the human GM-CSF promoter. We observed, however, that the mouse GM-CSF promoter was significantly more active than the human GM-CSF promoter and found that it supported a level of activity equivalent to the combination of the human GM-CSF promoter and the human GM-CSF enhancer. Consequently, the activity of mouse GM-CSF promoter was not significantly elevated in the presence of the mouse GM-CSF enhancer. Because the mouse GM-CSF enhancer is considerably less active than its human homologue we suggest that the mouse GM-CSF gene has evolved with less dependence upon the upstream enhancer for its activation.
TGF-beta 2 decreases migration of lymphocytes in vitro and homing of cells into the central nervous system in vivo.
Authors: Fabry Z , Topham D J , Fee D , Herlein J , Carlino J A , Hart M N , Sriram S
Published: December 2022

Migration of leukocytes through an in vitro, cell culture model of the blood-brain barrier (BBB) composed of murine brain microvessel endothelial (En) cells and astrocytes, and in vivo in experimental allergic encephalomyelitis (EAE), was investigated. We have recently shown that the adhesiveness of cultured murine brain microvascular endothelial cells for lymphocytes can be increased significantly by pretreatment with IL-1 beta, TNF-alpha, IFN-gamma, and LPS. In the present study, we investigated the role of TGF-beta 2 on the migration of leukocytes through the BBB. In vitro migration was assessed by measuring the percentage of 51Cr-labeled leukocytes migrating through the En/astrocyte monolayers. The basal level of migration was up-regulated significantly by treating the En/astrocyte monolayers with IL-1 alpha, IFN-gamma, TNF-alpha, and LPS. The ability of these cytokines to modulate migration was dose-dependent. Treatment of En cell/astrocyte monolayers with TGF-beta 2 down-regulated the level of leukocyte migration up-regulated by IL-1 alpha, IFN-gamma, and TNF-alpha in vitro in a dose-dependent manner. TGF-beta 2 also inhibited the migration of lymphocytes into the central nervous system (CNS) in vivo in a dose-dependent fashion. Taken together, these findings strongly suggest that TGF-beta plays an important role in the reduction of lymphocyte infiltration into the CNS in inflammatory demyelinating diseases such as EAE.
Phorbol ester-stimulated adherence of neutrophils to endothelial cells is reduced by adenosine A2 receptor agonists.
Authors: Felsch A , Stöcker K , Borchard U
Published: December 2022

In this study we investigated the effect of adenosine receptor agonists on the adherence of PMA-stimulated human neutrophils to cultured porcine aortic endothelial cells. Additionally, we studied the influence of adenosine analogues on the second messenger cAMP in neutrophils and cultured endothelial cells. In the presence of 10 ng/ml PMA, there was a rapid and stable increase on adherence of neutrophils to the endothelial layer. The adenosine A2 receptor agonists, 2-(p-(2-carboxylethyl)phenethylamino)-5' N-ethylcarboxamido-adenosine (CGS 21680) (0.01 to 1 microM) and 5' N-ethylcarboxamidoadenosine (NECA) (0.01 to 1 microM) decreased the adherence of PMA-stimulated neutrophils maximally by 43% and 34%, respectively. In contrast the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (0.01 to 1 microM) showed a 30% increase in PMA-stimulated adherence of neutrophils to endothelial cells. CGS 21680 (0.01 to 1 microM) and NECA (0.01 to 1 microM) were without detectable effect on the formation of cAMP in neutrophils and endothelial cells; however, in the presence of the phosphodiesterase inhibitor Ro 20-1724 (70 microM), CGS 21680 and NECA maximally increased cAMP level 20-fold and 10-fold, respectively, in neutrophils, and 1.8-fold and 2-fold, respectively, in cultured endothelial cells. However, addition of 70 microM Ro 20-1724 to the adherence assay did not potentiate the inhibitory effects of CGS 21680 and NECA on PMA-stimulated neutrophil adherence. On the other hand, 2-chloro-N6-cyclopentyladenosine (0.01 to 1 microM) did not significantly alter cAMP level in neutrophils and endothelial cells in the presence of 4(3-butoxy-4-methoxyphenyl)methyl)-2-imidazolidinone (Ro 20-1724). Our results indicate that adenosine A2 receptor agonists decrease phorbol ester-stimulated adherence of neutrophils to cultured endothelial cells. This effect is possibly independent of adenosine A2 receptor-mediated stimulation of adenylate cyclase in neutrophils and cultured endothelial cells.
Inhibitors of protein tyrosine kinase suppress TNF-stimulated induction of endothelial cell adhesion molecules.
Authors: Weber C , Negrescu E , Erl W , Pietsch A , Frankenberger M , Ziegler-Heitbrock H W , Siess W , Weber P C
Published: December 2022

We studied the role of tyrosine phosphorylation in the induction of vascular cell adhesion molecule 1 (VCAM-1), endothelial leukocyte adhesion molecule 1 (ELAM-1), and intercellular adhesion molecule 1 (ICAM-1) in HUVEC. Induction of VCAM-1 and ELAM-1 surface expression by TNF was dose-dependently reduced by pretreatment with the protein tyrosine kinase inhibitors herbimycin A (HMA, IC50 300 nM) or genistein (IC50 30 microM). Only genistein attenuated ICAM-1 induction. Genistein or HMA did not affect adhesion molecule up-regulation by PMA. U937 monocyte adhesion to TNF-stimulated HUVEC was markedly inhibited by a combination of anti-VCAM-1 and anti-ELAM-1 mAb, as well as by HMA or genistein, probably due to suppression of VCAM-1 and ELAM-1 up-regulation. HMA appeared to prevent VCAM-1 transcription, since it reduced induction of VCAM-1 mRNA by TNF. Gelshift analysis demonstrated inhibition of TNF-induced nuclear factor-kappa B (NF-kappa B) mobilization by HMA. TNF rapidly enhanced tyrosine phosphorylation of a protein migrating with an apparent molecular mass of 35 kDa. HMA and genistein suppressed constitutive tyrosine phosphorylation of all detectable proteins and prevented TNF-induced tyrosine phosphorylation of the 35 kDa protein with an IC50 and dose range, similar to inhibition of VCAM-1 and ELAM-1 induction. Our data suggest that specific phosphorylation following protein tyrosine kinase activation may be required for NF-kappa B mobilization and induction of VCAM-1 and ELAM-1 by TNF.
Costimulatory requirements of naive CD4+ T cells. ICAM-1 or B7-1 can costimulate naive CD4 T cell activation but both are required for optimum response.
Authors: Dubey C , Croft M , Swain S L
Published: December 2022

Efficient initiation of a CD4 T cell response requires both activation through the TCR and costimulation provided by molecules on APC with counterreceptors on the T cell. We investigated the relative contribution of the ICAM-1:LFA-1 and B7:CD28/CTLA-4 costimulatory pathways in naive T cell activation, using either anti-CD28 Ab or fibroblast cell lines transfected with I-Ek, which express either no costimulatory molecules, ICAM-1 alone, B7-1 alone, or ICAM-1 and B7-1 together. Peptide Ag or immobilized anti-CD3 was used to provide the TCR signal. CD4 T cells from mice transgenic for the V beta 3/V alpha 11 TCR, which recognize a peptide of pigeon cytochrome c complexed to I-Ek, were used as a source of naive T cells. Naive T cells stimulated with Ag or anti-CD3 responded well to high numbers of APC expressing either ICAM-1 alone or B7-1 alone. However, APC expressing both ICAM-1 and B7-1 were much better stimulators of proliferation and IL-2 secretion at low cell numbers, and were far superior inducers of IL-2 at higher numbers, indicating a synergy between the two pathways. Stimulation provided by ICAM-1 could not be solely attributed to adhesive strengthening of other pathways, since costimulation was seen when immobilized anti-CD3 was used and when ICAM-1 only APC were added, indicating that ICAM-1 was in fact acting as a classic costimulatory molecule. Both the magnitude of the response and the amount of costimulation required for response were dependent on the intensity of TCR interaction. These results suggest that an efficient naive T cell response requires both a strong TCR signal and more than one costimulatory signal that will synergize with the TCR signal. This offers an explanation as to why APC such as dendritic cells and activated B cells, which express high levels of multiple costimulatory/adhesion molecules, are the only APC that elicit naive T cell responses.
IL-12 regulation of parasite antigen-driven IgE production in human helminth infections.
Authors: King C L , Hakimi J , Shata M T , Medhat A
Published: December 2022

Recombinant IL-12 inhibits IgE synthesis by IL-4-stimulated lymphocytes from healthy persons and influences the development of Th subset selection involved in Ig isotype selection. Whether endogenous IL-12 production modulates IgE synthesis in patients with elevated serum IgE is unknown. To examine this question we studied the effects of neutralizing anti-IL-12 or recombinant IL-12 on parasite Ag-driven polyclonal IgE production and corresponding IFN-gamma and IL-4 synthesis by PBMC from helminth-infected individuals. The addition of neutralizing anti-IL-12 Ab significantly inhibited parasite Ag-driven IgE production in 11 of 12 individuals (p < 0.001). Recombinant IL-12 (1-10 U/ml) suppressed Ag-driven IgE production in a dose-dependent fashion up to 94% relative to untreated cultures. The effect of endogenous IL-12 on IgE production occurred, in part, by suppressing Ag-induced IL-4 and augmenting IFN-gamma production. IL-12 did not suppress IgE synthesis by purified B cells. An IFN-gamma-independent effect of IL-12 on Ag-driven IgE production was suggested by the ability of human rIL-12 to suppress IgE synthesis in the presence of neutralizing anti-IFN-gamma. This study demonstrates that IL-12 modulates helminth Ag-driven IgE production, in part, by regulating the relative quantities of IFN-gamma and IL-4 generated by Ag-specific lymphocytes.
Functional glucocorticoid receptor expression is required for cAMP-mediated apoptosis in a human leukemic T cell line.
Authors: Kiefer J , Okret S , Jondal M , McConkey D J
Published: December 2022

The involvement of the glucocorticoid receptor (GR) in cAMP-induced apoptosis in a GR-deficient derivative of the CEM.C7 human T-ALL line was investigated. Incubation of the parental CEM.C7 cells with agents that elevate cAMP levels (dibutyryl cAMP and forskolin) resulted in DNA fragmentation characteristic of apoptotic cell death, whereas the GR-deficient ICR.27 cells were insensitive to the cytolytic effects of cAMP. Reconstitution of GR expression by transfection not only restored glucocorticoid sensitivity to the ICR.27 cells, but also promoted sensitivity to induction of apoptosis by cAMP. Thus, cAMP-induced apoptosis in T cells appears to occur via ligand-independent stimulation of at least some aspects of glucocorticoid receptor function.