bluuebunny/crossref_metadata_2025 huggingface dataset:
The B Cell Antigen Receptor Activates the Akt (Protein Kinase B)/Glycogen Synthase Kinase-3 Signaling Pathway Via Phosphatidylinositol 3-Kinase
Authors: Gold Michael R. , Scheid Michael P. , Santos Lorna , Dang-Lawson May , Roth Richard A. , Matsuuchi Linda , Duronio Vincent , Krebs Danielle L.
Published: January 2023

We have previously shown that the B cell Ag receptor (BCR) activates phosphatidylinositol (PI) 3-kinase. We now show that a serine/threonine kinase called Akt or protein kinase B is a downstream target of PI 3-kinase in B cells. Akt has been shown to promote cell survival as well as the transcription and translation of proteins involved in cell cycle progression. Using an Ab that specifically recognizes the activated form of Akt that is phosphorylated on serine 473, we show that BCR engagement activates Akt in a PI 3-kinase-dependent manner. These results were confirmed using in vitro kinase assays. Moreover, BCR ligation also induced phosphorylation of Akt of threonine 308, another modification that is required for activation of Akt. In the DT40 chicken B cell line, phosphorylation of Akt on serine 473 was completely dependent on the Lyn tyrosine kinase, while the Syk tyrosine kinase was required for sustained phosphorylation of Akt. Complementary experiments in BCR-expressing AtT20 endocrine cells confirmed that Src kinases are sufficient for BCR-induced Akt phosphorylation, but that Syk is required for sustained phosphorylation of Akt on both serine 473 and threonine 308. In insulin-responsive cells, Akt phosphorylates and inactivates the serine/threonine kinase glycogen synthase kinase-3 (GSK-3). Inactivation of GSK-3 may promote nuclear accumulation of several transcription factors, including NF-ATc. We found that BCR engagement induced GSK-3 phosphorylation and decreased GSK-3 enzyme activity. Thus, BCR ligation initiates a PI 3-kinase/Akt/GSK-3 signaling pathway.
The HSC73 Molecular Chaperone: Involvement in MHC Class II Antigen Presentation
Authors: Panjwani Naveed , Akbari Omid , Garcia Sylvie , Brazil Melanie , Stockinger Brigitta
Published: January 2023

Heat shock proteins (HSP) are conserved proteins, many of which share the ability for indiscriminate peptide binding and ATPase-coupled peptide release. In this paper, we show that heat shock cognate protein (HSC)73, a constitutively expressed member of the HSP70 family, could be a candidate for chaperone activity within the MHC class II presentation pathway. HSC73 expression in macrophages was shown to overlap with expression of MHC class II; overexpression of HSC73 in stable transfectants of a macrophage line markedly enhanced their presentation of exogenous Ag without affecting presentation of processing independent peptide. Ag from an exogenous source was demonstrated to associate with HSC73 in macrophages, and this association was sensitive to ATP treatment and inhibited by deoxyspergualin, an immunosuppressive agent that has previously been shown to bind specifically to HSC73. Furthermore, deoxyspergualin reduced Ag presentation by macrophages in relation to the amount of HSC73 expressed in these cells. The data are consistent with a potential role for HSC73 in binding and protecting peptides from extensive degradation and/or facilitating the kinetics of peptide transfer to MHC class II molecules.
Regulatory Role of Peritoneal NK1.1+αβ T Cells in IL-12 Production DuringSalmonellaInfection
Authors: Naiki Yoshikazu , Nishimura Hitoshi , Kawano Tetsu , Tanaka Yujiro , Itohara Shigeyoshi , Taniguchi Masaru , Yoshikai Yasunobu
Published: January 2023

NK1.1+αβ T cells emerge in the peritoneal cavity after an i.p. infection with Salmonella choleraesuis in mice. To elucidate the role of the NK1.1+αβ T cells during murine salmonellosis, mice lacking NK1.1+αβ T cells by disruption of TCRβ (TCRβ−/−), β2m (β2m−/−), or Jα281 (Jα281−/−) gene were i.p. inoculated with S. choleraesuis. The peritoneal exudate T cells in wild type (wt) mice on day 3 after infection produced IL-4 upon TCRαβ stimulation, whereas those in TCRβ−/−, β2m−/−, or Jα281−/− mice showed no IL-4 production upon the stimulation, indicating that NK1.1+αβ T cells are the main source of IL-4 production at the early phase of Salmonella infection. Neutralization of endogenous IL-4 by administration of anti-IL-4 mAb to wt mice reduced the number of Salmonella accompanied by increased IL-12 production by macrophages after Salmonella infection. The IL-12 production by the peritoneal macrophages was significantly augmented in mice lacking NK1.1+αβ T cells after Salmonella infection accompanied by increased serum IFN-γ level. The aberrantly increased IL-12 production in infected TCRβ−/− or Jα281−/− mice was suppressed by adoptive transfer of T cells containing NK1.1+αβ T cells but not by the transfer of T cells depleted of NK1.1+αβ T cells or T cells from Jα281−/− mice. Taken together, it is suggested that NK1.1+αβ T cells eliciting IL-4 have a regulatory function in the IL-12 production by macrophages at the early phase of Salmonella infection.
AND-34, a Novel p130Cas-Binding Thymic Stromal Cell Protein Regulated by Adhesion and Inflammatory Cytokines
Authors: Cai Dongpo , Clayton Linda K. , Smolyar Alex , Lerner Adam
Published: January 2023

We have characterized a novel cDNA whose steady state mRNA levels rise in the thymus 2 to 6 h following the induction of CD4+CD8+ thymocyte apoptosis by in vivo cross-linking of CD3ε. This cDNA, AND-34-1, contains an open reading frame (ORF) encoding a protein with an amino-terminal Src homology 2 (SH2) domain and a carboxyl-terminal domain homologous to GDP-exchange factors (GEFs). Northern analysis demonstrates widespread expression of the AND-34 gene. Anti-CD3ε treatment induces up-regulation of the AND-34 mRNA levels in total thymic RNA but not in RNA from purified thymocytes, suggesting that this transcript is derived from a thymic stromal cell population. IL-1 and TNF increase AND-34 transcript levels in thymic cortical reticular, thymic nurse, and fibroblast cell lines. In the thymic cortical reticular cell line, IL-1 and TNF induce a protein of the predicted 93-kDa size reactive with anti-AND-34 peptide antisera. Fifteen minutes of serum stimulation of vanadate-pretreated AND-34-1-transfected NIH3T3 fibroblasts induces tyrosine phosphorylation of AND-34 as well as coprecipitating 95-, 125-, and 130-kDa proteins. One of these tyrosine phosphorylated proteins is identified as p130Cas (Crk-associated substrate), a signaling molecule previously known to bind to a GDP-exchange factor (C3G) and inducibly associate with the focal adhesion complex. Consistent with such an association, AND-34 tyrosine phosphorylation is induced following adherence of trypsinized fibroblasts to fibronectin or poly-l-lysine-coated surfaces.
Human IL-3 Stimulates Endothelial Cell Motility and Promotes In Vivo New Vessel Formation
Authors: Dentelli Patrizia , Sorbo Lorenzo Del , Rosso Arturo , Molinar Annarita , Garbarino Giovanni , Camussi Giovanni , Pegoraro Luigi , Brizzi Maria Felice
Published: January 2023

Angiogenesis is a critical process for growth of new capillary blood vessels from preexisting capillaries and postcapillary venules, both in physiological and pathological conditions. Endothelial cell proliferation is a major component of angiogenesis and it is regulated by several growth factors. It has been previously shown that the human hemopoietic growth factor IL-3 (hIL-3), predominantly produced by activated T lymphocytes, stimulates both endothelial cell proliferation and functional activation. In the present study, we report that hIL-3 is able to induce directional migration and tube formation of HUVEC. The in vivo neoangiogenetic effect of hIL-3 was also demonstrated in a murine model in which Matrigel was used for the delivery of the cytokine, suggesting a role of hIL-3 in sustaining neoangiogenesis. Challenge of HUVEC with hIL-3 lead to the synthesis of platelet-activating factor (PAF), which was found to act as secondary mediator for hIL-3-mediated endothelial cell motility but not for endothelial cell proliferation. Consistent with the role of STAT5 proteins in regulating IL-3-mediated mitogenic signals, we herein report that, in hIL-3-stimulated HUVEC, the recruitment of STAT5A and STAT5B, by the β common (βc) subunit of the IL-3R, was not affected by PAF receptor blockade.
Cyclic Tensile Stress Exerts Antiinflammatory Actions on Chondrocytes by Inhibiting Inducible Nitric Oxide Synthase
Authors: Gassner Robert , Buckley Michael J. , Georgescu Helga , Studer Rebecca , Stefanovich-Racic Maja , Piesco Nicholas P. , Evans Christopher H. , Agarwal Sudha
Published: January 2023

Continuous passive motion manifests therapeutic effects on inflamed articular joints by an as-yet-unknown mechanism. Here, we show that application of cyclic tensile stress (CTS) in vitro abrogates the catabolic effects of IL-1β on chondrocytes. The effects of CTS are mediated by down-regulation of IL-1β-dependent inducible NO production, and are directly attributed to the inhibition of inducible NO synthase (iNOS) mRNA expression and protein synthesis. The inhibition of iNOS induction by CTS is paralleled by abrogation of IL-1β-induced down-regulation of proteoglycan synthesis. Furthermore, CTS inhibits iNOS expression and up-regulates proteoglycan synthesis at concentrations of IL-1β frequently observed in inflamed arthritic joints, suggesting that the actions of CTS may be clinically relevant in suppressing the sustained effects of pathological levels of IL-1β in vivo. These results are the first to demonstrate that mechanisms of the intracellular actions of CTS in IL-1β-activated chondrocytes are mediated through inhibition of a key molecule in the signal transduction pathway that leads to iNOS expression.
Direct Adenoviral Gene Transfer of Viral IL-10 to Rabbit Knees with Experimental Arthritis Ameliorates Disease in Both Injected and Contralateral Control Knees
Authors: Lechman Eric R. , Jaffurs Daniel , Ghivizzani Steven C. , Gambotto Andrea , Kovesdi Imre , Mi Zhibao , Evans Christopher H. , Robbins Paul D.
Published: January 2023

IL-10, a cytokine produced primarily by macrophages, B lymphocytes, and Th2 cells, has both immunostimulatory and immunosuppressive properties. A homologue of IL-10 encoded by EBV, known as viral IL-10 (vIL-10), is also able to suppress the immune response, but may lack some of the immunostimulatory properties of IL-10. To evaluate the potential of vIL-10 to block the progression of rheumatoid arthritis, we have utilized a replication-defective adenovirus vector to deliver the gene encoding vIL-10 to the knee joints of rabbits with Ag-induced arthritis. Intraarticular expression of vIL-10 significantly reduced leukocytosis, cartilage matrix degradation, and levels of endogenous rabbit TNF-α, as well as the degree of synovitis, while maintaining high levels of cartilage matrix synthesis. Interestingly, an antiarthritic effect was also observed in opposing contralateral control knee joints that received only a marker gene. An adenoviral vector carrying the enhanced green fluorescent protein marker gene was used to demonstrate that a morphologically similar subset of cells infected in the injected knee joint are able to traffic to the uninjected contralateral knee joint. Our results suggest that direct, local intraarticular delivery of the vIL-10 gene may have polyarticular therapeutic effects.
A Null Mutation in the Inflammation-Associated S100 Protein S100A8 Causes Early Resorption of the Mouse Embryo
Authors: Passey Robert J. , Williams Elizabeth , Lichanska Agnieszka M. , Wells Christine , Hu Shengping , Geczy Carolyn L. , Little Melissa H. , Hume David A.
Published: January 2023

S100A8 (also known as CP10 or MRP8) was the first member of the S100 family of calcium-binding proteins shown to be chemotactic for myeloid cells. The gene is expressed together with its dimerization partner S100A9 during myelopoiesis in the fetal liver and in adult bone marrow as well as in mature granulocytes. In this paper we show that S100A8 mRNA is expressed without S100A9 mRNA between 6.5 and 8.5 days postcoitum within fetal cells infiltrating the deciduum in the vicinity of the ectoplacental cone. Targeted disruption of the S100A8 gene caused rapid and synchronous embryo resorption by day 9.5 of development in 100% of homozygous null embryos. Until this point there was no evidence of developmental delay in S100A8−/− embryos and decidualization was normal. The results of PCR genotyping around 7.5–8.5 days postcoitum suggest that the null embryos are infiltrated with maternal cells before overt signs of resorption. This work is the first evidence for nonredundant function of a member of the S100 gene family and implies a role in prevention of maternal rejection of the implanting embryo. The S100A8 null provides a new model for studying fetal-maternal interactions during implantation.
Preferential Localization of CD8+ αEβ7+ T Cells Around Acinar Epithelial Cells with Apoptosis in Patients with Sjögren’s Syndrome
Authors: Fujihara Tsutomu , Fujita Hiromi , Tsubota Kazuo , Saito Keiko , Tsuzaka Kensei , Abe Tohru , Takeuchi Tsutomu
Published: January 2023

The T lymphocytes that infiltrate the exocrine glands in Sjögren’s syndrome (SS) play a key role in damaging glandular epithelial cells, but the mechanisms of this damage by T lymphocytes are not fully understood. To determine the cellular basis of this phenomenon, we focused our attention on the T lymphocytes around acinar epithelial cells in SS. We showed that CD8+ but not CD4+ T lymphocytes were located around the acinar epithelial cells and that a majority of these CD8+ T lymphocytes possess an unique integrin, αEβ7 (CD103). The acinar epithelial cell adherent with αEβ7 (CD103)+ CD8+ T lymphocytes was apoptotic. Both the perforin/granzyme B and Fas/Fas ligand pathways were implicated in the process of programmed cell death in lacrimal glands. These results suggested that αEβ7 integrin, by interacting with E-cadherin, mediates the adhesion between CD8+ T lymphocytes and acinar epithelial cells in SS and participates in inducing epithelial cell apoptosis, leading to secretory dysfunction of exocrine glands, a hallmark of SS.
Increased Susceptibility to Immunologically Mediated Glomerulonephritis in IFN-γ-Deficient Mice
Authors: Ring Guido H. , Dai Zhenhua , Saleem Sohail , Baddoura Fady K. , Lakkis Fadi G.
Published: January 2023

It is postulated that IFN-γ confers susceptibility to immunologically mediated tissue injury. To test this hypothesis, we compared the intensity of accelerated anti-glomerular basement membrane glomerulonephritis between wild-type (IFN-γ+/+) and IFN-γ gene knockout (IFN-γ−/−) mice. This disease model is initiated by binding of heterologous (sheep) anti-glomerular basement membrane Abs to the glomeruli of mice preimmunized with sheep IgG. The secondary cellular and humoral immune responses to the planted Ag then lead to albuminuria and glomerular pathology. We found that IFN-γ−/− mice or IFN-γ+/+ mice injected with IFN-γ-neutralizing Ab develop worse albuminuria and glomerular pathology than IFN-γ+/+ mice. The humoral response to sheep IgG (serum mouse anti-sheep IgG titers and intraglomerular mouse IgG deposits) was comparable in the IFN-γ+/+ and IFN-γ−/− groups. In contrast, IFN-γ−/− mice mounted a stronger cellular immune response (cutaneous delayed-type hypersensitivity reaction) to sheep IgG than IFN-γ+/+ mice. These findings provide evidence that endogenous IFN-γ has a protective role in immunologically mediated glomerulonephritis initiated by foreign Ags.
Newborns Develop a Th1-Type Immune Response to Mycobacterium bovis Bacillus Calmette-Guérin Vaccination
Authors: Marchant Arnaud , Goetghebuer Tessa , Ota Martin O. , Wolfe Ingrid , Ceesay Serign J. , De Groote Donat , Corrah Tumani , Bennett Steve , Wheeler Jeremy , Huygen Kris , Aaby Peter , McAdam Keith P. W. J. , Newport Melanie J.
Published: January 2023

Data obtained in animals indicate that neonatal immune responses are biased toward Th2. This could reduce the efficacy of vaccines against viral and mycobacterial diseases. The ability of human newborns to develop a Th1 immune response upon immunization has not been studied. Since the vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) triggers a Th1-type response in adults, we investigated whether it induces a similar response in newborns and whether age at vaccination influences immunogenicity. We found that BCG vaccination at birth induces a memory Th1-type response of similar magnitude to that when given later in life. This study demonstrates that human newborns can be immunized against pathogens controlled by a Th1 immune response.
B7 Costimulation in the Development of Lupus: Autoimmunity Arises Either in the Absence of B7.1/B7.2 or in the Presence of Anti-B7.1/B7.2 Blocking Antibodies
Authors: Liang Bailin , Gee Renelle J. , Kashgarian Michael J. , Sharpe Arlene H. , Mamula Mark J.
Published: January 2023

Costimulatory molecules, termed B7.1 and B7.2, are present on the surfaces of APC and are important for the activation of T lymphocytes specific for both foreign Ags and autoantigens. We have examined the role of B7 costimulation in the MRL-lpr/lpr murine model of human systemic lupus erythematosus. MRL-lpr/lpr mice receiving both anti-B7.1 and anti-B7.2 Abs expressed significantly lower anti-small nuclear ribonucleoprotein particles (snRNP) and anti-dsDNA autoantibodies than did untreated mice. Anti-B7.2 Ab treatment alone inhibited anti-dsDNA autoantibody expression while having no effect on anti-snRNP autoantibody expression. Anti-B7.1 Ab treatment alone did not change the expression of either anti-snRNP or anti-dsDNA autoantibodies. Parallel studies performed in MRL-lpr/lpr mice genetically deficient in either B7.1 or B7.2 expressed autoantibody profiles comparable to those found in wild-type MRL-lpr/lpr mice. However, B7.1-deficient MRL-lpr/lpr mice exhibited distinct and more severe glomerulonephritis while B7.2-deficient MRL-lpr/lpr mice had significantly milder or absent kidney pathology as compared with age-matched wild-type mice. These studies indicate that each B7 costimulatory signal may control unique pathological events in murine systemic lupus erythematosus that may not always be apparent in autoantibody titers alone.
Cutting Edge: A Single, Essential Hydrogen Bond Controls the Stability of Peptide-MHC Class II Complexes
Authors: McFarland Benjamin J. , Beeson Craig , Sant Andrea J.
Published: January 2023

The binding of peptides to MHC class II molecules is mediated in part by a conserved array of intermolecular hydrogen bonds. We have evaluated the consequences of disrupting the hydrogen bond between β-His-81 of the class II molecule and bound peptide. These studies revealed that peptide dissociation rates were accelerated by factors ranging to 200-fold. The sensitivity of a peptide to loss of the hydrogen bond is inversely correlated with the inherent kinetic stability of the peptide-MHC complex. The same relationship has been observed between inherent kinetic stability and the susceptibility to DM. Given that the rate enhancement observed for MHC class II I-Ad protein mutated at position 81 in the β-chain is comparable with DM-catalyzed rates for other class II molecules, we suggest that DM could function by stabilizing a peptide-MHC intermediate in which one or more hydrogen bonds between the peptide and MHC, such as that contributed by the β-His-81 hydrogen bond, are disrupted.
Fibroblast-Secreted Macrophage Colony-Stimulating Factor Is Responsible for Generation of Biphenotypic B/Macrophage Cells from a Subset of Mouse B Lymphocytes
Authors: Borrello Melinda A. , Phipps Richard P.
Published: January 2023

Normal and malignant CD5+ B lymphocytes can develop macrophage-like characteristics. One stimulus of this phenotypic shift is culture of normal mouse splenic B lymphocytes with splenic fibroblasts or their conditioned media. These biphenotypic B/macrophage (B/Mφ) cells simultaneously display macrophage characteristics, such as phagocytosis and F4/80 expression, while retaining B cell features, including expression of surface Ig, CD5, B220, and rearranged Ig genes. The present study investigated the fibroblast-secreted factor that promotes this phenotypic change from B cell to B/Mφ cell. RT-PCR analysis demonstrated that mRNA for M-CSF is produced by splenic fibroblasts. Recombinant M-CSF (CSF-1) could replace fibroblast-conditioned medium to elicit the development and survival of B/Mφ cells from splenic B lymphocytes. In addition, neutralization of fibroblast-secreted M-CSF with specific mAbs abrogated the ability of conditioned supernatants to promote outgrowth of B/Mφ cells. The transition from B lymphocyte to B/Mφ cell was marked by the kinetic appearance of mRNA for the M-CSF receptor, c-fms, at day 3 following culture initiation. These results demonstrate that M-CSF is important in the development and physiology of mouse B/Mφ cells and potentially in the growth of human biphenotypic hematological malignancies. Interestingly, the presence of IFN-γ in splenic B lymphocyte cultures abrogated the effect of fibroblast-conditioned medium or M-CSF on outgrowth of B/Mφ cells. Furthermore, these findings suggest that a Th1 microenvironment favored by typical macrophages is detrimental to the outgrowth of B/Mφ cells.
Kinetic Analysis of Oral Tolerance: Memory Lymphocytes Are Refractory to Oral Tolerance
Authors: Chung Yeonseok , Chang Sun-Young , Kang Chang-Yuil
Published: January 2023

Oral administration of soluble Ag before immunization induces peripheral tolerance and is effective in suppressing animal models of autoimmune diseases. Although tolerance induction in primed animals is more clinically relevant, it is not well studied. Therefore, this study was designed to examine the feeding effects on different phases of the immune response. We observed that feeding a single high dose (250 mg) of OVA to OVA-primed BALB/c mice could induce OVA-specific suppression in the Ab production and T cell proliferation only at the naive and the activation phases of the immune response, whereas multiple high doses (100 mg/feed for 10 days) were effective at the effector phase. OVA-specific IL-4 production in culture supernatant was also suppressed in the tolerized groups. However, when the mice had resting memory lymphocytes, even multiple feeding regimens were not effective in tolerance induction, although multiple low doses (1 mg/feed for 10 days) partially suppressed Ab production. This phenomenon was confirmed by adoptive transfer study. Nevertheless, the reactivated memory response was suppressed partially by multiple high doses. Our findings have an important implication for understanding the mechanism of oral tolerance and for the therapeutic applications of oral tolerance to autoimmune diseases.
Tyrosine Phosphorylation of Crk-Associated Substrate Lymphocyte-Type Is a Critical Element in TCR- and β1 Integrin-Induced T Lymphocyte Migration
Authors: Ohashi Yoshiyuki , Iwata Satoshi , Kamiguchi Kenjiro , Morimoto Chikao
Published: January 2023

Crk-associated substrate (Cas) lymphocyte-type (Cas-L) is a 105-kDa cytoplasmic protein consisting of Src homology-3 domain and multiple YXXP motifs (substrate domain). Our previous studies showed that Cas-L is tyrosine-phosphorylated following the ligation of TCR and β1 integrins in T lymphocytes. Here we show that Cas-L is involved in T cell motility following the ligation of TCR and β1 integrin. Peripheral T lymphocytes showed a marked increase of migration on fibronectin (FN) after the ligation of TCR. In contrast, the migrating Jurkat cells, in which Cas-L was marginally expressed, were less than one-tenth in number on the same condition. Transfection of wild-type Cas-L into Jurkat cells resulted in restoring CD3 plus FN-induced cell migration. Furthermore, following the ligation of β1 integrin alone, the Cas-L transfectants significantly migrated better than the vector control. Mutational analysis of Cas-L revealed that the substrate domain is required for both FN- and CD3-induced tyrosine phosphorylation of Cas-L and cell migration caused by FN alone and CD3 plus FN. In contrast, the Src homology-3 domain is required only for the FN-induced tyrosine phosphorylation of Cas-L and cell migration, but not for CD3-induced tyrosine phosphorylation or CD3 plus FN-induced cell migration. These data strongly suggest that Cas-L is a key molecule in T cell migration induced by the ligation of CD3 and β1 integrins and that tyrosine phosphorylation of Cas-L is essential for T cell migration.
Expression of B7 Molecules in Recipient, Not Donor, Mice Determines the Survival of Cardiac Allografts
Authors: Mandelbrot Didier A. , Furukawa Yutaka , McAdam Alexander J. , Alexander Stephen I. , Libby Peter , Mitchell Richard N. , Sharpe Arlene H.
Published: January 2023

Blockade of the CD28/CTLA4/B7 costimulatory pathway using CTLA4-Ig has great therapeutic potential, and has been shown to prolong allograft survival in a variety of animal models. To gain further insight into the mechanism by which costimulatory blockade prevents allograft rejection, we studied cardiac allograft survival in the complete absence of B7 costimulation using mice lacking B7-1 and B7-2 (B7-1/B7-2−/− mice). To determine the role of B7 on donor vs recipient cells, we used B7-1/B7-2−/− mice as either donors or recipients of allografts. Wild-type (WT) recipients acutely reject fully allogeneic hearts from both WT and B7-1/B7-2−/− mice. In contrast, B7-1/B7-2−/− recipients allow long-term survival of grafts from both WT and B7-1/B7-2−/− mice, with minimal histologic evidence of either acute or chronic rejection in grafts harvested after 90 days. The B7-1/B7-2−/− mice acutely reject B7-1/B7-2−/− allografts if CD28 stimulation is restored by the administration of Ab to CD28 and can mount an alloresponse in mixed lymphocyte reactions. Therefore, B7-1/B7-2−/− mice are capable of generating alloresponses both in vivo and in vitro. Our results demonstrate that in the alloresponse to mouse heterotopic cardiac transplantation, B7 molecules on recipient cells rather than donor cells provide the critical costimulatory signals. The indefinite survival of allografts into B7-1/B7-2−/− recipients further shows that the absence of B7 costimulation alone is sufficient to prevent rejection.
IL-5 Mediates Eosinophilic Rejection of MHC Class II-Disparate Skin Allografts in Mice
Authors: Le Moine Alain , Surquin Murielle , Demoor François Xavier , Noël Jean Christophe , Nahori Marie-Anne , Pretolani Marina , Flamand Véronique , Braun Michel Y. , Goldman Michel , Abramowicz Daniel
Published: January 2023

CD4 T cells play a crucial role in the acute rejection of MHC class II-disparate skin allografts, mainly by Fas/Fas ligand-mediated cytotoxicity. Because recent observations indicate that eosinophils may be found within allografts rejected by CD4 T cells, we evaluated the role played by IL-5, the main eosinophil growth factor, and by eosinophils in the rejection of MHC class II-disparate skin grafts. C57BL/6 mice rapidly rejected MHC class II-disparate bm12 skin grafts. Rejected skins contained a dense, aggressive eosinophil infiltrate. Lymphocytes isolated from lymph nodes draining rejected bm12 skin were primed for IL-5 secretion, and IL-5 mRNA was present within rejected grafts. The IL-5/eosinophil pathway played an effector role in allograft destruction, because the rejection of bm12 skin was significantly delayed in IL-5-deficient mice as compared with wild-type animals. The role of the IL-5/eosinophil pathway was further investigated in MHC class II-disparate donor-recipient strains unable to establish Fas/Fas ligand interactions. Fas ligand-deficient gld/gld mice rejected bm12 skins, and bm12 mice rejected Fas-deficient lpr/lpr C57BL/6 skins. Neutralization of IL-5 prevented acute rejection in both combinations. We conclude that MHC class II-disparate skin allografts trigger an IL-5-dependent infiltration of eosinophils that is sufficient to result in acute graft destruction.
Elucidation and Role of Critical Residues of Immunodominant Peptide Associated with T Cell-Mediated Parasitic Disease
Authors: Hernandez Hector J. , Stadecker Miguel J.
Published: January 2023

Granulomatous inflammation in schistosomiasis is strictly dependent on CD4+ Th lymphocytes sensitized to egg Ags, but its intensity is genetically regulated. C3H and CBA (H-2k) are strains of mice that develop large granulomas; they also strongly respond to the major egg Ag Sm-p40. We now show that the immunodominant epitope recognized by CD4+ Th cells from infected H-2k mice is confined to 13-mer peptide 234–246 (PKSDNQIKAVPAS), which elicits an I-Ak-restricted Th1-type response. Using a panel of alanine-monosubstituted peptides, we identified Asp237 as the main contact residue with I-Ak. On the other hand, three TCR contact residues were essential to stimulate epitope-specific T cell hybridomas: for two hybridomas these were Asn238, Gln239, and Lys241; and for one, Asn238, Lys241, and Pro244. In one instance, alanine substitution for Gln239 generated an antagonist that blocked subsequent stimulation with wild-type peptide. Most importantly, replacement of Asn238, Gln239, or Lys241 caused a profound loss of polyclonal CD4+ T cell reactivity from schistosome-infected mice. This study identifies the critical residues of immunodominant peptide 234–246 involved in the T cell response against the Sm-p40 egg Ag and suggests that suitable altered peptides may be capable of precipitating its down-regulation.
Dendritic Cell (DC)-Based Anti-Infective Strategies: DCs Engineered to Secrete IL-12 Are a Potent Vaccine in a Murine Model of an Intracellular Infection
Authors: Ahuja Seema S. , Reddick Robert L. , Sato Naoko , Montalbo Elleanor , Kostecki Vannessa , Zhao Weiguo , Dolan Matthew J. , Melby Peter C. , Ahuja Sunil K.
Published: January 2023

Infections with intracellular pathogens such as Leishmania donovani and Mycobacterium tuberculosis pose serious health problems worldwide. Effective vaccines for these pathogens are not available. Furthermore, despite optimal therapy, disease progression is often seen with several intracellular infections. For these reasons, we initiated studies to develop novel anti-infective vaccine and treatment strategies that couple the potent Ag-presenting capacity of dendritic cells (DC) with paracrine delivery of potent anti-infective cytokines such as IL-12 to local immune response sites. We tested this strategy in a murine model of visceral leishmaniasis. Adoptive transfer of DCs pulsed ex vivo with soluble L. donovani Ags (SLDA) to naive mice induced the Ag-specific production of IFN-γ, and increased the percentage of activation markers on spleen lymphocytes. SLDA-pulsed DCs engineered by retroviral gene transfer techniques to secrete high levels of biologically active murine IL-12 augmented this immune response further. In several different vaccination and immunotherapy protocols, compared with sham-treated mice, animals receiving SLDA-pulsed DCs either before or following infection had 1–3 log lower parasite burdens, and this protection was associated with a pronounced enhancement in the parasite-specific IFN-γ response. The augmentation of this protection by IL-12-engineered DCs was striking. First, live parasites were not detected in the liver of mice vaccinated with IL-12-transduced, SLDA-pulsed DCs. Second, this parasitological response was associated with a nearly normal liver histology. In contrast, parasites and granulomas were found in mice vaccinated with SLDA-pulsed, nontransduced DCs. Collectively, these studies provide the rationale for the development of potent DC-based immunotherapies.
Human Toll-Like Receptors Mediate Cellular Activation by Mycobacterium tuberculosis
Authors: Means Terry K. , Wang Shuyan , Lien Egil , Yoshimura Atsutoshi , Golenbock Douglas T. , Fenton Matthew J.
Published: January 2023

Recent studies have implicated a family of mammalian Toll-like receptors (TLR) in the activation of macrophages by Gram-negative and Gram-positive bacterial products. We have previously shown that different TLR proteins mediate cellular activation by the distinct CD14 ligands Gram-negative bacterial LPS and mycobacterial glycolipid lipoarabinomannan (LAM). Here we show that viable Mycobacterium tuberculosis bacilli activated both Chinese hamster ovary cells and murine macrophages that overexpressed either TLR2 or TLR4. This contrasted with Gram-positive bacteria and Mycobacterium avium, which activated cells via TLR2 but not TLR4. Both virulent and attenuated strains of M. tuberculosis could activate the cells in a TLR-dependent manner. Neither membrane-bound nor soluble CD14 was required for bacilli to activate cells in a TLR-dependent manner. We also assessed whether LAM was the mycobacterial cell wall component responsible for TLR-dependent cellular activation by M. tuberculosis. We found that TLR2, but not TLR4, could confer responsiveness to LAM isolated from rapidly growing mycobacteria. In contrast, LAM isolated from M. tuberculosis or Mycobacterium bovis bacillus Calmette-Guérin failed to induce TLR-dependent activation. Lastly, both soluble and cell wall-associated mycobacterial factors were capable of mediating activation via distinct TLR proteins. A soluble heat-stable and protease-resistant factor was found to mediate TLR2-dependent activation, whereas a heat-sensitive cell-associated mycobacterial factor mediated TLR4-dependent activation. Together, our data demonstrate that Toll-like receptors can mediate cellular activation by M. tuberculosis via CD14-independent ligands that are distinct from the mycobacterial cell wall glycolipid LAM.
C-C Chemokines in Allergen-Induced Late-Phase Cutaneous Responses in Atopic Subjects: Association of Eotaxin with Early 6-Hour Eosinophils, and of Eotaxin-2 and Monocyte Chemoattractant Protein-4 with the Later 24-Hour Tissue Eosinophilia, and Relationship to Basophils and Other C-C Chemokines (Monocyte Chemoattractant Protein-3 and RANTES)
Authors: Ying Sun , Robinson Douglas S. , Meng Qiu , Barata Luis T. , McEuen Alan R. , Buckley Mark G. , Walls Andrew F. , Askenase Philip W. , Kay A. Barry
Published: January 2023

The relationship of expression of the C-C chemokines eotaxin, eotaxin 2, RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4 to the kinetics of infiltrating eosinophils, basophils, and other inflammatory cells was examined in allergen-induced, late-phase allergic reactions in the skin of human atopic subjects. EG2+ eosinophils peaked at 6 h and correlated significantly with eotaxin mRNA and protein, whereas declining eosinophils at 24 h correlated significantly with eotaxin-2 and MCP-4 mRNA. In contrast, no significant correlations were observed between BB1+ basophil infiltrates, which peaked at 24 h, and expression of eotaxin, eotaxin-2, RANTES, MCP-3, and MCP-4 or elastase+ neutrophils (6-h peak), CD3+ and CD4+ T cells (24 h), and CD68+ macrophages (72 h). Furthermore, 83% of eosinophils, 40% of basophils, and 1% of CD3+ cells expressed the eotaxin receptor CCR3, while eotaxin protein was expressed by 43% of macrophages, 81% of endothelial cells, and 6% of T cells (6%). These data suggest that 1) eotaxin has a role in the early 6-h recruitment of eosinophils, while eotaxin-2 and MCP-4 appear to be involved in later 24-h infiltration of these CCR3+ cells; 2) different mechanisms may guide the early vs late eosinophilia; and 3) other chemokines and receptors may be involved in basophil accumulation of allergic tissue reactions in human skin.
Dendritic cells generated in the presence of PGE2 promote IL-17 production in activated CD4+ T cells (100.10)
Authors: Khayrullina Tanzilya , Ganea Doina
Published: January 2023

Dendritic cells (DCs), the most potent antigen presenting cells, play an essential role both in the induction of the immune response and in maintaining tolerance to self antigens. The DCs phenotype and function depend on various factors, including prostaglandins. PGE2 plays an important role in inflammation and autoimmunity through changes in the proinflammatory cytokine profile. We reported previously that PGE2 induces IL-23 in immature DCs. The proinflammatory cytokine IL-23 is associated with autoimmune disorders such as EAE, RA and IBD, primarily through its effect on the proliferation/survival of Th17 effector cells. The purpose of this study was to determine whether generation of DC in the presence of PGE2 affects their response to inflammatory conditions, particularly in terms of the IL-12p70/IL-23 balance. DC were generated in the presence or absence of PGE2 (DCp and DC, respectively), stimulated with LPS, and characterized in terms of cytokine profile. DCp secreted higher levels of the proinflammatory cytokines IL-6, IL-1b and TNFa. In addition, PGE2 shifted the IL-23/IL-12 balance in favor of IL-23. The changes at protein level reflected changes in transcription, with PGE2 upregulating p19 (the IL-23 specific subunit) and downregulating p35 (the IL-12 specific subunit) expression. In addition, DC generated in the presence of PGE2 preferentially induced IL-17 production in activated CD4+ T cells, and administration of DCp to PLP-immunized SJL/J mice exacerbated EAE.

Grant support: 2RO1 AI0562306 (DG) and Dissertation Fellowship from Rutgers University (TK).
Targeting different isoforms of HMGB1 leads to different beneficial effects in preclinical models of sepsis and inflammatory arthritis (100.6)
Authors: An Ling-Ling , Xu Linda , Barber Arnita Sunhe , Mao Shenlan , Mao Su-Yau , LaRosa Gregory J. , Gao Changshou , McCarthy Michael P. , Wu Herren , Kiener Peter A. , Coyle Anthony J. , Audoly Laurent P.
Published: January 2023

HMGB1 is a multifunctional protein that exerts diverse proinflammatory effects when released into the extracellular milieu. Because several HMGB1 isoforms may be generated depending on the activation state of a cell, these isoforms may play distinct roles in different inflammatory disorders. To examine this, we generated a panel of fully human anti-HMGB1 monoclonal antibodies using a phage display approach. The two most potent mAbs, IA-4 and IA-6, displayed comparable binding affinity to HMGB1 (KD=30 and 39nM) and blockade of HMGB1/RAGE binding (IC50=0.9 and 1.2nM). However, only IA-4 but not IA-6 could detect HMGB1 actively secreted from LPS-treated cells. IA-4 and IA-6 were then tested in rodent models of inflammatory arthritis (rat AIA) and sepsis (mouse CLP). IA-4 reduced paw swelling in the rat AIA model (32% inhibition @ 10mg/kg, p<0.05 vs. isotype control, n=32) while IA-6 had little effect. In contrast, only IA-6 but not IA-4 prolonged survival in the mouse CLP model (@ 8mg/kg , p<0.05 vs. isotype control, n=72). IA-4 and IA-6 displayed similar pharmacokinetics and recognized recombinant rodent HMGB1 with equal affinity. In summary, these results suggest that different isoforms of HMGB1 may have distinct in vivo roles and that HMGB1 mAbs targeting these isoforms may provide tailored therapeutics.
Susceptibility of PIR-B deficient mice to Salmonella infection (101.1)
Authors: Torii Ikuko , Oka Satoshi , Hotomi Muneki , Benjamin William H. , Kearney John F. , Takai Toshiyuki , Briles David E. , Kubagawa Hiromi
Published: January 2023

Paired Ig-like receptors of activating (PIR-A) and inhibitory (PIR-B) isoforms are expressed by many blood cell types including B cells, dendritic cells, monocyte/macrophages, granulocytes, mast cells and megakaryocyte/platelets. To determine the role of PIR in infection, various doses of attenuated S. typhimurium (WB335) were inoculated i.v. into PIR-B−/− or wild-type mice. When given high doses of bacteria (>105 CFU), both types of mice died within 3 – 5 days post-infection. However, when given small doses (103– 104 CFU), all PIR-B−/− mice died within 1 – 3 weeks post-infection, whereas many control mice survived. The bacterial loads in liver, spleen and lung tissues during the 1st week of infection were much higher in PIR-B−/− mice than in control mice. PIR-B−/−, but not control, mice developed sepsis around day 7 post-infection. There were no significant differences in serum levels of cytokines (TNFα, IL1), liver enzymes (ALT, AST, ALK) and BUN or in splenic lymphoid and myeloid subsets between PIR-B−/− and control mice. Ingestion of bacteria by phagocytic cells ex vivo was also the same; however PIR-B−/− phagocytic cells exhibited tighter adherence to plates and released more nitrite than control phagocytic cells. These results suggest that PIR-B plays a role in regulating phagocytic cell responses against bacterial infection.

(Supported by NIH/NIAID grant AI042127)
Regulation of inflammatory disease by the fatty acid binding proteins A-FABP and E-FABP (101.12)
Authors: Reynolds Joseph Michael , Liu Qioahong , Brittingham Katherine C , Stout Robert D , Suttles Jill
Published: January 2023

The fatty acid binding protein (FABP) family is a group of highly homologous intracellular lipid receptors with the ability to bind and transport various hydrophobic compounds within the cell. Adipocyte and epidermal FABPs, A-FABP and E-FABP, respectively, are expressed in macrophages and we have previously reported that A-FABP-deficient macrophages display reduced inflammatory function. Subsequently, we have found that deficiency of E-FABP likewise results in reduced inflammatory potential of macrophages. We hypothesized that FABP function may contribute to the pathogenesis of autoimmune inflammatory diseases. Since a prominent role of macrophages in the pathogenesis of human multiple sclerosis (MS) has been well-documented, we evaluated the role of A- and E-FABPs in the development of experimental autoimmune encephalomyelitis, (EAE), the murine model of human MS. FABP-deficient mice exhibit reduced clinical symptoms of EAE, reduced proinflammatory cytokine expression in CNS tissue, and reduced induction of proinflammatory Th1 and Th17 responses. In addition we demonstrate that dendritic cells express high levels of both A- and E-FABPs, and that antigen presentation by FABP-deficient DC and macrophages does not promote proinflammatory T cell responses. Our studies demonstrate that A- and E-FABP act as positive regulators of inflammatory responses, impacting both innate and adaptive immunity.
Increased nitric oxide production by peripheral blood monocytes and selective elevation of serum IgE in patients with systemic lupus erythematosus (SLE). (101.13)
Authors: Khalifa Dalia , Chimezie Patricia , Ahn Soojin , Finkielstein Anna , Dimitrios George , Lee Susan , Ginzler Ellen , Durkin Helen G , Nowakowski Maja
Published: January 2023

Prospective studies of patients with SLE have reported a prevalence of clinically apparent coronary artery disease (CAD) of up to 20%, a marked increase compared with the estimated CAD prevalence rate of 3.5% among healthy females 20–35 years (AHA 1997 Heart and Stroke Statistical Update). Nitric oxide is an important mediator of inflammation associated with vascular damage. We measured nitric oxide production by cultures of peripheral blood monocytes (N= 26) from patients with SLE and determined serum immunoglobulin concentrations (N=19). Elevated immunoglobulin concentrations were observed in sera from 8 patients (42%), and 4 individuals had a selective increase in IgE (>100 IU/ml), with other immunoglobulin isotypes in the normal range. Nitric oxide (NO) production by adherent monocyte cultures from patients with SLE was determined on day 7 using a modified colorimetric Griess reaction to measure nitrite, the stable NO metabolite. The results showed activation in 31% of the patients, with nitrite concentrations exceeding the baseline of 2.8μM more than threefold, reaching 8.8 μM. The mechanism of this in vivo activation of blood monocytes among some patients with SLE remains to be established. Activated monocyte-derived nitric oxide may contribute to the development of atherosclerosis and cardiovascular complications seen in many patients with SLE.
Modulation of apoptosis and the production of reactive oxygen species in human neutrophils with epigallocatechin-3-gallate (EGCG) (101.6)
Authors: Babcock George F , Osterburg Andrew R , Schwemberger Sandy , Hyon Suong-Hyu , Koizumi Takeo
Published: January 2023

Our laboratory has demonstrated that following thermal injury, neutrophils (PMNs) display altered immune responsiveness. Decreased apoptosis was associated with the expression of Heat Shock Protein 72 (HSP72), while increased apoptosis is associated with the production of reactive oxygen species. EGCG is an anti-oxidant found in green tea which possesses immunomodulatory properties. In this study, we investigated the effects of EGCG on PMN apoptosis and the production of reaction oxygen species. Human PMNs (HSP 72 + or −) were treated with tumor necrosis factor (TNF[alpha]) or phorbol myristate acetate (PMA), with or without EGCG. PMNs were analyzed for apoptosis, the production of superoxide anion (O2−), and hydrogen peroxide (H2O2). PMNs treated with EGCG had a significant reduction in the percentage of cells undergoing apoptosis with either agonist (by 78 and 53%, respectively). While PMNs expressing HSP72 had decreased apoptosis compared to controls, treatment with EGCG further decreased the percentage of HSP 72+ PMNs undergoing apoptosis. Treatment with EGCG significantly reduced the production of H2O2 and O2− by PMNs treated with TNF[alpha]or PMA. The data suggests that EGCG significantly reduces apoptosis, H2O2 and O2− in activated PMNs. Due to its low toxicity, ECGC may be an ideal agent to modulate the inflammatory response associated with burn injury.

Supported in part by a grant from Shriners of North America
Recipient Conditioning with Mesenchymal Stem Cells and Interleukin-10 Prolonged Cardiac Allograft Survival (102.1)
Authors: Salgar Shashikumar , Li S , Hernandez M , Wei Y , Mateu D , Vazquez-Padron R , Mathew J , Pham S
Published: January 2023

Mesenchymal Stem Cells (MSC) and viral interleukin-10 (vIL-10) have immunosuppressive properties. In this study, we tested their ability to prevent cardiac allograft rejection. Bone marrow derived MSC from Lewis rat were expanded ex vivo and transduced with rvIL-10-retrovirus. Autologous MSC or vIL-10 transduced MSC were injected (~25 x 106; i.v.) into irradiated (4 Gy) rat (RT1.Al). Six weeks later heterotopic heart (RT1.An) transplantation (Tx) was performed. MSC therapy prolonged (P<0.05) cardiac allograft survival (14±1 days; n= 4) compared to untreated controls (7±1 days; n=4). Also, vIL-10-MSC treatment prolonged (P<0.01) graft survival (38±1 days; n=6) compared to empty vector treated group (15±1 days; n=4). In vIL-10-MSC preconditioned animals that received donor bone marrow after heart Tx, graft survival was 22±2 days (n=8). Intragraft expression of co-stimulatory molecule (CD80) and cytokines (IL-2, IFN-γ) as determined by RT-PCR was lower (P<0.03) in vIL-10-MSC treated grafts compared to untreated control grafts. Ex vivo expanded MSC were CD34−, CD45+(5 %), CD29+(90%), CD80 (0%), CD 86 (8%), CD90+(93%), MHC Class I+(23–57%), and MHC Class II- as determined by FACS. Both vIL-10 and empty vector engineered MSC expressed CD29 (>95%) but not other molecules. The vIL-10-MSC produced ~ 6 ng/ml of vIL-10 ex vivo. vIL-10-MSC addition in MLR cultures inhibited lymphoproliferative response (P<0.05). TGF-β expression in stimulated (TNF-α or IL1-β) and unstimulated MSC was 46 – 65% of β-actin. IL-2, IL-4, IL-10, IFN-γ, and TNF-α expression was negligible in MSC (0–7% of β-actin). VEGF and HGF mRNA expression in MSC was 58% and 5% of β-actin, respectively. TgGFP+ MSC were demonstrable in various tissues for >28 days. Both MSC and vIL-10 conditioning to promote allograft survival seems promising.