bluuebunny/crossref_metadata_2025 huggingface dataset:
Fas-mediated liver damage in MRL hemopoietic chimeras undergoing lpr-mediated graft-versus-host disease.
Authors: Bobé P , Bonardelle D , Reynès M , Godeau F , Mahiou J , Joulin V , Kiger N
Published: January 2023

Fas is an apoptosis-signaling receptor that triggers cell death upon binding to its ligand (FasL). Autoimmune-prone MRL/lpr mice, characterized by a spontaneous mutation of Fas, exhibit a defect in the activation-induced cell death of mature T cells through a Fas-mediated pathway. As a consequence of this defect, activated T cells accumulating in this strain overexpress the FasL and can therefore mediate in vitro Fas-dependent cytotoxicity. To determine whether hepatic injury could be the result of an interaction between T lymphocytes bearing FasL and Fas-expressing liver cells, the livers of lethally irradiated MRL+/+ recipients reconstituted with MRL/lpr lymphoid cells were studied. After transfer of MRL/lpr spleen cells, livers were infiltrated by polyclonal CD8+ T lymphocytes of lpr origin with a peak on day 21 postgrafting. These donor-derived intrahepatic lymphocytes overexpressed the FasL and exerted in vitro Fas-mediated cytotoxicity against Fas+ thymocytes, which was specifically inhibited by soluble recombinant Fas in a dose-dependent manner. These intrahepatic lymphocytes induced apoptosis in vitro, irrespective of MHC restriction, in Fas-expressing primary cultured hepatocytes. Histologic examination of the liver revealed severe endothelialitis as well as periportal and intralobular infiltrations of activated lymphocytes with apoptotic hepatocytes in their vicinity. Simultaneously, liver damage was ascertained by elevated serum transaminase levels. These observations support the notion that an Ag-independent mechanism involving FasL may play a role in certain liver pathologies.
B cell antigen receptor-evoked calcium influx is enhanced in CD22-deficient B cell lines.
Authors: Nadler M J , McLean P A , Neel B G , Wortis H H
Published: January 2023

CD22 is a B cell membrane glycoprotein that, upon Ag receptor engagement, becomes rapidly tyrosyl phosphorylated and associates with several signaling molecules including Lyn, Syk, PLCgamma1, and the protein-tyrosine phosphatase, SHP-1. Two allelic forms of murine CD22 exist: CD22.1 is expressed in strains such as NZB and DBA/2, whereas CD22.2 is expressed in BALB/c and most other strains. WEHI-231 cells, which derive from a (BALB/c x NZB)F1 mouse, express one copy of each allele. Previous studies have proposed both positive and negative functions for CD22. We explored the role of CD22 in surface IgM Ag receptor signal transduction by examining signaling in three clonally independent WEHI-231 variants that have lost expression of the CD22.2 allele. This experimental design allowed us to assess the signaling functions of CD22 independent of its developmental role. These variants, which exhibit a 50% reduction of total surface CD22, are hyper-responsive to Ag receptor stimulation: several cellular proteins are hyperphosphorylated on tyrosyl residues and surface IgM-mediated calcium flux is markedly increased. Interestingly, the increased calcium response observed in CD22-deficient cells is due largely to enhanced calcium influx. Reconstitution of CD22 expression reduces these changes. The SHP-1/CD22 association is reduced in CD22-deficient cell lines and is restored by re-expression of CD22. Our results demonstrate that CD22 is a cell autonomous negative regulator of B cell Ag receptor signaling, and suggest that it regulates calcium entry via a mechanism downstream from or independent of calcium release from intracellular stores.
Regulation of B cell functions by C3a and C3a(desArg): suppression of TNF-alpha, IL-6, and the polyclonal immune response.
Authors: Fischer W H , Hugli T E
Published: January 2023

Regulation of the humoral immune response by fragments of complement component C3 has been shown to play an important role in host defense. In this study, we investigated expression of the C3a receptor (C3aR) by human B cells and the effects of C3a and C3a(desArg) on IgG, TNF-alpha, and IL-6 production in Staphylococcus aureus Cowan strain I (SAC)/IL-2-activated B cells. Here we report that tonsil-derived human B cells express a C3aR that is indistinguishable at both the protein (by flow cytometry) and mRNA (by reverse transcription-PCR) levels from that on human neutrophils or on C3aR-transfected mouse L cells. Incubation of SAC/IL-2-activated B cells with C3a or C3a(desArg) resulted in a dose-dependent suppression of the polyclonal immune response. Under these same experimental conditions, IL-6 and TNF-alpha release was suppressed in a dose-dependent manner. Kinetic studies with SAC/IL-2-activated B cells revealed that C3a must be present at initiation of the culture to exert its suppressive effect on IgG and IL-6 production. These results demonstrate that tonsil-derived B cells express the C3aR and that C3a as well as C3a(desArg) have a direct immunomodulatory effect on these cells.
Rigidification of the alpha2 helix of an MHC class I molecule by a valine to proline mutation in position 165 does not prevent peptide-specific antigen presentation.
Authors: Plaksin D , Polakova K , Mage M G , Margulies D H
Published: January 2023

Although classical MHC class I glycoproteins bind peptide Ags for display at the cell surface, some MHC class I-related molecules such as the neonatal Fc receptor (FcRn) execute their function without binding peptide ligands. The three-dimensional structure of the FcRn suggested that a substitution of the conserved valine at position 165 of the alpha2 helix by proline contributed to a kink in the position of this helix relative to the alpha1 helix, and resulted in closing of the potential peptide-binding cleft. To test the contribution of proline 165 to the occlusion of the cleft and the binding of potential antigenic peptides, we introduced this mutation into the classical murine MHC class I molecule, H-2Dd, and characterized the ability of such a mutant to present peptide Ags to either a peptide-specific, H-2Dd-restricted T cell hybridoma (B4.2.3), or an allospecific, peptide-dependent, T cell hybridoma (3DT52.5.8). We show that the V165P mutation, expressed at the cell surface either in H-2Dd or in a single chain membrane version of H-2Dd, fails to eliminate recognition of the peptide/MHC complexes by two different T cells. Evaluation of a panel of synthetic substituted peptides suggests that subtle differences in the fine specificity of presentation can be discerned. Thus, the proline substitution at position 165 of FcRn and some other class I-like molecules is not the sole cause of the lack of peptide presentation.
T cell receptor-mediated signaling is defective in T cells obtained from patients with primary intracranial tumors.
Authors: Morford L A , Elliott L H , Carlson S L , Brooks W H , Roszman T L
Published: January 2023

It has been well established that patients with malignant glioblastomas exhibit T cell anergy. In this report, we further investigate the nature of this T cell anergy. The results demonstrate that tumor size but not location correlates with decreased mitogen or anti-CD3 mAb responsiveness of T cells obtained from patients. Stimulation of the TCR/CD3 complex on these patients' T cells revealed defects in early transmembrane signaling. Both PHA and anti-CD3 mAb activated PBL and T cells obtained from patients exhibited a marked decrease in the tyrosine phosphorylation of a number of proteins. In particular, decreased phosphorylation of pp100 and phospholipase Cgamma1 (PLCgamma1) was observed. In addition, PLCgamma1 and p56(lck) protein levels were dramatically reduced in T cells obtained from patients harboring a glioma. In contrast, the protein levels of p59(fyn) were normal or only slightly reduced in T cells obtained from patients with gliomas. Quantitation of free intracellular calcium concentrations ([Ca2+]i) after mitogen (PHA) stimulation or ionomycin treatment of T cells obtained from patients revealed that they mobilize less calcium than do T cells obtained from normal subjects. Stimulation of T cells obtained from patients with PMA and ionomycin, which should bypass the requirement for PLCgamma1 activation as well as directly activate the p21(ras) signaling pathway, did not restore the proliferative capacity of these T cells to normal levels. These results indicate that the anergy observed in T cells obtained from these patients is a consequence of one or more defects in the early transmembrane signaling events associated with TCR/CD3 stimulation.
TNF Receptor-Deficient Mice Reveal Striking Differences Between Several Models of Thymocyte Negative Selection
Authors: Page Dawne M. , Roberts Edda M. , Peschon Jacques J. , Hedrick Stephen M.
Published: January 2023

Central tolerance depends upon Ag-mediated cell death in developing thymocytes. However, the mechanism of induced death is poorly understood. Among the known death-inducing proteins, TNF was previously found to be constitutively expressed in the thymus. The role of TNF in thymocyte negative selection was therefore investigated using TNF receptor (TNFR)-deficient mice containing a TCR transgene. TNFR-deficient mice displayed aberrant negative selection in two models: an in vitro system in which APC are cultured with thymocytes, and a popular in vivo system in which mice are treated with anti-CD3 Abs. In contrast, TNFR-deficient mice displayed normal thymocyte deletion in two Ag-induced in vivo models of negative selection. Current models of negative selection and the role of TNFR family members in this process are discussed in light of these results.
Detection and Delineation of CXCR-4 (Fusin) as an Entry and Fusion Cofactor for T Cell-Tropic HIV-1 by Three Different Monoclonal Antibodies
Authors: Hori Toshiyuki , Sakaida Hitoshi , Sato Akihiko , Nakajima Toshihiro , Shida Hisatoshi , Yoshie Osamu , Uchiyama Takashi
Published: January 2023

A chemokine receptor, CXCR-4, has been identified as an entry cofactor for T cell line-tropic (T-tropic) HIV-1. To detect expression of CXCR-4 at the single cell level and dissect postbinding events of HIV-1 infection, we generated three mAbs against human CXCR-4. These mAbs inhibited SDF-1-induced intracellular Ca2+ mobilization, and one of the mAbs immunoprecipitated a specific 47-kDa component from CXCR-4+ cells. Flow cytometric analysis showed that most human cell lines examined expressed CXCR-4. A fraction of normal PBMC expressed CXCR-4, but neutrophils were negative. Two-color analysis revealed that the majority of T cells, virtually all B cells, and all monocytes expressed CXCR-4, while it was only weakly present on NK cells. Thus, expression of CXCR-4 is not ubiquitous but cell type specific in hemopoietic cells. The three mAbs were shown to suppress cell fusion mediated by envelope proteins of a T-tropic NL432 virus but not by those of an M-tropic JRCSF virus. Likewise, they suppressed infection of NL432 but not that of an M-tropic NL162 virus. In both cases it was noted that the suppressive activity varied considerably among the mAbs. These data confirmed that CXCR-4 is directly involved in env-mediated entry and fusion of T-tropic HIV-1 and suggest that the epitopes on CXCR-4 recognized by the three mAbs may have different roles in interaction with the envelope proteins of T-tropic HIV-1.
Staphylococcal Enterotoxin D Is a Promiscuous Superantigen Offering Multiple Modes of Interactions With the MHC Class II Receptors
Authors: Al-Daccak Reem , Mehindate Khalil , Damdoumi Farida , Etongué-Mayer Pierre , Nilsson Helen , Antonsson Per , Sundström Michael , Dohlsten Mikael , Sékaly Rafick-Pierre , Mourad Walid
Published: January 2023

Dimerization of MHC class II molecules on the cell surface of human THP-1 monocytic cell line is a requirement for staphylococcal superantigen (SAG)-induced cytokine gene expression. The capacities of various SAG to induce this response are governed by their modes of interaction with MHC class II molecules. Staphylococcal enterotoxin A (SEA), with its two binding sites, dimerizes MHC class II molecules and subsequently induces cytokine gene expression in THP-1 cells. Here, we demonstrate that staphylococcal enterotoxin D (SED) and staphylococcal enterotoxin E (SEE) induce, similarly, IL-1β and TNF-α gene expression in these cells. Using mutated toxins that lost their binding site with the MHC class II α- or β-chain, we demonstrate that this response is also mediated by the dimerization of MHC class II molecules through two binding sites. Furthermore, SED forms Zn2+-dependent homodimers that allow multiple modes of MHC class II clustering, including ligation of α-chains (α/α), β-chains (β/β), or the α- and β-chains of two different class II molecules. The β/β interaction following Zn2+-dependent SED/SED homodimer formation seems to be mediated by the appearance of a novel binding site on SED that interacts with histidine 81 of the MHC class II β-chain. The different modes of SED interactions also influence SED-induced T cell activation where simultaneous ligation of the α- and β-chains is essential for optimal response. These various modes of SED binding may be used to preserve bivalency regardless of variability in the MHC class II α/β/peptide complexes.
A Two-Step Mechanism for Recruitment of Pip by PU.1
Authors: Perkel Jeffrey M. , Atchison Michael L.
Published: January 2023

Transcription of the Ig κ light chain gene is controlled in part by the 3′ κ enhancer. Two of the proteins that bind to the 3′ enhancer, PU.1 and Pip, show tissue-restricted expression and may be responsible for the tissue specificity of 3′ enhancer activity. PU.1 alone can bind to DNA; however, Pip cannot bind to its 3′ enhancer site in electrophoretic mobility shift assays, unless recruited by PU.1. Previously, we showed that the PU.1 PEST domain (rich in the amino acids proline, glutamate, serine, and threonine; sequences 118–160) is necessary for Pip recruitment to DNA. Here we used detailed mutagenic analyzes of PU.1 to more precisely identify sequences required for Pip recruitment by electrophoretic mobility shift assay. We found that mutation of three segments within the PU.1 PEST domain (118–125, 133–139, and 141–147) modulated the efficiency of Pip recruitment, while mutation of sequences between residues 88–118 and 154–168 had no effect. Interestingly, we found that the PU.1 ETS domain (residues 170 to 255) is both necessary and sufficient for Pip interaction in solution and that other ETS domain proteins can physically interact with Pip as well. Our results suggest that Pip recruitment to DNA by PU.1 occurs via a two-step mechanism. First, a physical interaction that is not sufficient to recruit Pip occurs via the PU.1 ETS domain. Second, a conformational change in the PU.1 PEST domain, apparently mediated by serine phosphorylation, induces a conformational change in Pip enabling it to bind to DNA. We also show that the PU.1 PEST domain does not target PU.1 for rapid turnover.
The Role of Recombinant Murine IL-12 and IFN-γ in the Pathogenesis of a Murine Systemic Candida albicans Infection
Authors: Lavigne Liz M. , Schopf Lisa R. , Chung Charles L. , Maylor Rich , Sypek Joseph P.
Published: January 2023

Studies on murine candidiasis suggest that resistance to disease is linked to a Th1 response and production of IFN-γ, while failure to elicit protection is associated with a Th2 response and production of IL-4 and IL-10. Experimental infection of C57BL/6 mice, IL-12 treatment of these mice, or both infection and IL-12 treatment resulted in a characteristic Th1 cytokine mRNA profile as measured by quantitative competitive PCR. Specifically, little or no IL-4 transcripts were detected, while IFN-γ message was elevated, particularly with IL-12 treatment. Despite its role in driving increased IFN-γ expression and production, IL-12 treatment, paradoxically, promoted disease progression in our model. Therefore, we examined the effect of IFN-γ neutralization on IL-12-induced susceptibility to infection. None of the systemically infected mice receiving IL-12 alone survived, while IL-12- and anti-IFN-γ-treated mice had a 70% survival rate, similar to that after infection alone. These results suggested that IFN-γ induced by IL-12 treatment contributed to lethality. However, in separate studies, IFN-γ knockout mice were more susceptible to infection than their wild-type counterparts, suggesting that IFN-γ is required for resistance. Nonetheless, infected IFN-γ knockout mice treated with recombinant murine IL-12 exhibited enhanced resistance, suggesting that the toxicities observed with IL-12 are directly attributable to IFN-γ and that an optimal immune response to Candida infections necessitates a finely tuned balance of IFN-γ production. Thus, we propose that although IFN-γ can drive resistance, the overproduction of IFN-γ during candidiasis, mediated by IL-12 administration, leads to enhanced susceptibility.
Accommodation and T-Independent B Cell Tolerance in Rats With Long Term Surviving Hamster Heart Xenografts
Authors: Lin Yuan , Vandeputte Michel , Waer Mark
Published: January 2023

It was previously reported that treatment with leflunomide (LF; 10 mg/kg/day) together with cyclosporine (CsA; 10 mg/kg/day) resulted in long term survival of hamster heart xenografts (Xg) in rats and that LF could be withdrawn 2 to 4 wk after transplantation. To study the mechanisms allowing withdrawal of LF, second hamster heart Xgs were transplanted 6 wk after the first xenograft. Only the rats that received LF for 4 wk accepted second Xgs (>30 days; n = 5). Hence, after 4 wk of LF, the rats developed partial B cell tolerance, as they were unable to produce T-independent (CsA-resistant) XAbs. Rejection of second Xgs (2–4 days; n = 5) in the 2-wk LF group resulted in the formation of IgM xenoantibodies (XAbs) localizing together with complement within rejected grafts. However, these XAbs did not affect first Xgs, suggesting that the latter Xgs became resistant to this IgM XAb-mediated rejection, a phenomenon referred to as accommodation. Accommodation was further confirmed as adoptive transfer of IgM XAbs, which resulted in hyperacute Xg rejection in naive rats (<1 h; n = 5), did not cause rejection in long term survivors (>30 days; n = 4). This was associated with a down-regulation of the expression on the graft endothelial cells of adhesion molecules (believed to be important expressers of xenogeneic epitopes), such as P- and E-selectins. Interestingly, these adhesion molecules reappeared after retransplanting the accommodated Xgs to naive recipients. In conclusion, depending on the duration of the LF treatment, long term survival of hamster hearts in CsA-treated rats is based in part on accommodation and in part on T-independent B cell tolerance.
Inhibition of Proinflammatory Molecule Production by Adenovirus-Mediated Expression of a Nuclear Factor κB Super-Repressor in Human Intestinal Epithelial Cells
Authors: Jobin Christian , Panja Asit , Hellerbrand Claus , Iimuro Yuji , Didonato Joseph , Brenner David A. , Sartor R. Balfour
Published: January 2023

NF-κB plays a major role in the transcriptional regulation of many proinflammatory genes in multiple cell lineages, including intestinal epithelial cells (IEC). Activation of NF-κB requires both phosphorylation and degradation of its natural cytoplasmic inhibitor, IκB. We tested whether a super-repressor of NF-κB activity, which is a mutated nondegradable IκBα resistant to phosphorylation and degradation, could be delivered into IEC using an adenoviral vector (Ad5IκB) and determined the anti-inflammatory potential of this inhibitor following different stimuli. We showed for the first time that recombinant adenovirus efficiently infected (>80%) transformed as well as primary IEC. Cytoplasmic levels of the NF-κB super-repressor protein were more than 50-fold higher than those of endogenous IκB, and this mutated IκB was resistant to IL-1β-induced degradation. Immunofluorescent RelA nuclear staining was strongly inhibited in Ad5IκB-infected IEC compared with control Ad5LacZ, and NF-κB, but not AP-1 binding activity, was reduced by more than 70% as measured by electrophoretic mobility shift assay (EMSA). Induction of inducible nitric-oxide synthase (iNOS), IL-1β, and IL-8 genes by IL-1β, TNF-α, or PMA was blocked in Ad5IκB-infected cells but not in Ad5LacZ controls as assayed by RT-PCR and ELISA. In addition, IL-1β-induced IL-8 secretion was totally inhibited by Ad5IκB in primary colonic IEC. We conclude that an adenoviral vector efficiently transfers a nondegradable IκB in both transformed and native IEC. The strong inhibition of NF-κB activity and the resulting down-regulation of multiple proinflammatory molecules by Ad5IκB suggests an exciting approach for in vivo intestinal gene therapy and illustrates the key role of NF-κB in transcriptional regulation of the inflammatory phenotype of IEC.
Importance of MEK in Neutrophil Microbicidal Responsiveness
Authors: Downey Gregory P. , Butler Jeffrey R. , Tapper Hans , Fialkow Lea , Saltiel Alan R. , Rubin Barry B. , Grinstein Sergio
Published: January 2023

Exposure of neutrophils to inflammatory stimuli such as the chemoattractant FMLP leads to activation of responses including cell motility, the oxidative burst, and secretion of proteolytic enzymes. A signaling cascade involving sequential activation of Raf-1, mitogen-activated protein kinase (MEK), and extracellular signal regulated kinase (ERK) is also rapidly activated after agonist exposure. The temporal relationship between these events suggests that the kinases may be involved in triggering the effector functions, but direct evidence of a causal relationship is lacking. To assess the role of the MEK/ERK pathway in the activation of neutrophil responses, we studied the effects of PD098059, a potent and selective inhibitor of MEK. Preincubation of human neutrophils with 50 μM PD098059 almost completely (>90%) inhibited the FMLP-induced activation of MEK-1 and MEK-2, the isoforms expressed by neutrophils. This dose of PD098059 virtually abrogated chemoattractant-induced tyrosine phosphorylation and activation of ERK-1 and ERK-2, implying that MEKs are the predominant upstream activators of these mitogen-activated protein kinases. Pretreatment of neutrophils with the MEK antagonist inhibited the oxidative burst substantially and phagocytosis only moderately. In addition, PD098059 antagonized the delay of apoptosis induced by exposure to granulocyte-macrophage CSF. However, the effects of PD098059 were selective, as it failed to inhibit other responses, including chemoattractant-induced exocytosis of primary and secondary granules, polymerization of F-actin, chemotaxis, or activation of phospholipase A2. We conclude that MEK and ERK contribute to the activation of the oxidative burst and phagocytosis, and participate in cytokine regulation of apoptosis.
CD8+ Myelin Peptide-Specific T Cells Can Chemoattract CD4+ Myelin Peptide-Specific T Cells: Importance of IFN-Inducible Protein 10
Authors: Biddison William E. , Cruikshank William W. , Center David M. , Pelfrey Clara M. , Taub Dennis D. , Turner Richard V.
Published: January 2023

The demyelination process that occurs in the central nervous system (CNS) of patients with multiple sclerosis (MS) is due, in part, to an inflammatory response in which CD4+ and CD8+ T cells and macrophages infiltrate white matter. While it is thought that the inflammatory and demyelination process in MS is the product of Th1-associated cytokines secreted by CD4+ myelin protein-specific T cells present in the CNS, the mechanisms that are responsible for the recruitment and maintenance of these myelin-reactive CD4+ T cells in the CNS have not been elucidated. We have shown previously that CD8+CTL that recognize peptides derived from sequences of the myelin proteolipid protein (PLP) presented by HLA class I molecules can be generated in vitro, and that these PLP-specific CD8+CTL secrete the proinflammatory chemokines macrophage-inflammatory protein-1α and -1β, IL-16, and IP-10. In this study, we demonstrate that soluble products of these PLP-specific CD8+CTL can chemoattract CD4+ T cells that are specific for a myelin basic protein peptide and a PLP peptide, and that the majority of this chemotactic activity is mediated by IFN-inducible protein 10. These results demonstrate that PLP-specific CD8+ T cells can play a role in the recruitment and retention of myelin-derived peptide-specific CD4+ T cells, and indicate that they may play a proinflammatory role in the pathogenesis of MS.
Adhesion Molecule Mechanisms Mediating Monocyte Migration Through Synovial Fibroblast and Endothelium Barriers: Role for CD11/CD18, Very Late Antigen-4 (CD49d/CD29), Very Late Antigen-5 (CD49e/CD29), and Vascular Cell Adhesion Molecule-1 (CD106)
Authors: Shang Xiao-zhou , Lang Bianca J. , Issekutz Andrew C.
Published: January 2023

Monocytes migrate through vascular endothelium, and then in connective tissue. As a model of this process, we investigated adhesion molecules involved in monocyte migration through HUVEC and a barrier of human synovial fibroblasts (HSF). Minimal spontaneous monocyte migration (6–7%) occurred through either cell barrier, but this increased markedly (27–35% of added monocytes) when a C5a chemotactic gradient was present. Migration across unstimulated HUVEC was partially inhibited (40%) by mAb to CD18 (β2 integrin) and completely blocked by anti-CD18 plus anti-α4 (CD49d; very late Ag-4 (VLA-4)) mAbs. In contrast, migration across HSF induced by C5a or monocyte chemoattractant protein-1 was not inhibited by mAb to CD18 and was only partially inhibited (33%) in combination with anti-α4 mAb. The CD18- and VLA-4-independent migration across HSF was completely inhibited by mAb to α5 of VLA-5. The inhibitory effect of mAbs to VLA-4 and VLA-5 was on the monocyte and required blockade of CD11/CD18 to be observed. In contrast to HSF, no role for VLA-5 in monocyte transendothelial migration was detected. Both HSF and IL-1-stimulated HUVEC expressed vascular cell adhesion molecule-1 (VCAM-1). However, VLA-4-mediated monocyte migration across HSF was only partially dependent on VCAM-1, in contrast to transendothelial migration, which was completely blocked by anti-VCAM-1 mAbs. In conclusion, unlike transendothelial migration, for which VLA-4 is the alternative mechanism to CD11/CD18 on monocytes, both VLA-4 and VLA-5 can mediate monocyte migration through fibroblast barriers. In addition to VCAM-1, other ligand(s) on HSF are also involved in the VLA-4-mediated migration.
In SCID-hu Mice, Passive Transfer of a Humanized Antibody Prevents Infection and Atrophic Change of Medulla in Human Thymic Implant due to Intravenous Inoculation of Primary HIV-1 Isolate
Authors: Okamoto Yukari , Eda Yasuyuki , Ogura Atsuo , Shibata Shinwa , Amagai Takashi , Katsura Yoshimoto , Asano Toshihiko , Kimachi Kazuhiko , Makizumi Keiichi , Honda Mitsuo
Published: January 2023

Using SCID-hu mice, it was tested whether humanized mAb Rμ5.5 could prevent infection by HIV-1 i.v. inoculation. The Ab that recognizes the IHIGPGRAFYT motif in the principal neutralizing determinant (PND) of HIVMN, as well as the original mouse mAb μ5.5, neutralized HIVMN with high activity. Seven primary field isolates from Japanese hemophiliacs seropositive for HIV-1 clade B were compared for their reactivities to Rμ5.5. Rμ5.5 was effective, particularly against the viruses that matched amino acid sequences of the PND region of HIV-1, and it completely neutralized primary isolates. Moreover, the passive transfer of the Ab elicited protection against challenge by the primary isolates in SCID-hu or hu-PBL-SCID mice after i.v. inoculation with the virus by both quantitative PCR and PBMC-based virus isolation in vitro. Further, inoculation with the Ab also prevented the atrophic change in the medulla of the thymic transplant that was induced by i.v. inoculation of the virus. Thus, the humanized neutralizing Ab Rμ5.5 appears to protect SCID-hu mice from infection by primary field isolates.
Differential Effects of the Rejection of Bone Marrow Allografts by the Depletion of Activating Versus Inhibiting Ly-49 Natural Killer Cell Subsets
Authors: Raziuddin Arati , Longo Dan L. , Mason Llewellyn , Ortaldo John R. , Bennett Michael , Murphy William J.
Published: January 2023

Natural killer cells mediate the specific rejection of bone marrow cell (BMC) allografts in lethally irradiated mice. The Ly-49 family of molecules present on subsets of murine NK cells appears capable of binding class I MHC molecules, resulting in transmission of an inhibitory signal to the NK cell. These Ly-49 family members have been shown to have an immunoreceptor tyrosine-based inhibitory motif that is responsible for the inhibitory signal. However, a new Ly-49 family member was found that lacks this motif, Ly-49D, and evidence suggests that this may be an activating receptor. We therefore compared the role of the activating Ly-49 member with NK cells bearing inhibitory Ly-49 receptors in BMC rejection. Depletion of Ly-49D+ NK cells in H-2b mice abrogated their ability to reject H-2d BMC allografts. Similarly, Ly-49C+ NK cells also were shown to mediate the specific rejection of H-2d BMC. When both subsets were depleted, an additive enhancement of BMC engraftment was observed, indicating that both subsets play a role in the rejection of allogeneic H-2-homozygous H-2d BMC. However, rejection of H-2b × d or D8 (H-2b, Dd transgene) BMC allografts was unaffected by Ly-49C+ NK cell depletion in H-2b mice. In marked contrast, depletion of Ly-49D+ NK cells in H-2b mice totally abrogated the rejection of H-2b × d heterozygous BMC in support of in vitro data suggesting that Ly-49D+ NK cells receive activating signals. Therefore, NK subsets demonstrate a differential ability to reject H-2 homozygous and heterozygous BMC.
Cutting Edge: Homologous Recombination of the MHC Class I K Region Defines New MHC-Linked Diabetogenic Susceptibility Gene(s) in Nonobese Diabetic Mice
Authors: Hattori Masakazu , Yamato Eiji , Itoh Naoto , Senpuku Hidenobu , Fujisawa Tomomi , Yoshino Masayasu , Fukuda Masahiro , Matsumoto Eisaku , Toyonaga Tetsushi , Nakagawa Ichiro , Petruzzelli Maria , McMurray Armand , Weiner Howard , Sagai Tomoko , Moriwaki Kazuo , Shiroishi Toshihiko , Maron Ruth , Lund Torben
Published: January 2023

To localize the MHC-linked diabetogenic genes in the nonobese diabetic (NOD) mouse, a recombinational hotspot from the B10.A(R209) mouse was introduced to the region between the MHC class I K and class II A of the NOD mouse with the recombinational site centromeric to the Lmp2/Tap1 complex by breeding the two strains. Replacement of the NOD region centromeric to the recombinational site with the same region in R209 mice prevented the development of diabetes (from 71 to 3%) and insulitis (from 61 to 15%) in the N7 intra-MHC recombinant NOD mice. Similarly, the replacement of the NOD class II A, E and class I D region with the same region in R209 mice prevented the diseases (diabetes, from 71 to 0%; insulitis, from 61 to 3%). In addition to the MHC class II genes, there are at least two MHC-linked diabetogenic genes in the region centromeric to Lmp2.
Spontaneous Human Monocyte Apoptosis Utilizes a Caspase-3-Dependent Pathway That Is Blocked by Endotoxin and Is Independent of Caspase-1
Authors: Fahy Ruairi J. , Doseff Andrea I. , Wewers Mark D.
Published: January 2023

Apoptosis is an important mechanism for regulating the numbers of monocytes and macrophages. Caspases (cysteine-aspartate-specific proteases) are key molecules in apoptosis and require proteolytic removal of prodomains for activity. Caspase-1 and caspase-3 have both been connected to apoptosis in other model systems. The present study attempted to delineate what role these caspases play in spontaneous monocyte apoptosis. In serum-free conditions, monocytes showed a commitment to apoptosis as early as 4 h in culture, as evidenced by caspase-3-like activity. Apoptosis, as defined by oligonucleosomal DNA fragmentation, was prevented by a generalized caspase inhibitor, z-VAD-FMK, and the more specific caspase inhibitor, z-DEVD-FMK. The caspase activity was specifically attributable to caspase-3 by the identification of cleavage of procaspase-3 to active forms by immunoblots and by cleavage of the fluorogenic substrate DEVD-AFC. In contrast, a caspase-1 family inhibitor, YVAD-CMK, did not protect monocytes from apoptosis, and the fluorogenic substrate YVAD-AFC failed to show an increase in activity in apoptotic monocytes. When cultured with LPS (1 μg/ml), monocyte apoptosis was prevented, as was the activation of caspase-3. Unexpectedly, LPS did not change baseline caspase-1 activity. These findings link spontaneous monocyte apoptosis to the proteolytic activation of caspase-3.
Switch Recombination in a Transfected Plasmid Occurs Preferentially in a B Cell Line That Undergoes Switch Recombination of Its Chromosomal Ig Heavy Chain Genes
Authors: Stavnezer Janet , Bradley Sean P. , Rousseau Norman , Pearson Todd , Shanmugam Ananth , Waite Debra J. , Rogers Paul R. , Kenter Amy L.
Published: January 2023

Ab class switching is induced upon B cell activation in vivo by immunization or infection or in vitro by treatment with mitogens, e.g. LPS, and results in the expression of different heavy chain constant region (CH) genes without a change in the Ab variable region. This DNA recombination event allows Abs to alter their biological activity while maintaining their antigenic specificity. Little is known about the molecular mechanism of switch recombination. To attempt to develop an assay for enzymes, DNA binding proteins, and DNA sequences that mediate switch recombination, we have constructed a plasmid DNA substrate that will undergo switch recombination upon stable transfection into the surface IgM+ B cell line (I.29μ), a cell line capable of undergoing switch recombination of its endogenous genes. We demonstrate that recombination occurs between the two switch regions of the plasmid, as assayed by PCRs across the integrated plasmid switch regions, followed by Southern blot hybridization. Nucleotide sequence analysis of the PCR products confirmed the occurrence of Sμ-Sα recombination in the plasmid. Recombination of the plasmid in I.29μ cells does not require treatment with inducers of switch recombination, suggesting that recombinase activity is constitutive in I.29μ cells. Recombination does not require high levels of transcription across the switch regions of the plasmid. Fewer recombination events are detected in four different B and T cell lines that do not undergo switch recombination of their endogenous genes.
Tumor Therapy with Bispecific Antibody: The Targeting and Triggering Steps Can Be Separated Employing a CD2-Based Strategy
Authors: Wild Martin K. , Strittmatter Wolfgang , Matzku Siegfried , Schraven Burkhart , Meuer Stefan C.
Published: January 2023

For tumor therapy with unprimed effector cells, we developed a novel combination of a CD2 × tumor Ag bispecific targeting Ab and an anti-CD2 triggering Ab. These Ab constructs were derived from two novel CD2 mAbs, termed M1 and M2 that, together, but not individually activate T cells. Unlike many other CD2 Abs, M1 and M2 do not interfere with TCR/CD3 triggering nor do they inhibit binding of CD2 to its ligand CD58, thus preserving the physiological functions of these important effector cell molecules. M2 was chemically conjugated with an Ab recognizing the epidermal growth factor-receptor (EGF-R). Incubation of unprimed peripheral blood mononuclear cells with the bispecific F(ab′)2 construct (M2xEGF-R) in the presence of trigger Ab M1 led to efficient selective lysis of EGF-R-positive targets by CTL and NK cells. Importantly, the need for trigger Ab M1 for effector cell stimulation allowed to separate targeting from triggering steps in vitro and should thus enable to focus immune responses to sites of target Ag expression in vivo.
BCL-6-Deficient Mice Reveal an IL-4-Independent, STAT6-Dependent Pathway That Controls Susceptibility to Infection by Leishmania major
Authors: Dent Alexander L. , Doherty T. Mark , Paul William E. , Sher Alan , Staudt Louis M.
Published: January 2023

The BCL-6 gene negatively regulates Th2 responses as shown by the finding that BCL-6-deficient (BCL-6−/−) mice develop a lethal Th2-type inflammatory disease. The response of inbred mouse strains to infection with Leishmania major is under genetic control; BALB/c mice are susceptible and develop a progressive parasite burden, whereas most other common laboratory strains of mice are resistant to infection. We found that BCL-6−/− mice on a resistant genetic background (C57BL/6 × 129 intercrossed mice) were highly susceptible to L. major infection; they resembled BALB/c mice in terms of lesion size, parasite load, and the production of Th2 cytokines. BCL-6−/−IL-4−/− double-mutant mice were also susceptible to L. major infection and produced 10-fold higher levels of the Th2 cytokine IL-13 than IL-4−/− littermate controls. By contrast, BCL-6−/−STAT6−/− double-mutant mice were resistant to L. major infection despite also producing elevated levels of IL-13. These results show that STAT6 is required for susceptibility to L. major infection and suggest that IL-13 signaling through STAT6 may contribute to a nonhealing, exacerbated L. major infection.
Structure and Function of the Glycosaminoglycan Binding Site of Chemokine Macrophage-Inflammatory Protein-1β
Authors: Koopmann Witte , Ediriwickrema Chandrika , Krangel Michael S.
Published: January 2023

The ability of chemokines to bind to glycosaminoglycans (GAGs) on cell surfaces and in the extracellular matrix is thought to play a crucial role in chemokine function. We investigated the structural basis for chemokine binding to GAGs by using in vitro mutagenesis to identify amino acids of chemokine macrophage-inflammatory protein-1β (MIP-1β) that contribute to its interaction with the model GAG heparin. Among six basic residues that are organized into a single basic domain in the folded MIP-1β monomer, three (R18, K45, and R46) were found to contribute significantly to heparin binding. Of these, R46 was found to play a dominant role, and proved essential for the interaction of MIP-1β with both heparin and heparan sulfate in physiological salt. The results of this mutational analysis have implications for the structure of the MIP-1β-heparin complex, and a comparison of these results with those obtained by mutational analysis of the MIP-1α-heparin interaction suggests a possible structural difference between the MIP-1β-heparin and MIP-1α-heparin complexes. To determine whether GAG binding plays an important role in receptor binding and cellular activation by MIP-1β, the activities of wild-type MIP-1β and R46-substituted MIP-1β were compared in assays of T lymphocyte chemotaxis. The two proteins proved equipotent in this assay, arguing that interaction of MIP-1β with GAGs is not intrinsically required for functional interaction of MIP-1β with its receptor.
Up-Regulation of ICAM-1 by Cytokines in Human Tracheal Smooth Muscle Cells Involves an NF-κB-Dependent Signaling Pathway That Is Only Partially Sensitive to Dexamethasone
Authors: Amrani Yassine , Lazaar Aili L. , Panettieri Reynold A.
Published: January 2023

Although the precise mechanisms by which steroids mediate their therapeutic effects remain unknown, steroids have been reported to abrogate cytokine-induced activation of the transcription factor NF-κB. In some cell types, NF-κB activation is necessary to regulate cytokine-mediated cellular functions. However, compelling evidence suggests that the steroid inhibition of NF-κB is complex and cell specific. Using EMSA, we show that stimulation with TNF-α or IL-1β induces NF-κB DNA-binding activity in human airway smooth muscle cells. TNF-α and IL-1β also increased luciferase activity in airway smooth muscle cells transfected with a reporter plasmid containing κB enhancer elements. Cytokines activated NF-κB by rapidly degrading its cytosolic inhibitor IκBα, which was then regenerated after 60 min. Cytokine-mediated IκBα reappearance was completely blocked by the protein synthesis inhibitor cycloheximide. Inhibition of cytokine-mediated IκBα proteolysis using the protease inhibitors N-tosyl-l-phenylalanine chloromethyl ketone and N-acetyl-l-leucinyl-l-leucinyl-norleucinal also inhibited cytokine-mediated early expression of ICAM-1. Although dexamethasone partially inhibited IL-1β- and TNF-α-induced up-regulation of ICAM-1 at 4 h, dexamethasone had no effect on cytokine-induced ICAM-1 expression at 18–24 h. In addition, neither cytokine-induced degradation or resynthesis of IκBα nor NF-κB DNA-binding activity were affected by dexamethasone. In cells transfected with the luciferase reporter, dexamethasone did not affect TNF-α-induced NF-κB-dependent transcription. Interestingly, cytokine-mediated expression of cyclooxygenase-2 was completely abrogated by dexamethasone at 6 h. Together, these data demonstrate that cytokine-mediated NF-κB activation and ICAM-1 expression involve activation of a steroid-insensitive pathway.
Persistent Expression of Serum Amyloid A During Experimentally Induced Chronic Inflammatory Condition in Rabbit Involves Differential Activation of SAF, NF-κB, and C/EBP Transcription Factors
Authors: Ray Alpana , Ray Bimal K.
Published: January 2023

The serum amyloid A (SAA) protein has been implicated in the pathogenesis of several chronic inflammatory diseases. Its induction mechanism in response to a chronic inflammatory condition was investigated in rabbits following multiple s.c. injections of AgNO3 over a period of 35 days. During unremitting exposure to inflammatory stimulus, a persistently higher than normal level of SAA2 expression was seen in multiple tissues. Induction of SAA was correlated with higher levels of several transcription factor activities. Increased SAA-activating factor (SAF) activity was detected in the liver, lung, and brain tissues under both acute and chronic inflammatory conditions. In the heart, kidney, and skeletal muscle tissues, this activity remained virtually constant. In contrast, CCAAT enhancer binding protein (C/EBP) DNA-binding activity was transiently induced in selective tissues. Higher than normal NF-κB DNA-binding activity was detected in the lung and to a lesser extent in the liver and kidney tissues under both acute and chronic conditions. This result suggested that C/EBP, SAF, and NF-κB are required for transient acute phase induction of SAA whereas SAF and NF-κB activities are necessary for persistent SAA expression during chronic inflammatory conditions.
CD40 Ligand-Activated Human Monocytes Amplify Glomerular Inflammatory Responses Through Soluble and Cell-to-Cell Contact-Dependent Mechanisims
Authors: Kuroiwa Takashi , Lee Eric G. , Danning Carol L. , Illei Gabor G. , McInnes Iain B. , Boumpas Dimitrios T.
Published: January 2023

Monocytes/macrophages play a critical role in the initiation and progression of a variety of glomerulonephritides. We sought to define the interactions between physiologically activated human monocytes and glomerular mesangial cells (MC) by employing a cell culture system that permits the accurate assessment of the contribution of soluble factors and cell-to-cell contact. Human peripheral blood monocytes, primed with IFN-γ and GM-CSF, were activated with CD40 ligand (CD40L) or TNF-α and cocultured with MC. CD40L-activated monocytes induced higher levels of IL-6, monocyte chemoattractant protein-1 (MCP-1) and ICAM-1 synthesis by MC. Separation of CD40L-activated monocytes from MC by a porous membrane decreased the mesangial synthesis of IL-6 by 80% and ICAM-1 by 45%, but had no effect on MCP-1. Neutralizing Abs against the β2 integrins, LFA-1 and Mac-1, decreased IL-6 production by 40 and 50%, respectively. Ligation of mesangial surface ICAM-1 directly enhanced IL-6, but not MCP-1, production. Simultaneous neutralization of soluble TNF-α and IL-1β decreased MCP-1 production by 55% in membrane-separated cocultures of MC/CD40L-activated monocytes. Paraformaldehyde-fixed CD40L-activated monocytes (to preserve membrane integrity but prevent secretory activity), cocultured with MC at various ratios, induced IL-6, MCP-1, and ICAM-1 synthesis by MC. Plasma membrane preparations from activated monocytes also induced mesangial IL-6 and MCP-1 synthesis. The addition of plasma membrane enhanced TNF-α-induced mesangial IL-6 production by ∼4-fold. Together, these data suggest that the CD40/CD40L is essential for optimal effector function of monocytes, that CD40L-activated monocytes stimulate MC through both soluble factors and cell-to-cell contact mediated pathways, and that both pathways are essential for maximum stimulation of MC.
Leukocyte Entry into Sites of Inflammation Requires Overlapping Interactions Between the L-Selectin and ICAM-1 Pathways
Authors: Steeber Douglas A. , Tang Mimi L. K. , Green Nathan E. , Zhang Xiu-Qin , Sloane Jennifer E. , Tedder Thomas F.
Published: January 2023

Leukocyte interactions with vascular endothelium during inflammation depend on cascades of adhesion molecule engagement, particularly during selectin-mediated leukocyte rolling. Leukocyte rolling is also facilitated by members of the integrin and Ig families. Specifically, leukocyte rolling velocities during inflammation are significantly increased in ICAM-1-deficient mice, with ICAM-1 expression required for optimal P- and L-selectin-mediated rolling. Elimination of ICAM-1 expression in L-selectin-deficient mice significantly reduces leukocyte rolling. Whether disrupted leukocyte rolling in L-selectin and ICAM-1 double-deficient (L-selectin/ICAM-1−/−) mice affects leukocyte entry into sites of inflammation in vivo was assessed in the current study by using experimental models of inflammation; thioglycollate-induced peritonitis, chemokine-induced neutrophil migration to the skin, delayed-type hypersensitivity responses, rejection of allogeneic skin grafts, and septic shock. In many cases, the loss of both L-selectin and ICAM-1 expression dramatically reduced leukocyte migration into sites of inflammation beyond what was observed with loss of either receptor alone. In fact, the effects from loss of both L-selectin and ICAM-1 effectively eliminated multiple chronic inflammatory responses in L-selectin/ICAM-1−/− mice. By contrast, the combined loss of L-selectin and ICAM-1 expression had minimal effects on the generation of Ag-specific T cell responses or humoral immunity. Thus, members of the selectin and Ig families function synergistically to mediate optimal leukocyte rolling and entry into tissues, which is essential for the generation of effective inflammatory responses in vivo.
Soluble Fas Ligand Induces Epithelial Cell Apoptosis in Humans with Acute Lung Injury (ARDS)
Authors: Matute-Bello Gustavo , Liles W. Conrad , Steinberg Kenneth P. , Kiener Peter A. , Mongovin Stephen , Chi Emil Y. , Jonas Mechthild , Martin Thomas R.
Published: January 2023

The goals of this study were to determine whether the Fas-dependent apoptosis pathway is active in the lungs of patients with the acute respiratory distress syndrome (ARDS), and whether this pathway can contribute to lung epithelial injury. We found that soluble Fas ligand (sFasL) is present in bronchoalveolar lavage (BAL) fluid of patients before and after the onset of ARDS. The BAL concentration of sFasL at the onset of ARDS was significantly higher in patients who died. BAL from patients with ARDS induced apoptosis of distal lung epithelial cells, which express Fas, and this effect was inhibited by blocking the Fas/FasL system using three different strategies: anti-FasL mAb, anti-Fas mAb, and a Fas-Ig fusion protein. In contrast, BAL from patients at risk for ARDS had no effect on distal lung epithelial cell apoptosis. These data indicate that sFasL is released in the airspaces of patients with acute lung injury and suggest that activation of the Fas/FasL system contributes to the severe epithelial damage that occurs in ARDS. These data provide the first evidence that FasL can be released as a biologically active, death-inducing mediator capable of inducing apoptosis of cells of the distal pulmonary epithelium during acute lung injury