bluuebunny/crossref_metadata_2025 huggingface dataset:
Differentiation of human lambda I variable regions with a monoclonal antibody to a defined, germ-line-encoded idiotope correlates with V lambda Ia chemical structure.
Authors: Suleyman S , Hosono O , Sioud M , Randen I , Thompson K M , Førre O , Natvig J B
Published: December 2022

This article describes the characterization of a mouse monoclonal anti-idiotypic antibody (1H7B) prepared against a human monoclonal rheumatoid factor (RFSJ2) whose L chain utilized a V lambda I subgroup gene. The mAb 1H7B reacted with 6 of the tested 12 human V lambda I proteins, as well as with a newly produced lambda mAb whose V lambda gene usage has not as yet been determined. Because six of the 1H7B-positive mAb were heterogeneous with respect to both their VH gene utilization and antigenic specificity, and because mAb 1H7B did not react with any of the tested 43 kappa or 12 lambda proteins belonging to various subgroups other than V lambda I, mAb 1H7B appeared to be a V lambda I subgroup-specific reagent. The L chain specificity of mAb 1H7B was confirmed by Western blotting, and the inhibition of RFSJ2 binding to human Fc gamma by 1H7B provided additional evidence for the V region specificity of mAb 1H7B. The 11 sequenced V lambda I proteins used in this study were assigned to sub-subgroups by comparison with the previously published germ-line V lambda Ia, V lambda Ib, and V lambda Ic sequences. The mAb 1H7B only appeared to recognize V lambda Ia proteins as it reacted with five of the seven V lambda Ia, but not with three V lambda Ib or with one V lambda Ic protein. Because mAb 1H7B reacted with at least one V lambda Ia sequence in germ-line configuration, it appeared to be a marker for V lambda Ia sub-subgroup germ-line gene(s). The idiotope recognized by 1H7B was localized to the first framework region by inhibiting its binding to RFSJ2 with a synthetic peptide to the 11-24 amino acid region of V lambda Ia L chains. Comparison of the V lambda 1-24 region sequence of RFSJ2 with those of the two 1H7B-negative V lambda Ia mAb revealed a single amino acid difference at position 17, suggesting that the idiotope recognized by 1H7B encompassed this position.
Leishmania antigens presented by GM-CSF-derived macrophages protect susceptible mice against challenge with Leishmania major.
Authors: Doherty T M , Coffman R L
Published: December 2022

Leishmania major, a causative agent of leishmaniasis, in humans is also capable of infecting mice. Several strains of mice, including the BALB/c strain, are unable to mount appropriate T cell responses to the parasite and develop a fatal, disseminated infection. We present evidence that injection of granulocyte-macrophage-CSF derived bone marrow macrophages (GMM phi), previously incubated with L. major antigens, into BALB/c mice before infection, induced a Th1-dominated response and subsequent healing. Injection of BALB/c mice with GMM phi pulsed with irrelevant Ag, or other macrophages pulsed with L. major Ag, failed to protect against L. major challenge. Protection induced by L. major Ag-bearing GMM phi correlated with the induction of a Th1-like response with the production of high levels of IFN-gamma, delayed-type hypersensitivity reactivity and long-lived resistance to reinfection. GMM phi-T cell interaction, rather than parasite killing by GMM phi, appeared to be a crucial step and there was a strong correlation between ability to function as APC in vitro and induction of protective immunity in vivo. These data suggest that presentation of Ag by a population of L. major Ag-bearing GMM phi can activate Th1 cells in BALB/c mice, leading to a protective immune response to parasite invasion. This implies that the nature of the APC population which presents Ag may influence the response to that Ag in vivo.
Immunosuppressive roles for IL-10 and IL-4 in human infection. In vitro modulation of T cell responses in leprosy.
Authors: Sieling P A , Abrams J S , Yamamura M , Salgame P , Bloom B R , Rea T H , Modlin R L
Published: December 2022

IL-10 and IL-4 have been shown to exert an inhibitory effect on cell-mediated immune responses. Our previous studies of leprosy demonstrated that IL-10 and IL-4 mRNA were preferentially expressed in lesions from lepromatous patients, those immunologically unresponsive individuals that manifest widespread infection. To define more precisely the regulatory roles of these two cytokines in the immune response to infection, we studied in vitro responses to Mycobacterium leprae. M. leprae triggered IL-10 release from PBMC of patients and healthy donors; the predominant source of the IL-10 was found to be monocytes/macrophages. Stimulation of PBMC in the presence of neutralizing anti-IL-10 mAb indicated that endogenous IL-10 production inhibits PBMC proliferation and release of TNF-alpha, GM-CSF, and IFN-gamma. Paradoxically, studies using neutralizing anti-IL-4 mAb indicated that endogenous IL-4 production enhances PBMC proliferative responses most strikingly in lepromatous patients. We found that rIL-4 expanded CD8+ T cells from lepromatous patients in vitro. CD8+ T cells from lepromatous patients have been shown to suppress CD4+ T cell responses, in part by the release of IL-4. Our study indicated that endogenous IL-4 production inhibited IL-10 secretion and, concomitantly, increased TNF-alpha and GM-CSF release. The present data suggest that, on balance, IL-4 and IL-10 contribute to immunosuppression in human infectious disease.
Identification of a major encephalitogenic epitope of proteolipid protein (residues 56-70) for the induction of experimental allergic encephalomyelitis in Biozzi AB/H and nonobese diabetic mice.
Authors: Amor S , Baker D , Groome N , Turk J L
Published: December 2022

Native proteolipid (PLP) and synthetic 15- or 16-mer peptides of the whole PLP molecule, with eight amino acid overlaps, were screened for their ability to induce experimental allergic encephalomyelitis in Biozzi AB/H (H-2dq1) mice. Clinical and histological evidence of experimental allergic encephalomyelitis developed after sensitization with native PLP and with the PLP sequence 56-70 (DYEYLINVIHAFQYV) but not with the other synthetic PLP peptides used. Nonobese diabetic mice that share similar MHC determinants (H-2Anod) with Biozzi AB/H mice, could be induced to develop experimental allergic encephalomyelitis after sensitization with either mouse spinal cord homogenate or the PLP 56-70 peptide. Although the PLP 56-70 overlaps with the encephalitogenic epitope of PLP (residues 43-64) in PL/J (H-2u) mice the Biozzi AB/H mice did not exhibit disease with either PLP 43-64 peptide or the nonapeptide PLP 56-64 that overlaps both the Biozzi AB/H and PL/J encephalitogenic peptides. The identification of a novel major encephalitogenic epitope of PLP for Biozzi AB/H mice, increases the repertoire of encephalitogenic epitopes of PLP and supports a role for PLP as a target Ag in autoimmune demyelinating diseases.
Regulation of IFN-gamma and tumor necrosis factor-alpha expression in vivo. Effects of cycloheximide and cyclosporine in normal and lipopolysaccharide-treated mice.
Authors: Cockfield S M , Ramassar V , Halloran P F
Published: December 2022

Despite accumulating information about cytokine expression in vitro, relatively little is known about the regulation and biologic relevance of these mediators in vivo. In order to study the effects of inhibition of protein synthesis and cyclosporine in vivo, we made use of systemically administered LPS, which induces the expression of a variety of cytokines. The expression of IFN-gamma and TNF-alpha mRNA in normal and LPS-treated mice was examined by Northern blot analysis and amplification using the polymerase chain reaction. IFN-gamma activity was monitored using the biologic end point of MHC induction. TNF-alpha activity in serum was assessed using a L929 cytotoxicity assay. Messenger RNA for IFN-gamma and TNF-alpha could not be reliably detected by Northern analysis in spleens or kidneys of normal mice. After treatment with cycloheximide, a protein synthesis inhibitor, IFN-gamma and TNF-alpha mRNA could be detected in both sites in otherwise normal mice. The level of both IFN-gamma and TNF-alpha mRNA increased after LPS, although the temporal patterns of expression were different. The concurrent administration of cycloheximide led to marked superinduction of both cytokine mRNA levels. Similar effects were seen in T cell-deficient nude mice, suggesting that these responses are T cell independent. Cyclosporin A blocked induction of IFN-gamma in a dose dependent manner, but failed to significantly inhibit TNF-alpha mRNA or protein expression. Thus at least part of the immunosuppressive effect of cyclosporin A in vivo may be caused by its ability to inhibit the expression of certain cytokine genes, as has been found in vitro systems. However, the cellular target for this effect may extend to cell populations other than T cells.
Human IL-10 is produced by both type 1 helper (Th1) and type 2 helper (Th2) T cell clones and inhibits their antigen-specific proliferation and cytokine production.
Authors: Del Prete G , De Carli M , Almerigogna F , Giudizi M G , Biagiotti R , Romagnani S
Published: December 2022

IL-10 gene transcription and IL-10 protein production was assessed in both type 1 (Th1) and type 2 (Th2) CD4+ human T cell clones by polymerase chain reaction and ELISA, respectively. Although Th2 clones apparently showed higher IL-10 mRNA levels, IL-10 mRNA expression was consistently found in Th1 clones, as well. Likewise, measurable IL-10 levels were found in the supernatants of both Th1 and Th2 clones. The effect of human IL-10 (h-IL-10) and viral IL-10 (v-IL-10) on the proliferative response and cytokine production by Th1 and Th2 human clones was also investigated. Addition in culture of h-IL-10 and v-IL-10 significantly reduced the proliferation of both Th1 and Th2 clones in response to the specific Ag and to PHA, but it had no inhibitory effect on the proliferative response of Th1 and Th2 clones to IL-2. h-IL-10 and v-IL-10 also inhibited the Ag-induced production of gamma-interferon (IFN-gamma) by Th1 clones and the production of IL-4 and IL-5 by Th2 clones, whereas they had no effect on the cytokine synthesis by the same clones stimulated with PMA plus anti-CD3 antibody. Preincubation of APC, but not of clonal T blasts, with h-IL-10 resulted in the inhibition of Ag-induced proliferation of both Th1 and Th2 clones, supporting the view that h-IL-10 primarily affects APC. These data demonstrate that, unlike the murine system where IL-10 is a product of Th2 (but not Th1) cells and seems to mainly down-regulate the Th1 response, in the human system, IL-10 is produced by, and down-regulates the function of, both Th1 and Th2 cells.
Targets of B lymphocyte antigen receptor signal transduction include the p21ras GTPase-activating protein (GAP) and two GAP-associated proteins.
Authors: Gold M R , Crowley M T , Martin G A , McCormick F , DeFranco A L
Published: December 2022

Cross-linking membrane Ig (mIg) on B cells stimulates tyrosine phosphorylation of proteins involved in signal transduction including the mIg-associated proteins Ig-alpha and Ig-beta, the tyrosine kinases p53/p56lyn, p55blk, p59fyn, and PTK72, phosphatidylinositol 3-kinase, phospholipase C gamma 1 and gamma 2, and the mitogen-activated protein kinase. We now show that the p21ras GTPase-activating protein (GAP) is also a substrate for mIg-activated tyrosine kinases. p21ras is a key regulator of cell growth and GAP may act as both a regulator of p21ras activity and as a downstream effector of p21ras. We found that mIg cross-linking caused a rapid increase in tyrosine phosphorylation of GAP in the immature B cell line WEHI-231, the mature B cell lines BAL 17 and Daudi, and the IgG-bearing B cell line A20. In fibroblasts, tyrosine kinase activation causes GAP to associate with two other tyrosine-phosphorylated proteins, p62 and p190, which have homologies to an RNA-binding protein and a transcriptional repressor, respectively. Similarly, mlg cross-linking induced the association of GAP with a 62-kDa tyrosine-phosphorylated protein in BAL 17, WEHI-231, and Daudi cells. Anti-Ig treatment also increased the amount of a 190-kDa tyrosine-phosphorylated protein associated with GAP in WEHI-231 and Daudi cells. After separation by SDS-PAGE and transfer to nitrocellulose, the tyrosine-phosphorylated p62 and p190 present in anti-GAP immunoprecipitates from B cells were capable of binding radiolabeled recombinant GAP, as previously reported for the GAP-associated p62 and p190 from fibroblasts. The amount of p62 that could be detected in this way after immunoprecipitation with antiphosphotyrosine antibodies was much greater from anti-IgM-treated BAL 17 cells than from unstimulated BAL 17 cells. This probably reflects anti-Ig-induced tyrosine phosphorylation of p62. In any case, GAP, p62, and/or p190 may be involved in signal transduction by mIg in B cells.
Origin, differentiation, and repertoire selection of CD3+CD4-CD8- thymocytes bearing either alpha beta or gamma delta T cell receptors.
Authors: Suda T , Zlotnik A
Published: December 2022

It has been widely accepted that CD3+CD4-CD8- T cells expressing TCR-alpha beta or TCR-gamma delta (found in the thymus as well as in the periphery) represent lineages distinct from either CD4+CD8- and CD4-CD8+ single-positive T cells expressing TCR-alpha beta. However, the origin, differentiation pathway, and TCR-repertoire selection of CD3+CD4-CD8- T cells remain controversial. We demonstrate that CD3+CD4-CD8- thymocytes can be separated into three subsets based on their expression of heat-stable Ag (HSA) and CD44. Our results further suggest the following: 1) the HSA+ subset represents a pre-selection population, although the HSA- subset is a postselection subset; 2) the high incidence of V beta 8.2 usage among CD3+CD4-CD8- thymocytes is a result of positive selection, rather than a predetermined event at a precursor cell level; 3) the maturation of CD3+CD4-CD8- thymocytes proceeds along the following differentiation pathway: HSA+CD44(-)-->HSA-CD44(-)-->HSA-CD44+. Both TCR-alpha beta +CD4-CD8- and TCR-gamma delta +CD4-CD8- thymocytes show similar differentiation processes; 4) CD3+CD4-CD8-cells directly differentiate from CD25+CD3-CD4-CD8- thymocytes which include precursor cells for both the CD3+CD4-CD8- and the CD4+CD8-/CD4-CD8+ lineages. Taken together, these results suggest that the CD25+CD3-CD4-CD8- stage of thymocyte differentiation represents a branching point for either the CD4+CD8- or CD4-CD8+ single-positive lineages or the CD3+CD4-CD8- lineages.
Molecular cloning and characterization of human eosinophil Charcot-Leyden crystal protein (lysophospholipase). Similarities to IgE binding proteins and the S-type animal lectin superfamily.
Authors: Ackerman S J , Corrette S E , Rosenberg H F , Bennett J C , Mastrianni D M , Nicholson-Weller A , Weller P F , Chin D T , Tenen D G
Published: December 2022

We have isolated and sequenced a 598-bp full length cDNA clone for the human Charcot-Leyden crystal (CLC) protein (eosinophil lysophospholipase), the unique and prominent constituent of human eosinophils and basophils that forms the hexagonal bipyramidal crystals classically observed in tissues and secretions from sites of eosinophil-associated inflammation. A 426-bp open reading frame encoded a 142-amino acid polypeptide with a predicted molecular mass of 16.5 kDa and isoelectric point of 7.28. The deduced amino acid sequence of CLC protein showed 20 to 30% similarity over regions of approximately 100 amino acids with the carboxyl-terminal domains of four IgE-binding proteins, including the 31-kDa human and rat IgE-binding proteins, the 35-kDa mouse carbohydrate binding protein (CBP35), Mac-2, the murine macrophage cell surface protein that is identical to CBP35, and the human homologue of Mac-2. These proteins are members of a superfamily of beta-galactoside binding S-type animal lectins, which includes a group of highly conserved 14-kDa lectins isolated from human lung, heart, placenta, bovine heart, chicken skin, mouse fibroblasts, and the electric organ of the electric eel; CLC protein also showed sequence similarities to these 14-kDa animal lectins, including conservation of 7 of 16 invariant amino acid residues thought to comprise the carbohydrate-binding domain of these proteins, with conservative amino acid changes at others; thus, CLC protein could potentially possess carbohydrate or IgE-binding activities. Northern analyses revealed an approximately 900-bp mRNA species that was present in peripheral blood eosinophils from patients with eosinophilia, basophils from patients with chronic myelogenous leukemia, and in HL-60 cells induced towards eosinophilic differentiation with B cell growth factor-II (IL-5) or granulocytic differentiation with DMSO, but was absent in neutrophils, monocytes, T cells, B cells, or HL-60 cells induced towards monocytic differentiation with vitamin D3. Southern analyses revealed a gene of approximately 5 to 6 kb in length. The cDNA clone and complete amino acid sequence data for CLC protein will facilitate structure-function analyses of its unusual hydrophobic properties, unique propensity for crystallization, lysophospholipase, and potential lectin-like activities.
The analysis of internal image-bearing anti-idiotypic monoclonal antibody in relation to carcinoembryonic antigen.
Authors: Tsujisaki M , Hinoda Y , Tokuchi S , Hanzawa Y , Arimura Y , Masuya J , Kitagawa H , Okochi E , Shimamura T , Hamuro J
Published: December 2022

Five anti-Id mAb (Ab2) were prepared from a BALB/c mouse immunized with anti-carcinoembryonic Ag (CEA) mAb MA208 (Ab1) in a syngeneic system. These anti-Id mAb appear to recognize unique idiotopes at the combining site of mAb MA208, because they were specifically reactive with mAb MA208 and showed the inhibitory activity against the binding of mAb MA208 to CEA. These anti-Id mAb were divided into three groups: group 1 (M7-625), group 2 (M7-413, M7-914), and group 3 (M7-049, M7-418), according to the analysis of anti-anti-Id antibodies (Ab3) induced with each anti-Id mAb (Ab2). Anti-anti-Id mAb M7-625 antisera (Ab3) reacted with purified CEA in binding assay and in Western blot analysis, and competed with Ab1 binding to CEA. Furthermore, the binding of anti-Id mAb M7-625 (Ab2) to mAb MA208 (Ab1) was inhibited with CEA, indicating that Ab2 mimicks the structure of the epitope in CEA which was recognized with Ab1. These serologic findings suggest that anti-Id mAb M7-625 carries the internal image of the Ag. According to the amino acid sequences of CDR 1, 2, and 3 of the mAb M7-625 variable region, there exists a homology of amino acid sequences between CDR2 in the H chain (5 amino acids of 10) and CDR3 in the L chain (3 amino acids of 9) of mAb M7-625 and domain III of CEA (545-554).
Regulation of mouse peritoneal mast cell secretory function by stem cell factor, IL-3 or IL-4.
Authors: Coleman J W , Holliday M R , Kimber I , Zsebo K M , Galli S J
Published: December 2022

We examined whether three cytokines that promote mouse mast cell development, the c-kit ligand stem cell factor (SCF), IL-3, or IL-4, also can directly stimulate or modulate mouse peritoneal mast cell (PMC) mediator release. Challenge of purified PMC with rat rSCF164 at 20 to 100 ng/ml for 30 min induced a modest release of serotonin (5-HT), whereas IL-3 or IL-4 did not directly stimulate 5-HT release. Experiments in which PMC were exposed to each cytokine for 15 min, and then to DNP-HSA Ag or anti-IgE antibody for a further 15 min, showed that SCF, but not IL-3 or IL-4, had an additive effect on the 5-HT release induced by either of the IgE cross-linking agents. In longer term experiments, SCF (0.16 to 500 ng/ml), IL-3 (2.5 to 100 ng/ml), or IL-4 (0.06 to 2.5 ng/ml) was added to peritoneal cell cultures for 48 h, during which the cells were passively sensitized with IgE anti-DNP antibody. Incubation of either unfractionated or highly purified PMC preparations with each of the three cytokines resulted in a concentration-related increase in 5-HT release upon subsequent challenge of the cells with DNP-HSA Ag. However, after pretreatment of peritoneal cells for 48 h with each cytokine, only IL-4 (10 ng/ml) enhanced release of 5-HT induced by calcium ionophore A23187 (0.25 microM); IL-3 (100 ng/ml) had no effect, whereas SCF (100 ng/ml) significantly inhibited ionophore-induced release. Although IL-3 or SCF up-regulate responsiveness to IgE-dependent stimuli, we detected no effect of these cytokines on the binding of [125I]IgE to PMC. This suggests that the enhancing effects of SCF or IL-3 on IgE-dependent 5-HT release did not simply reflect changes in the amount of IgE bound to the cells. In conclusion, we found that SCF, IL-3, or IL-4 each exerted a different spectrum of stimulatory, costimulatory, or regulatory effects on the secretory function of mouse PMC.
Activation of the C4 and C2 components of complement by a proteinase in serum bactericidal factor, Ra reactive factor.
Authors: Ji Y H , Fujita T , Hatsuse H , Takahashi A , Matsushita M , Kawakami M
Published: December 2022

Ra-reactive factor (RaRF) is a C-dependent bactericidal factor that binds specifically to LPS of Ra chemotype strains of Salmonella and kills the bacteria by triggering the C cascade. In the present study, we investigated the components of mouse RaRF that activate C4 and C2. The RaRF bound to LPS-coated E, and activated the C4 on the surface of E, causing the C4 to bind to the cells. Diisopropyl fluorophosphate (DFP) bound to RaRF and inhibited its ability to activate C4 and C2. Cleavage of the alpha-chain of C4 by RaRF generated a polypeptide with a size similar to that of the alpha'-chain of C4b, which is known to be a product of the cleavage of C4 by C1s subcomponent of C1. A fraction with the ability to activate C4 and C2 was separated from RaRF by gel-permeation chromatography in the presence of EDTA and acetonitrile. This fraction contained a DFP-binding polypeptide with an apparent m.w. of 100,000. This polypeptide is not the C1s in mouse C1 because the sizes of this polypeptide and of the fragments produced by its reduction were different from those of DFP-binding proteinases in mouse C1. These results indicate that mouse RaRF contains a C1s-like serine proteinase that is capable of activating C4 and, probably, C2.
CD1 molecule expression on human monocytes induced by granulocyte-macrophage colony-stimulating factor.
Authors: Kasinrerk W , Baumruker T , Majdic O , Knapp W , Stockinger H
Published: December 2022

In this paper we demonstrate that granulocyte-macrophage CSF (GM-CSF) specifically induces the expression of CD1 molecules, CD1a, CD1b and CD1c, upon human monocytes. CD1 molecules appeared upon monocytes on day 1 of stimulation with rGM-CSF, and expression was up-regulated until day 3. Monocytes cultured in the presence of LPS, FMLP, PMA, recombinant granulocyte-CSF, rIFN-gamma, rTNF-alpha, rIL-1 alpha, rIL-1 beta, and rIL-6 remained negative. The induction of CD1 molecules by rGM-CSF was restricted to monocytes, since no such effect was observed upon peripheral blood granulocytes, PBL, and the myeloid cell lines Monomac1, Monomac6, MV4/11, HL60, U937, THP1, KG1, and KG1A. CD1a mRNA was detectable in rGM-CSF-induced monocytes but not in those freshly isolated. SDS-PAGE and immunoblotting analyses of CD1a mAb VIT6 immunoprecipitate from lysate of rGM-CSF-activated monocytes revealed an appropriate CD1a polypeptide band of 49 kDa associated with beta 2-microglobulin. Expression of CD1 molecules on monocytes complements the distribution of these structures on accessory cells, and their specific induction by GM-CSF strengthens the suggestion that CD1 is a family of crucial structures required for interaction between accessory cells and T cells.
Mouse 3T3 fibroblasts induce rat basophilic leukemia (RBL-2H3) cells to acquire responsiveness to compound 48/80.
Authors: Swieter M , Midura R J , Nishikata H , Oliver C , Berenstein E H , Mergenhagen S E , Hascall V C , Siraganian R P
Published: December 2022

2H3 subline of rat basophilic leukemia (RBL-2H3) cells are mast cell analogs that lack responsiveness to nonimmunologic stimuli such as compound 48/80 and substance P. To determine if fibroblasts can influence this responsiveness, RBL-2H3 cells were cocultured with confluent monolayers of mouse 3T3 fibroblasts and assayed for secretagogue-induced histamine release. After 1 wk in coculture, RBL-2H3 cells began to respond to compound 48/80. Responsiveness reached a maximum at 2 wk in coculture and remained at this level for an additional 2 wk. Histamine release was specific, noncytotoxic, dose-dependent, and occurred even in the absence of extracellular Ca2+. No soluble factor from 3T3 cells was found that induced these alterations. Moreover, neither recombinant rat or mouse steel factor, at concentrations up to 250 ng/ml, was able to alter RBL-2H3 cell reactivity to compound 48/80. By 2 wk in coculture, RBL-2H3 cells also became responsive to substance P, although no changes in histamine content, Alcian blue+/safranin- staining or type of serine protease were detected. These results show that 3T3 fibroblasts cause an alteration in the functional repertoire of RBL-2H3 cells and that soluble steel factor cannot duplicate the effect.
Identification of a novel granulocyte chemotactic protein (GCP-2) from human tumor cells. In vitro and in vivo comparison with natural forms of GRO, IP-10, and IL-8.
Authors: Proost P , De Wolf-Peeters C , Conings R , Opdenakker G , Billiau A , Van Damme J
Published: December 2022

Stimulated human osteosarcoma cells (MG-63) were used as a source of granulocyte chemotactic protein (GCP). In addition to the previously isolated GCP-1/IL-8, natural forms of GRO alpha, GRO gamma, and IP-10 were purified and identified by amino acid sequence analysis. Further, a novel GCP, GCP-2, was isolated in its natural form (6 kDa) and was found to be structurally related to the other members of the IL-8 family. GRO alpha, IP-10, and GCP-2 showed heterogeneity, in that several forms of each protein were recovered. These differed in truncation at the amino terminus. Reverse phase HPLC allowed us to separate four such different forms of GCP-2. These tumor-derived factors were compared in granulocyte activation and chemotaxis assays. IL-8 induced neutrophil gelatinase B release at 2 nM, but GRO alpha and GCP-2 showed a 5- to 10-fold lower specific activity. When the migration of granulocytes through polycarbonate micropore membranes was measured, GCP-2 and GRO alpha had a maximal chemotactic index comparable to that of IL-8. The minimal effective dose for GCP-2 and GRO alpha was 3 to 10 nM, whereas the specific activity of IL-8 was at least 10-fold higher. IP-10 was not active in this assay at doses up to 100 nM. Finally, in vivo chemotaxis was measured by using granulocyte recruitment in the rabbit skin model. After intradermal injection of 200 ng/site, GCP-2 provoked a significant granulocyte infiltration, albeit to a lesser extent than did IL-8 and GRO alpha. GCP-2 did not attract monocytes in vivo nor did it induce the cells in vitro to migrate or to produce enzyme. In conclusion, this study reveals a new member of the IL-8 family and shows that these related inflammatory mediators possess different potencies and efficacies towards granulocytes.
T lymphocytes capable of activating endothelial cells in vitro are present in rats with autoimmune diabetes.
Authors: Doukas J , Mordes J P
Published: December 2022

Endothelial activation as evidenced by increased expression of leukocyte adhesion molecules occurs during immune-mediated inflammatory processes. One such process is insulitis, the pancreatic islet inflammation that leads to autoimmune insulin-dependent diabetes mellitus (IDDM). To determine if the induction of IDDM correlates with the presence of T lymphocytes capable of activating endothelial cells (EC), we studied the diabetes resistant BB (DR) rat. These animals become diabetic after in vivo depletion of T cells expressing the RT6 alloantigen. Various populations of purified DR T lymphocytes were cocultured with MHC compatible rat EC. We observed: 1) RT6- T cells from diabetic animals induced maximal endothelial MHC Ag expression. 2) The ability of RT6- T cells to activate EC increased with the duration of in vivo RT6 depletion. It was acquired before the onset of insulitis but subsided after the onset of diabetes. 3) In contrast, neither unsorted total T cells nor in vitro-purified RT6- T cells activated EC. 4) Older DR rats depleted of RT6+ T cells did not become diabetic and their RT6- T cells did not activate EC. 5) T cell IFN-gamma production correlated with the intensity of EC activation. 6) direct T cell-EC contact was required for maximal IFN-gamma production and EC activation. We conclude that RT6- T cells capable of activating EC are generated during the induction of IDDM in DR rats. We hypothesize that such T cell activity may lead to endothelial activation in vivo and contribute to immune-mediated insulitis, beta-cell destruction, and IDDM.
Appearance of granulocyte-macrophage colony-stimulating factor activity at allergen-challenged cutaneous late-phase reaction sites.
Authors: Massey W , Friedman B , Kato M , Cooper P , Kagey-Sobotka A , Lichtenstein L M , Schleimer R P
Published: December 2022

The cytokines IL-3 and granulocyte-macrophage CSF (GM-CSF) activate and/or prime monocytes, basophils, and eosinophils for a number of proinflammatory events in vitro. It was hypothesized that IL-3 and GM-CSF might also participate in the local inflammatory cascades that occur at cutaneous blister sites after Ag challenge in vivo. The M-07e megakaryocytic leukemia cell line, which proliferates in response to IL-3 or GM-CSF, was used to determine whether these cytokines were present in fluids derived after Ag challenge in the cutaneous blister chamber model. Fluids from blister chambers after either Ag (timothy grass, orchard grass, or ragweed) or vehicle control challenge were collected hourly for 12 h from nine patients with allergic rhinitis. Cytokine (IL-3/GM-CSF) activity was modestly elevated at 4 h after Ag challenge compared to control with the median of maximal proliferation 4% (range, 2 to 22%) vs 2% (range, 1 to 14%), respectively (Ag vs control, p < 0.03). Activity peaked at 7 h (Ag = 10%, range 1 to 12%, vs control = 1%, range 1 to 9%, p < 0.02) and then steadily declined. No increase in cytokine activity over control was seen in Ag-challenged nonatopics (n = 5, p = NS), indicating that release did not result from a nonspecific effect of the Ag solution. Neutralization of cytokine bioactivity in pooled late phase reaction (LPR) fluids from h 4 to 12 (n = 5) with anti-IL-3, anti-GM-CSF, or both antisera revealed that the majority of the activity was GM-CSF. To better quantify cytokine levels, pooled LPR fluids prepared from an additional 11 subjects were concentration-dialyzed (10x) and tested for cytokine activity. Pooled fluids from Ag-challenged sites contained a median of 625 pg/ml (range, 30 to 1250 pg/ml) GM-CSF equivalents, whereas those from the vehicle control-challenged sites contained a median 30 pg/ml (range, 30 to 300 pg/ml) GM-CSF equivalents (p < 0.004 Ag vs control groups, n = 11). Concentrated fluid from Ag- and control-challenged sites in two nonatopic subjects contained < 10 pg/ml cytokine activity. To evaluate the IL-3 and GM-CSF activity with a separate technique, ELISA were performed on separately pooled blister fluids from six atopic subjects. Although no IL-3 activity was detected after Ag challenge in these six subjects, all of them demonstrated levels of GM-CSF at Ag-challenged sites comparable to that found in the bioassay.(ABSTRACT TRUNCATED AT 400 WORDS)
Unmodified pancreatic islet allograft rejection results in the preferential expression of certain T cell activation transcripts.
Authors: O'Connell P J , Pacheco-Silva A , Nickerson P W , Muggia R A , Bastos M , Kelley V R , Strom T B
Published: December 2022

Polymerase chain reaction-assisted reverse transcription was used to study the temporal course of rejection in unmodified recipients of murine pancreatic islet cell allografts (DBA/2-->B6AF1) by using syngeneic tissues as controls. The histologic appearance of the grafts was analyzed in parallel. Preproinsulin and constant region of the TCR-beta chain transcripts were studied as markers of graft integrity and infiltrating T cell mass, respectively. The participation of certain cytokines and CTL were analyzed by the detection of IL-2, IFN-gamma, IL-4, and CTL-specific serine protease (granzyme B) transcripts. The time-related disappearance of intragraft preproinsulin transcripts correlated with graft destruction, whereas the intensity of intragraft TCR-beta chain transcript levels correlated with the magnitude of mononuclear leukocyte infiltration in allografts. In unmodified allografts, the magnitude of IL-2 and IFN-gamma intragraft mRNA levels correlated with the intense mononuclear leukocyte infiltrate found on histologic examination at day 8. Only after stable IL-2 gene transcription on day 8 does evidence of graft destruction become apparent, indicating that IL-2 gene activation is closely related to and probably required for expression of alloimmune cytopathic processes. In contrast, IL-4 transcripts were absent or detected in low copy number throughout this time course. Intragraft expression of granzyme B mRNA, a CTL-specific transcript, peaked from day 8 to day 12 in allografts compared with syngeneic grafts or normal tissue. In syngeneic grafts IL-2 and/or IL-4 mRNA was essentially not detected. Although IFN-gamma and granzyme B transcripts were detected in syngeneic grafts, after 4 days the levels of detected transcripts were far less than those noted in allografts. In vivo detection of intragraft IL-2 transcripts in the relative absence of detectable IL-4 transcripts strongly suggests IL-2-dependent immune effector mechanisms are associated with, and perhaps responsible, for allograft rejection. Apparently IL-4-dependent effector mechanisms are not necessary for allograft rejection.
Control of lymphocyte recirculation in man. II. Differential regulation of the cutaneous lymphocyte-associated antigen, a tissue-selective homing receptor for skin-homing T cells.
Authors: Picker L J , Treer J R , Ferguson-Darnell B , Collins P A , Bergstresser P R , Terstappen L W
Published: December 2022

Recent work indicates that a novel homing receptor (HR)/endothelial ligand pair--the cutaneous lymphocyte-associated Ag (CLA) and E-selectin--is involved in targeting a unique skin-associated subpopulation of memory T cells to cutaneous sites of chronic inflammation. To investigate the regulatory mechanisms responsible for the generation of a memory T cell subset with skin-selective homing capability, we used multiparameter flow cytometry to assess the expression of CLA on T cells during initial T cell activation in secondary lymphoid tissues (the virgin to memory transition), and upon reactivation in skin. Our analyses indicate that in vivo induction of CLA (and E-selectin-binding ability) first occurs during the virgin to memory transition, and is regulated in a highly tissue-selective manner. The frequency of CLA expression on CD45RA+/RO+ transitional T cells within skin-associated peripheral lymph nodes was > fivefold higher than within the mucosal microenvironment of the appendix. In keeping with its role as a skin-selective homing receptor, levels of cell-surface CLA on T cells obtained from cutaneous blisters overlying delayed-type hypersensitivity sites were a mean 23-fold higher than on corresponding peripheral blood T cells, including all time points of lesional evolution. However, comparisons of CLA expression on resting vs activated skin blister T cells (HLA-DR - vs +, respectively) indicated that CLA expression, which is already quite high on the resting subset, appeared to be further up-regulated upon activation in skin. The critical involvement of local microenvironments in the regulation of CLA expression was also supported by in vitro studies demonstrating the necessity of specific secondary signals for the induction of CLA glycoproteins on mitogen-activated T cells. CLA up-regulation on both the virgin and memory T cell subsets was dependent on the presence of TGF-beta 1 (or, to a lesser extent, IL-6), but not a wide variety of other cytokines. Thus, the development of the CLA+ memory T cell subset is likely a product of the cumulative experience of those T cells with respect to local microenvironments at previous sites of activation, perhaps involving differential availability of bioactive TGF-beta 1 and/or IL-6 (both cytokines produced in skin). Repeated activation in skin or skin-associated peripheral lymph nodes may act to reinforce CLA expression on T cells functionally-associated with skin, and thus enhance the functional efficiency of these cells by preferentially focusing their recirculation to the skin or related sites.
IFN-alpha induces homotypic adhesion and Leu-13 expression in human B lymphoid cells.
Authors: Evans S S , Collea R P , Leasure J A , Lee D B
Published: December 2022

IFN-alpha influences the recirculation and growth of normal and malignant B lymphocytes, although the mechanisms involved are not currently known. Lymphocyte recirculation is fundamentally dependent on cell-to-cell interactions that are mediated by cell surface adhesion molecules. In this report, we examined the relationship between the effect of IFN-alpha on cell-to-cell adhesion processes and induction of the Leu-13 cell surface protein in established human Daudi B lymphoid cell lines that are either sensitive or resistant to the antiproliferative activity of IFN-alpha. IFN-alpha directly triggered homotypic adhesion of IFN-sensitive Daudi B cells in a time- and dose-dependent manner. In contrast, IFN-alpha had no effect on the cell-to-cell adhesion of IFN-resistant Daudi B cells. The capacity of IFN-alpha to trigger homotypic aggregation correlated directly with the level of induction of the cell surface protein Leu-13 and could be potentiated by anti-Leu-13 mAb. Other cytokines also known to influence the proliferation, differentiation, or recirculation of B lymphocytes such as IFN-gamma, IL-2, IL-4, IL-6, TNF-alpha, and low molecular weight B cell growth factor did not induce either Leu-13 expression or homotypic aggregation of Daudi B cells. The adhesion pathway triggered by the IFN-inducible protein Leu-13 required metabolic energy and an intact cytoskeleton but was not dependent on: 1) new protein synthesis; 2) protein kinase C, protein kinase A, or tyrosine kinase activities; or 3) the function of known adhesion molecules including LFA-1, ICAM-1, CD44, or VLA-4. Taken together, these studies demonstrate a fundamental role for IFN-alpha and the IFN-inducible protein Leu-13 in regulating a novel homotypic adhesion pathway in B lymphocytes, and provide insight into the possible mechanisms by which IFN-alpha regulates biologic processes including recirculation.
Activity of stem cell factor and IL-7 in combination on normal bone marrow B lineage cells.
Authors: Funk P E , Varas A , Witte P L
Published: December 2022

The production of B cells is regulated by soluble and cell contact signals presumably provided by bone marrow stromal cells. Among these is IL-7, a well characterized proliferative stimulus for a subset of pre-B cells. Stem cell factor (SCF), a stromal cell-derived cytokine with broad hemopoietic effects, has been reported to synergize with IL-7 to drive the proliferation and differentiation of B220- bone marrow cells into B220+ B cell precursors in long term culture. A subsequent report has cast doubt on this result by showing that SCF and IL-7 were incapable of producing mu+ pre-B cells after short term culture. Here, using the cell sorter to assure discrete separation of B220+ and B220- cells followed by soft agar culture to prevent interaction with accessory cells, we demonstrate that the combination of SCF and IL-7 does not stimulate the expansion or differentiation of B220- lymphoid precursors but can act synergistically in the clonal proliferation of B220+ cells.
Influence of protein-quaternary structure on antigen processing.
Authors: Rouas N , Christophe S , Housseau F , Bellet D , Guillet J G , Bidart J M
Published: December 2022

T cell recognition of the quaternary structure of human chorionic gonadotropin (hCG), resulting from the association between its alpha (hCG-alpha) and beta (hCG-beta) subunits, was analyzed using hCG-alpha and hCG-beta T cell hybridomas produced in BALB/c mice. First, the fine specificity of these T cell hybridomas was determined, enabling us to divide hCG-alpha-specific T cell hybridomas into two groups. Group I recognized the hCG-alpha(61-81) region, and group II responded to the hCG-alpha(50-70) part of the molecule. Two groups of hCG-beta-specific T cell hybridomas, designated groups III and IV, were analyzed and found to respond to the C- and the N-terminal parts of the hCG-beta(1-22) peptide, respectively. Moreover, we observed that the nature of APC influenced Ag recognition by hCG-beta T cell hybridomas from group IV, but not by other selected T cell hybridomas. We then showed that recognition of the hCG alpha/beta dimer by alpha-specific T cell hybridomas was dramatically reduced compared to both free hCG-alpha and heat-dissociated hCG alpha/beta molecules. In contrast, hCG-beta hybridomas exhibited comparable responses to the free beta subunit and the hCG dimer. Experiments using a dimeric molecule assembled from the alpha-subunit of human follicle-stimulating hormone, which is identical to hCG-alpha, and the beta-subunit of human follicle-stimulating hormone, which is homologous to hCG-beta, confirmed that the three-dimensional structure of the complex rather than the primary structure of the beta-subunit plays a critical role in the processing pathway. Finally, kinetic experiments showed that the presentation of hCG-alpha T cell epitopes differed depending upon whether the alpha-subunit was in its free or combined form. In contrast, the kinetic expression of hCG-beta T cell epitopes appeared to be independent of the quaternary structure of hCG. Thus, conformational alterations resulting from the alpha/beta subunit association mainly influenced processing of the alpha-subunit in its complexed form, rather than processing of the combined beta-subunit. The effect of protein-quaternary structure on T cell recognition may represent a new element in our understanding of the processing and presentation of oligomeric molecules.
Molecular structure of a cross-reactive idiotype on autoantibodies recognizing parenchymal self.
Authors: Karp S L , Kieber-Emmons T , Sun M J , Wolf G , Neilson E G
Published: December 2022

The autoimmune B cell repertoire in anti-tubular basement membrane (alpha TBM) disease is focused on the nephritogenic P1 domain of the 3M-1 target Ag in kidney and normally expresses a disease-modifying cross-reactive Id (IdX). The molecular structure of this Id was determined from a library of rat mAb alpha TBM/alpha 3M-1 by using anchor polymerase chain reactions. Our findings provide the first alignment of V region sequences for rat IgG and reveal that specificity for the P1 domain among alpha 3M-1 antibodies is derived from several recurring germ-line VH genes which have not undergone somatic mutation. The IdX in this repertoire localizes to the H chain on Western blot, and to the CDR3 region as deduced from the cDNA encoding several informative clones. Computer modeling of the Id reveals a conformational structure largely dependent on hydroxyl groups in or near turn position 4 of the H chain CDR3 region. These findings demonstrate that regulatory elements protective of autoimmunity are not encoded in the germ line as IdX, but rather emerge from a recombinatorial diversity engaged by the recognition of parenchymal self.
CD4+ subset expression in murine candidiasis. Th responses correlate directly with genetically determined susceptibility or vaccine-induced resistance.
Authors: Romani L , Mencacci A , Cenci E , Spaccapelo R , Mosci P , Puccetti P , Bistoni F
Published: December 2022

Previous work has shown that in hybrid (BALB/cCr x DBA/2Cr)F1 mice the development of a fatal disseminated disease by systemic infection with virulent Candida albicans is associated with the detection of strong Th2-like responses. However, a predominant Th1-like response and long-lived antifungal protection are induced by vaccinating these mice with live blastospores of attenuated C. albicans strains. When injected into DBA/2Cr mice, one such live vaccine strain was found in the present study to result in a progressive disease characterized by strong Th2 responses. Elevated serum IgG1, IgA, and IgE responses, weak or absent footpad reactions, sustained production in vitro of Th2 (IL-4 and IL-10) but not Th1 (IL-2 and IFN-gamma) cytokines by CD4+ cells, and eosinophilia were all detected in DBA/2 mice after infection with the attenuated vaccine. This was in marked contrast with the development of strong Th1 responses and persistent anticandidal protection in similarly infected, H-2-compatible BALB/cCr mice. Therefore, our data suggest that the type of Th response that predominates in mice after C. albicans infection correlates with genetically determined susceptibility or vaccine-induced resistance. Moreover, the genetic control of this resistance may not be associated with the H-2 complex.
CD28- T lymphocytes. Antigenic and functional properties.
Authors: Azuma M , Phillips J H , Lanier L L
Published: December 2022

A subset of CD8+ alpha beta-TCR/CD3+ T lymphocytes in adult human peripheral blood lacks expression of CD28, a membrane receptor for the B7/BB1 B cell differentiation Ag that is involved in T cell activation. CD28-8+ T cells were not observed in the thymus and were present at only low frequency in cord blood, suggesting that these cells may represent a type of "memory" population. Consistent with this interpretation, CD28-8+ T cells were morphologically large, granular lymphocytes and expressed CD54 (intercellular adhesion molecule-1), CD58 (lymphocyte function-associated Ag-3 (LFA)), and high levels of CD11a (LFA-1), but did not express Ag associated with acute activation (e.g., HLA-DR, CD25, CD69). Freshly isolated CD28-8+ T lymphocytes mediated potent anti-CD3 redirected cytotoxicity against FcR-bearing targets, demonstrating that the CD3/TCR complex is functional and that these cells possess cytolytic activity. However, the anti-CD3-induced proliferative response of CD28-8+ T cells was substantially less than CD28+8+ T cells and this deficiency could not be overcome by addition of exogenous IL-2. A large panel of T cell clones were produced from the CD28+8+ and CD28-8+ T cell populations by single cell sorting using a flow cytometer. CD28 expression was stable on clones derived from CD28+8+ T lymphocytes, whereas CD28 expression was quite variable and apparently reexpressed on some clones generated from the CD28-8+ T cell population. As with the freshly isolated cells, CD28-8+ T cell clones were cytotoxic, but anergic to anti-CD3- induced proliferation and could not be costimulated using B7 or CD58 (LFA-3) murine L cell transfectants. These results indicate that CD28- and CD28+ T cells may play different roles in an immune response.