bluuebunny/crossref_metadata_2025 huggingface dataset:
Engagement of ICAM-3 Activates Polymorphonuclear Leukocytes: Aggregation Without Degranulation or β2 Integrin Recruitment
Authors: Feldhaus Michael J. , Kessel Julie M. , Zimmerman Guy A. , McIntyre Thomas M.
Published: January 2023

ICAM-3 is a preferred counterreceptor for the leukocyte αLβ2 integrin. It activates T cells through outside-in signaling, but polymorphonuclear leukocytes (PMN) are reported to be refractory to ICAM-3 stimulation. We found that engagement of ICAM-3 by a mAb (CAL3.10), which binds in the region where αLβ2 integrin binds, activates PMN homotypic aggregation and adhesion to surfaces. These functional changes were due to ICAM-3 outside-in signaling because aggregation and adhesion were β2 integrin-dependent, tyrosine kinase and protein kinase C activities were activated, and there was a reorganization of the cytoskeleton. This reorganization and kinase activity was required for ICAM-3-, but not FMLP-, induced aggregation. This is not an Fc-mediated event as an appropriate anti-ICAM-3 F(ab′)2 fragment still induced aggregation. Another anti-ICAM-3 Ab (HP2/19), which activates T cells, did not activate PMN. Strikingly, anti-ICAM-3 did not induce degranulation or cause an increase in surface β2 integrin expression, so adhesion and aggregation were due solely to the activation of the constitutively expressed β2 integrins. Aggregation in response to ICAM-3, but not FMLP, was compromised at lower cell densities, showing that β2 integrin recruitment enhances aggregation under suboptimal conditions. We conclude that engagement of ICAM-3 stimulates PMN as well as T cells, but that the appropriate epitope varies between these two cells. ICAM-3 outside-in signaling reorganizes the cytoskeleton without causing degranulation, induces serine and tyrosine kinase activation, and activates existing surface β2 integrins to a proadhesive state.
Oral Delivery of Group A Streptococcal Cell Walls Augments Circulating TGF-β and Suppresses Streptococcal Cell Wall Arthritis
Authors: Chen Wanjun , Jin Wenwen , Cook Melissa , Weiner Howard L. , Wahl Sharon M.
Published: January 2023

Oral administration of autoantigens can influence the outcome of experimental autoimmune diseases, yet little is known about nonself Ag-induced tolerance. In this study, we administered group A streptococcal cell wall (SCW) peptidoglycan-polysaccharide complexes orally and monitored the impact on SCW-induced erosive polyarthritis. Oral administration of low dose SCW (3 μg/day), initiated 7 days before an arthritogenic dose of systemic SCW, virtually eliminated the joint swelling and destruction typically observed during both the acute and chronic phases of the arthritis. High (300 μg), but not intermediate (30 μg), dose regimens also profoundly inhibited the disease. Most previous studies have demonstrated that prior feeding is required for efficacy, yet oral feeding of low dose SCW suppressed the evolution of arthritis even when administration was begun 10–15 days after induction of the arthritis. While the synovial inflammatory cell infiltration and expression of proinflammatory cytokines were markedly suppressed, no local enhancement of the regulatory cytokines IL-4, IL-10, and TGF-β was detected. Oral administration of low dose SCW, however, up-regulated circulating levels of TGF-β, concomitant with decreased circulating TNF-α and suppression of chronic arthritis. Moreover, IL-10 was increased in tolerized spleen lymphocytes, and unexpectedly, this SCW-specific IL-10 production was TGF-β dependent. These data support a pivotal role for TGF-β, although not necessarily in the joint, in the regulation of specific immune tolerance responsible for suppressed synovial inflammation and matrix destruction. The distant induction and up-regulation of regulatory cytokines and/or cells may contribute to the inhibition of the immune response through blunted infiltration of inflammatory cells to the joint.
Substance P Regulates Somatostatin Expression in Inflammation
Authors: Blum Arthur M. , Elliott David E. , Metwali Ahmed , Li Jie , Qadir Khurram , Weinstock Joel V.
Published: January 2023

Substance P (SP) and somatostatin (SOM) are made at mucosal surfaces and sites of inflammation. There is a SP/SOM immunoregulatory circuit that modulates the IFN-γ response in murine schistosomiasis. SP enhances, while SOM decreases, IFN-γ secretion. Various inflammatory mediators induce macrophages to make SOM, but no known factor limits this expression. It was discovered that SP regulates SOM synthesis. Splenocytes from normal, uninfected mice cultured with LPS, IFN-γ, or IL-10 for 4 h strongly expressed SOM mRNA, but failed to do so in the presence of SP. The inhibition with 10−9 M SP was >85% shown by quantitative PCR. Also, splenocyte SOM content decreased from 1048 ± 275 to <10 pg/4 × 108 cells following SP exposure. Immunohistochemistry identified SOM solely within splenic macrophages following cytokine stimulation. Mice infected with Schistosoma mansoni form granulomas in the liver and intestines resulting from deposition of parasite eggs in these organs. The granulomas contain macrophages that make SOM constitutively. SP at 10−8 M decreased SOM mRNA expression >90% in dispersed granuloma cells cultured for 4 h or longer. Specific SP receptor antagonists blocked SP suppression of SOM expression in splenocytes and dispersed granuloma cells, showing that an authentic SP receptor mediated the regulation. Additional studies revealed that IL-4 antagonized the SP effect in the spleen. It is concluded that in granulomas and splenocytes from mice with schistosomiasis and in splenocytes from uninfected animals that 1) SP inhibits macrophage SOM induction and ongoing expression at the mRNA and protein levels acting through the SP receptor, and 2) IL-4 can antagonizes this SP effect.
CDw150 Associates with Src-Homology 2-Containing Inositol Phosphatase and Modulates CD95-Mediated Apoptosis
Authors: Mikhalap Svitlana V. , Shlapatska Larisa M. , Berdova Anna G. , Law Che-Leung , Clark Edward A. , Sidorenko Svetlana P.
Published: January 2023

CDw150, a receptor up-regulated on activated T or B lymphocytes, has a key role in regulating B cell proliferation. Patients with X-linked lymphoproliferative disease have mutations in a gene encoding a protein, DSHP/SAP, which interacts with CDw150 and is expressed in B cells. Here we show that CDw150 on B cells associates with two tyrosine-phosphorylated proteins, 59 kDa and 145 kDa in size. The 59-kDa protein was identified as the Src-family kinase Fgr. The 145-kDa protein is the inositol polyphosphate 5′-phosphatase, SH2-containing inositol phosphatase (SHIP). Both Fgr and SHIP interact with phosphorylated tyrosines in CDw150’s cytoplasmic tail. Ligation of CDw150 induces the rapid dephosphorylation of both SHIP and CDw150 as well as the association of Lyn and Fgr with SHIP. CD95/Fas-mediated apoptosis is enhanced by signaling via CDw150, and CDw150 ligation can override CD40-induced rescue of CD95-mediated cell death. The ability of CDw150 to regulate cell death does not correlate with serine phosphorylation of the Akt kinase, but does correlate with SHIP tyrosine dephosphorylation. Thus, the CDw150 receptor may function to regulate the fate of activated B cells via SHIP as well as via the DSHP/SAP protein defective in X-linked lymphoproliferative disease patients.
Tumor Antigens are Constitutively Presented in the Draining Lymph Nodes
Authors: Marzo Amanda L. , Lake Richard A. , Lo David , Sherman Linda , McWilliam Andrew , Nelson Delia , Robinson Bruce W. S. , Scott Bernadette
Published: January 2023

Tumor growth is rarely associated with a strong specific CTL response, suggesting that the immune system is ignorant of the presence of tumor because the Ags are not readily available to or are sequestered from potential effector cells. We studied the in vivo activation of naive TCR transgenic hemagglutinin (HA)-specific CD8+ T cells adoptively transferred into mice bearing HA-expressing tumor using 5,6-carboxy-succinimidyl-fluorescein-ester labeling, which allows the identification of proliferating HA-specific T cells. We demonstrate that tumor Ags are constitutively presented in the lymph nodes draining tumors and are powerfully mitogenic for responding T cells despite the absence of anti-tumor CTL responses. Importantly, this proliferative signal occurs throughout tumor growth and is still detectable 6 mo after tumor inoculation when tumor is not palpable. These results provide the first evidence that there is constitutive presentation of tumor Ags in draining lymph nodes.
Pertussis Toxin-Sensitive Signal Controls the Trafficking of Thymocytes Across the Corticomedullary Junction in the Thymus
Authors: Suzuki Gen , Sawa Hirofumi , Kobayashi Yoshiyasu , Nakata Yukiko , Nakagawa Ken-ichi , Uzawa Akiko , Sakiyama Hisako , Kakinuma Shizuko , Iwabuchi Kazuya , Nagashima Kazuo
Published: January 2023

We investigated a role of chemokines in thymocyte trafficking. Genes encoding stromal cell-derived factor-1 and its receptor CXCR4 were detected in the cortex by in situ hybridization. Early immigrant cells did not express CXCR4, whereas their descendant CD44+CD25+CD4−CD8− cells did. CXCR4 expression was down-modulated when CD4+CD8+ double-positive cells became CD4+CD8− or CD4−CD8+ single-positive (SP) cells. Positively selected CD69+CD3intermediate cells gained CCR4, of which ligand, thymus activation-regulated chemokine, was expressed in the medulla. At the next developmental stage, CD69−CD3high cells lost CCR4 but gained CCR7. These results suggest that thymocytes use different chemokines along with their development. Blockade of chemokine receptor-mediated signaling by pertussis toxin perturbed the normal distribution of SP cells and resulted in the accumulation of SP cells in the cortex. Thus, a pertussis toxin-sensitive event controls the trafficking of SP cells across the corticomedullary junction.
CD8+ CTLs Are Essential for Protective Immunity Against Encephalitozoon cuniculi Infection
Authors: Khan Imtiaz A. , Schwartzman Joseph D. , Kasper Lloyd H. , Moretto Magali
Published: January 2023

Encephalitozoon cuniculi is a protozoan parasite that has been implicated recently as a cause of opportunistic infection in immunocompromised individuals. Protective immunity in the normal host is T cell-dependent. In the present study, the role of individual T cell subtypes in immunity against this parasite has been studied using gene knockout mice. Whereas CD4−/− animals resolved the infection, mice lacking CD8+ T cells or perforin gene succumbed to parasite challenge. The data obtained in these studies suggest that E. cuniculi infection induces a strong and early CD8+ T response that is important for host protection. The CD8+ T cell-mediated protection depends upon the CTL activity of this cell subset, as the host is rendered susceptible to infection in the absence of this function. This is the first report in which a strong dependence upon the cytolytic activity of host CD8+ T cells has been shown to be important in a parasite infection.
The Surface Protein Superfamily ofTrypanosoma cruziStimulates a Polarized Th1 Response That Becomes Anergic
Authors: Millar Amanda E. , Wleklinski-Lee Monika , Kahn Stuart J.
Published: January 2023

Trypanosoma cruzi is an obligate intracellular parasite that chronically infects mammals. Extracellular mammalian stage trypomastigotes simultaneously express and release multiple members of the parasite’s surface protein superfamily; these extracellular proteins should stimulate MHC class II-restricted CD4 T cells. The surface protein superfamily, however, encodes variant epitopes that may inhibit this CD4 response. In this report the surface protein-specific CD4 response was investigated. CD4 cells isolated from acutely and chronically infected mice did not proliferate when stimulated with surface proteins. Adoptive transfer of surface protein-specific CD4 clones or immunization with a peptide encoding a surface protein T cell epitope protected mice during T. cruzi infection. These data strongly suggested that surface proteins were expressed and presented to CD4 cells during infection. Limiting dilution analysis identified an expanded population of surface protein-specific CD4 cells during the acute and chronic infection. These surface protein-specific CD4 cells did not produce IL-2 or IL-4, but did produce IFN-γ. Enzyme-linked immunospot analyses confirmed that many of the surface protein-specific CD4 cells produce IFN-γ. Together these results suggest that during T. cruzi infection a potentially protective CD4 response becomes anergic. It is possible that this anergy is induced by variant T cell epitopes encoded by the surface protein superfamily.
TNF-Induced Haptoglobin Release from Human Neutrophils: Pivotal Role of the TNF p55 Receptor
Authors: Berkova Nadia , Gilbert Caroline , Goupil Serge , Yan Ju , Korobko Vyatcheslav , Naccache Paul H.
Published: January 2023

Haptoglobin (Hp), TNF-α, and neutrophils are parts of a highly interactive ensemble participating in inflammatory processes. Hp is taken up by neutrophils, stored within a cytoplasmic granular compartment, and is secreted during phagocytosis by those cells. In the present study, the effects of TNF-α on the release of Hp from human neutrophils were investigated. Incubation of neutrophils with TNF-α induced the release of Hp from cells in a time- and concentration-dependent manner as revealed by Western blot analysis and immunofluorescence. The release of Hp induced by TNF-α was not due to nonspecific lysis of the cells. TNF-α is a highly pleiotropic cytokine that mediates its effects by binding to two distinct receptors (p55 and p75). Administration of TNF-α mutants binding specifically either to the p55 or to the p75 TNF receptors showed that there is a preference of TNF-α for the p55 receptor in the mediation of Hp release by neutrophils. A stimulated release of Hp was also induced by the chemotactic tripeptide fMLP. The TNF-α-induced release of Hp from neutrophils was inhibited by erbstatin, a tyrosine kinase inhibitor. These findings suggest that TNF-α may promptly increase the level of Hp at sites of infection or injury, leading to the modulation of the acute inflammatory response.
The β2-Adrenergic Agonist Salbutamol Is a Potent Suppressor of Established Collagen-Induced Arthritis: Mechanisms of Action
Authors: Malfait Anne-Marie , Malik Angela S. , Marinova-Mutafchieva Lilia , Butler Debra M. , Maini Ravinder N. , Feldmann Marc
Published: January 2023

The therapeutic potential of salbutamol, a β2-adrenergic agonist, was explored in collagen-induced arthritis. This study was based on a report that salbutamol, by elevating intracellular cAMP, inhibits IL-12 production by macrophages and dendritic cells, thus preventing Th1 development. Ten-week-old male DBA/1 mice were immunized by intradermal injection of type II collagen in CFA. Arthritis developed 15–30 days later and the mice were treated after onset of disease with salbutamol, 200 μg i.p. After 10 days, the mice were sacrificed, and the hind paws were evaluated histologically. Salbutamol, 200 μg daily or every other day, had a profound therapeutic effect on the clinical progression of arthritis, as assessed by clinical score and paw thickness. The therapeutic effect was dose dependent. Daily administration of 200 μg of salbutamol offered the best protection against joint damage, as assessed by histology. In vitro, salbutamol reduced IL-12 and TNF-α release by peritoneal macrophages in a dose-dependent manner, as well as TNF release by synovial cells from arthritic mice. Ex vivo, draining lymph node cells of the salbutamol-treated arthritic mice showed a diminished CII-specific IFN-γ production and proliferation. In vivo, salbutamol specifically blocked mast cell degranulation in joint tissues. In conclusion, salbutamol has important effects on the immunoinflammatory response and a significant therapeutic action in collagen-induced arthritis.
Cutting Edge: Contrasting Roles of TNF Receptor-Associated Factor 2 (TRAF2) and TRAF3 in CD40-Activated B Lymphocyte Differentiation
Authors: Hostager Bruce S. , Bishop Gail A.
Published: January 2023

In B lymphocytes, CD40 signals contribute to the activation of Ab secretion, isotype switching, T cell costimulation, and immunological memory. TRAF proteins appear to be important components of the CD40 signal transduction complex, but their roles in the activation of B cell effector functions are poorly understood. We examined the contributions of TNF receptor-associated factor 2 (TRAF2) and TRAF3 to CD40-activated differentiation in mouse B cells transfected with inducible TRAF and dominant-negative TRAF cDNAs. We find that binding of TRAF2 and TRAF3 to CD40 is not required for the induction of Ab secretion, but that both TRAF molecules can regulate the activation process. We demonstrate a negative regulatory role for TRAF3 and that this activity is dependent on the availability of an intact TRAF3-binding site in the cytoplasmic domain of CD40. In contrast, TRAF2 appears to play a positive role in B cell differentiation, and this activity is apparent even when its binding site on CD40 is disrupted.
Potential Role of CD4+ T Cell-Mediated Apoptosis of Activated Astrocytes in Theiler’s Virus-Induced Demyelination
Authors: Palma JoAnn P. , Yauch Robert L. , Lang Sharon , Kim Byung S.
Published: January 2023

Intracerebral inoculation of Theiler’s murine encephalomyelitis virus (TMEV) into susceptible mouse strains results in a chronic, immune-mediated demyelinating disease similar to human multiple sclerosis. Here, we examined the role of astrocytes as an APC population in TMEV-induced demyelination and assessed the potential consequences of T cell activation following Ag presentation. IFN-γ-pretreated astrocytes were able to process and present all the predominant T cell epitopes of TMEV to virus-specific T cell hybridomas, clones, as well as bulk T cells. Despite low levels of proliferation of T cells due to prostaglandins produced by astrocytes, such Ag presentation by activated astrocytes induced the production of IFN-γ, a representative proinflammatory cytokine, in TMEV-specific Th cell clones derived from the CNS of virus-infected mice. Furthermore, these Th cell clones mediate lysis of the astrocytes in vitro in a Fas-dependent mechanism. TUNEL staining of CNS tissue demonstrates the presence of apoptotic GFAP+ cells in the white matter of TMEV-infected mice. These results strongly suggest that astrocytes could play an important role in the pathogenesis of TMEV-induced demyelination by activating T cells, subsequently leading to T cell-mediated apoptosis of astrocytes and thereby compromising the blood-brain barrier.
ICAM-2 and a Peptide from Its Binding Domain Are Efficient Activators of Leukocyte Adhesion and Integrin Affinity
Authors: Kotovuori Annika , Pessa-Morikawa Tiina , Kotovuori Pekka , Nortamo Pekka , Gahmberg Carl G.
Published: January 2023

Cell adhesion mediated by the CD11/CD18 integrins and their ligands, the ICAMs, is required for many leukocyte functions. In resting cells the integrins are nonadhesive, but when activated they become adhesive for their ligands. Previous findings have shown that a peptide derived from the first Ig domain of ICAM-2 (P1) binds to LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) and activates leukocyte aggregation. Because its mechanism of action has remained poorly understood, we have now studied the peptide-induced ligand binding in detail. Here we show that P1 was able to induce CD11/CD18-dependent adhesion of human T lymphocytes to immobilized, purified ICAM-1, -2, and -3. The optimal peptide concentration was 150 μg/ml, whereas concentrations higher than 400 μg/ml did not have any stimulatory effect. The increase in adhesion was detectable within 10 min of treatment with the peptide; it was dependent on energy, divalent cations, temperature, and an intact cytoskeleton but was unaffected by protein kinase C and protein tyrosine kinase inhibitors. Peptide treatment resulted in strong stimulation of the binding of soluble, recombinant ICAMs to T lymphocytes, showing that the integrin affinity toward its ligands was increased. Importantly, soluble ICAM-2Fc was also able to induce T lymphocyte adhesion to purified ICAM-1, -2, and -3, and it was a more potent stimulatory molecule than ICAM-1Fc or ICAM-3Fc.
IL-12 Delivery from Recombinant Vaccinia Virus Attenuates the Vector and Enhances the Cellular Immune Response Against HIV-1 Env in a Dose-Dependent Manner
Authors: Gherardi M. Magdalena , Ramirez Juan C. , Rodríguez Dolores , Rodríguez Juan R. , Sano Gen-Ichiro , Zavala Fidel , Esteban Mariano
Published: January 2023

To develop vaccination strategies against HIV-1 infection aimed to specifically enhance the cell-mediated immunity (CMI), we have engineered vaccinia virus (VV) recombinants expressing HIV-1 Env (rVVenv) and murine IL-12 (rVVlucIL-12) genes or coexpressing both genes (rVVenvIL-12). In mice inoculated with rVVlucIL-12 there is a rapid clearance of the virus, and this correlates with the induction of high levels of IL-12 and IFN-γ in serum and spleen early after infection. Enzyme-linked immunospot analysis of mice inoculated with rVVlucIL-12, revealed a nearly 2-fold increase in the number of specific anti-VV CD8+ T cells compared with that in mice given control rVV, and the serum Ab response was biased in favor of a Th1 response. An enhancement of about 2-fold in the number of anti-gp160 IFN-γ-secreting CD8+ T cells was observed in mice inoculated with rVVenvIL-12, when a dose of 1 × 107 PFU/mouse was used, but this enhancement was not observed when mice were given 5 × 107 PFU. This variation with virus dosage was confirmed in mice immunized simultaneously with different multiplicities of rVV expressing singly the env or IL-12 genes. The highest specific CMI was obtained in mice coadministered a low dose (2 × 104 PFU) of rVVlucIL-12 and 1 × 107 PFU of rVVenv. Our findings provide evidence for specific enhancement of the CMI to HIV-1 Env by the differential expression of IL-12 and env genes delivered from VV recombinants. This approach can be of wide vaccination interest as a means to improve immune responses to other Ags.
Differential Regulation of the IL-12 p40 Promoter and of p40 Secretion by CpG DNA and Lipopolysaccharide
Authors: Cowdery John S. , Boerth Nancy J. , Norian Lyse A. , Myung Peggy S. , Koretzky Gary A.
Published: January 2023

Challenge of macrophages with DNA containing an internal CpG motif results in IL-12 p40 secretion. In the presence of IFN-γ, CpG DNA induces more p40 secretion than does LPS. In the RAW 264 macrophage cell line, both CpG DNA and LPS activate a p40 promoter-reporter construct, and the promoter response to either agent is augmented 2- to 5-fold by IFN-γ. While either LPS or CpG DNA induces p40 promoter activity, only CpG DNA induces an increase in p40 mRNA or protein secretion. Even though IFN-γ augmented LPS-driven p40 promoter activity in RAW 264 cells, the combination of IFN-γ and LPS induced less p40 mRNA or protein secretion than the combination of IFN-γ and CpG DNA. The ability of IFN-γ to augment LPS or CpG DNA-induced p40 promoter activation was observed with truncation mutants of the IL-12 promoter containing as few as 250 bp 5′ of the TATA box. Although LPS alone is a poor inducer of p40 transcription, both LPS and CpG DNA induce similar nuclear translocation of NF-κB. This binding is not augmented by costimulation with IFN-γ. Thus, CpG DNA induces p40 transcription by a mechanism that includes NF-κB translocation; however, CpG DNA appears to induce other factor(s) necessary for p40 transcription. These results illustrate fundamental differences between CpG DNA and LPS with respect to activation of IL-12 p40 secretion.
Lysosomal Accumulation and Recycling of Lipopolysaccharide to the Cell Surface of Murine Macrophages, an In Vitro and In Vivo Study
Authors: Forestier Claire , Moreno Edgardo , Pizarro-Cerda Javier , Gorvel Jean-Pierre
Published: January 2023

In this study, we detailed in a time-dependent manner the trafficking, the recycling, and the structural fate of Brucella abortus LPS in murine peritoneal macrophages by immunofluorescence, ELISA, and biochemical analyses. The intracellular pathway of B. abortus LPS, a nonclassical endotoxin, was investigated both in vivo after LPS injection in the peritoneal cavity of mice and in vitro after LPS incubation with macrophages. We also followed LPS trafficking after infection of macrophages with B. abortus strain 19. After binding to the cell surface and internalization, Brucella LPS is routed from early endosomes to lysosomes with unusual slow kinetics. It accumulates there for at least 24 h. Later, LPS leaves lysosomes and reaches the macrophage cell surface. This recycling pathway is also observed for LPS released by Brucella S19 following in vitro infection. Indeed, by 72 h postinfection, bacteria are degraded by macrophages and LPS is located inside lysosomes dispersed at the cell periphery. From 72 h onward, LPS is gradually detected at the plasma membrane. In each case, the LPS present at the cell surface is found in large clusters with the O-chain facing the extracellular medium. Both the antigenicity and heterogenicity of the O-chain moiety are preserved during the intracellular trafficking. We demonstrate that LPS is not cleared by macrophages either in vitro or in vivo after 3 mo, exposing its immunogenic moiety toward the extracellular medium.
IL-18, a Novel Immunoregulatory Cytokine, Is Up-Regulated in Crohn’s Disease: Expression and Localization in Intestinal Mucosal Cells
Authors: Pizarro Theresa T. , Michie Miette H. , Bentz Marcia , Woraratanadharm Jan , Smith Michael F. , Foley Eugene , Moskaluk Christopher A. , Bickston Stephen J. , Cominelli Fabio
Published: January 2023

IL-18, a novel immunoregulatory cytokine with potent IFN-γ-inducing activities, may play an important role in Th1-mediated chronic inflammatory disorders. The aim of the present study was to characterize the expression and localization of IL-18 in colonic specimens and isolated mucosal cell populations from patients with Crohn’s disease (CD), a prototypic Th1-mediated disorder. Using a semiquantitative RT-PCR protocol, IL-18 mRNA transcripts were found to be increased in freshly isolated intestinal epithelial cells (IEC) and lamina propria mononuclear cells (LPMC) from CD compared with ulcerative colitis (UC) and noninflamed control (cont) patients, and were more abundant in IEC compared with LPMC. Immunohistochemical analysis of surgically resected colonic tissues localized IL-18 to both LPMC (specifically, macrophages and dendritic cells) as well as IEC. Staining was more intense in CD compared with UC and cont, and in involved (inv) vs noninvolved (n inv) areas. Western blot analysis revealed that an 18.3-kDa band, consistent with both recombinant and mature human IL-18 protein, was found predominantly in CD vs UC intestinal mucosal biopsies; a second band of 24 kDa, consistent with the inactive IL-18 precursor, was detected in n inv areas from both CD and UC biopsies and was the sole form found in noninflamed cont. To our knowledge, this report is the first describing increased expression of IL-18 in a human Th1-mediated chronic inflammatory disease. In addition, our studies further support the concept that IEC and dendritic cells may possess important immunoregulatory functions in both normal, as well as pathological, mucosal immunity.
Does IgE Bind to and Activate Eosinophils from Patients with Allergy?
Authors: Kita Hirohito , Kaneko Masayuki , Bartemes Kathleen R. , Weiler Deborah A. , Schimming Andrew W. , Reed Charles E. , Gleich Gerald J.
Published: January 2023

Human eosinophils have been reported to express both the mRNA and protein for the high affinity IgE receptor (FcεRI); it is speculated that this receptor plays a role in eosinophil mediator release in allergic diseases. However, questions still remain. How much of the FcεRI protein is actually expressed on the cell surface of the eosinophil? If they are present, are these IgE receptors associated with effector functions of eosinophils? To address these issues, we studied blood eosinophils from patients with ragweed hay fever. A high level of low affinity IgG receptor (FcγRII, CD32), but no expression of FcεRI, was detectable on the eosinophil surface by standard FACS analysis. However, after in vitro sensitization with biotinylated chimeric IgE (cIgE), cell-bound cIgE was detected by PE-conjugated streptavidin. This cIgE binding was partially inhibited by anti-FcεRI mAb, suggesting that eosinophils do express minimal amounts of FcεRI detectable only by a sensitive method. Indeed, FACS analysis of whole blood showed that eosinophils express ∼0.5% of the FcεRI that basophils express. When stimulated with human IgE or anti-human IgE, these eosinophils did not exert effector functions; there was neither production of leukotriene C4 or superoxide anion nor any detectable degranulation response. In contrast, eosinophils possessed membrane-bound human IgG and showed functional responses when stimulated with human IgG or anti-human IgG. Thus, IgG and/or cytokines, such as IL-5, appear to be more important for eosinophil activation in allergic diseases than IgE.
Cutting Edge: The CD45 Tyrosine Phosphatase Is an Inhibitor of Lck Activity in Thymocytes
Authors: D’Oro Ugo , Ashwell Jonathan D.
Published: January 2023

A widely accepted model for regulation of the Lck tyrosine kinase is that it is activated by CD45-mediated dephosphorylation of its COOH-terminal negative regulatory tyrosine (Tyr505). Previous work from our laboratory, however, found that despite hyperphosphorylation of Tyr505, the activity of Lck from CD45− T cell lines was actually increased due to hyperphosphorylation of the positive regulatory tyrosine, residue 394. To avoid potential complications introduced by transformed cells, in this study we have characterized the effect of CD45 on Lck activity in normal cells. Lck in thymocytes from CD45−/− mice was hyperphosphorylated on tyrosine residues. Importantly, and in disagreement with the model that CD45 only activates Lck in vivo, the kinase activity of Lck from cells lacking CD45 was substantially increased. These results support a model in which CD45 dephosphorylates both Tyr505 and Tyr394, the net effect in normal thymocytes being a decrease in enzymatic activity.
Fas Ligand and Fas Are Expressed Constitutively in Human Astrocytes and the Expression Increases with IL-1, IL-6, TNF-α, or IFN-γ
Authors: Choi Chulhee , Park Joo Young , Lee Jeonggi , Lim Jung-Hee , Shin Eui-Cheol , Ahn Young Soo , Kim Chul-Hoon , Kim Se-Jong , Kim Joo-Deuk , Choi Il Saing , Choi In-Hong
Published: January 2023

Fas ligand (FasL) and Fas are mediators of apoptosis, which are implicated in the peripheral deletion of autoimmune cells, activation-induced T cell death, and cytotoxicity mediated by CD8+ T cells. Fas is also believed to be involved in several central nervous system diseases, but until now, the effector cells expressing FasL in the brain have not been identified. We investigated the expression levels of Fas and FasL with the stimulation of cytokines and the possible effector cells targeting Fas-bearing cells. Our data demonstrated that: 1) FasL is expressed constitutively on astrocytes taken from a fetus or an adult and that its expression increases when these cells are treated with IL-1, IL-6, or TNF-α in which the pretreatment of IFN-γ triggers astrocytes to express more FasL; 2) astrocytes induce apoptosis in MOLT-4 cells through FasL; 3) Fas is also expressed constitutively and is up-regulated by IL-1, IL-6, or TNF-α in which the pretreatment of IFN-γ triggers astrocytes to express more Fas; 4) apoptosis occurs when fetal astrocytes are treated with agonistic anti-Fas IgM Ab after culture with IFN-γ and TNF-α; and 5) TNF-related apoptosis inducing ligand is up-regulated in fetal astrocytes with stimuli of IL-1 or TNF-α. These findings suggest a possible role of astrocytes in the induction of apoptosis in central nervous system diseases.
The Role of CD80, CD86, and CTLA4 in Alloimmune Responses and the Induction of Long-Term Allograft Survival
Authors: Judge Thomas A. , Wu Zihou , Zheng Xiang-Guang , Sharpe Arlene H. , Sayegh Mohamed H. , Turka Laurence A.
Published: January 2023

Blocking the interaction of the CD28 costimulatory receptor with its ligands, CD80 and CD86, inhibits in vivo immune responses, such as allograft rejection, and in some instances induces tolerance. Previously, we found that CTLA4Ig, which blocks the CD28/CTLA-4 (CD152) ligands CD80 and CD86, can be used to induce transplantation tolerance to vascularized allografts. Recent data suggest that an intact CD152-negative signaling pathway is essential for induction of tolerance to nominal Ags. Here, we show that blockade of CD152 using an anti-CD152 mAb at the time of transplantation prevents the induction of long-term allograft survival by agents that target CD80 and CD86. In contrast, CD152 signals are not required for the maintenance of established graft survival. We also report for the first time that blockade of CD86 alone can induce long-term graft survival. This requires that anti-CD86 mAb is given on the day of transplantation and also depends upon an intact CD152 pathway. This result, plus experiments using CD80-deficient mice, suggests a dominant role for CD80 molecules on donor cells as the relevant ligand for CD152. We additionally find that blockade of CD152 at the time of transplantation does not interfere with the effectiveness of anti-CD154 mAbs, suggesting distinct mechanisms for inhibition of graft rejection by blocking the CD28 vs CD154 pathways.
Identification of Tyrosine Phosphorylation Sites in the CD28 Cytoplasmic Domain and Their Role in the Costimulation of Jurkat T Cells
Authors: Sadra Ali , Cinek Tomas , Arellano Jerry L. , Shi Jia , Truitt Kenneth E. , Imboden John B.
Published: January 2023

The cytoplasmic domain of CD28 contains four tyrosine residues. Because signal transduction by CD28 appears to involve its tyrosine phosphorylation, we determined sites of CD28 tyrosine phosphorylation using mutants of mouse CD28 that retained tyrosine at one position, with the remaining three positions mutated to phenylalanine. When expressed in Jurkat cells and stimulated by mAb, only the mutants with tyrosine at position 170 or 188 were tyrosine phosphorylated. Phosphorylation of Tyr170 recruits phosphatidylinositol 3-kinase to CD28. Tyr188 has not been associated with any specific signaling event, but we found that ligation of CD28 by the natural ligand B7.2 also induced phosphorylation of Tyr188, suggesting that this event is of physiological importance. Consistent with that possibility, mutation of Tyr188 to phenylalanine severely impaired the ability of mouse CD28 to deliver a costimulus for the expression of CD69 and the production of IL-2. The functional consequences of the mutation of Tyr188 were unique; mutation of the other three tyrosines, individually or in combination, did not impair costimulation. Therefore, of the four CD28 tyrosine residues only Tyr188 is required for signaling in Jurkat cells, suggesting that its phosphorylation is a key event in the costimulation of T cells.
Evidence of Positive Cross-Regulation on Th1 by Th2 and Antigen-Presenting Cells: Effects on Th1 Induced by IL-4 and IL-12
Authors: Oriss Timothy B. , McCarthy Susan A. , Campana Martha A. K. , Morel Penelope A.
Published: January 2023

The response of Th cells to cytokines is normally strictly regulated, such that following antigenic stimulation, Th cells respond for only a short period of time, after which they become refractory to cytokine-mediated effects. IL-12, a costimulator of Th1 having no proliferation-inducing capacity of its own, allows Th1 clones and lines to respond to IL-4 when they would otherwise be unable to respond to this cytokine. Cells that have proliferated in response to IL-4 plus IL-12 are fully able to be subsequently activated by specific Ag and APC. Additionally, the response to IL-4 of Th1 effector cells derived from normal murine spleen is enhanced significantly by IL-12. Furthermore, in the presence of IL-12, stimulated Th2 can induce proliferation of Th1 via IL-4 production, in a dual chamber culture system. We hypothesize that the effects of IL-4 and IL-12 represent a novel, positive cross-regulatory pathway that acts on Th1, and is mediated by Th2 (the IL-4 source) and APC (the IL-12 source). We propose this as a way for a Th2 immune response to positively influence an ongoing or waning Th1 response.
Positive Recognition of MHC Class I Molecules by the Ly49D Receptor of Murine NK Cells
Authors: George Thaddeus C. , Mason Llewellyn H. , Ortaldo John R. , Kumar Vinay , Bennett Michael
Published: January 2023

Members of the murine Ly49 family of receptors have been shown to inhibit and activate NK cell function. Subsets of Ly49-expressing NK cells mediate the rejection of bone marrow cell allografts and the lysis of allogeneic lymphoblasts. In this report we have studied Ly49-mediated positive and negative signaling in an in vitro cytotoxicity assay using sorted NK cell subsets as effectors and a panel of 51Cr-labeled Con A lymphoblasts as targets in the presence or the absence of Abs to Ly49 and/or class I molecules. Our results demonstrate that the activating receptor Ly49D delivers stimulatory signals for target cell lysis upon interacting with H2-Dd, Dr, and Dsp2, but not H2b or H2k class I Ags. On the other hand, the inhibitory receptor Ly49G2 delivers negative signals for target cell lysis upon interacting with Dd, Dr, and H2k, but not H2b or Dsp2, class I Ags. Furthermore, Ly49-mediated negative signaling dominates Ly49D-mediated positive signaling. Thus, lysis of class I MHC-bearing targets by NK cells is not merely the consequence of the absence of an Ly49-mediated negative signal, but also requires positive recognition of class I molecules by certain Ly49 receptors. Activation of NK cells by nonself class I molecules was not predicted by the missing self hypothesis.
Functional Expression of Receptors for Calcitonin Gene-Related Peptide, Calcitonin, and Vasoactive Intestinal Peptide in the Human Thymus and Thymomas from Myasthenia Gravis Patients
Authors: Marie J.-C. , Wakkach A. , Coudray A.-M. , Chastre E. , Berrih-Aknin S. , Gespach C.
Published: January 2023

The molecular and functional expression of serpentine membrane receptors for vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), and calcitonin (CT) were characterized in human thymus and thymomas from myasthenia gravis (MG) patients and thymic epithelial cells either in primary culture (PTEC) or transformed by the siman virus 40 large T (SV40LT) oncogene (LT-TEC). Using RT-PCR combined with Southern analysis, we identified the PCR products corresponding to the receptor (-R) transcripts for VIP, CGRP, and CT in thymus from control subjects and MG patients with either hyperplasia or thymoma. Similar expressions of the VIP- and CGRP-R transcripts were observed in PTEC, whereas the CT-R message was not detected. In LT-TEC, the signals for VIP-R, CGRP-R, and CT-R transcripts were seen with a lower intensity than those in control and MG thymus. In agreement with our molecular analysis, 1) VIP was the most potent peptide among VIP-related peptides (VIP > PACAP > PHM > PHV) to stimulate cAMP production through specific type 1 VIP receptors in both PTEC and LT-TEC; 2) cAMP generation was induced by CGRP in PTEC and by CT in LT-TEC; 3) in frozen thymic sections and by flow cytometry, type 1 VIP-R, CGRP-R, and CT-R were localized in epithelial cells; and 4) in parallel, the transcription of the acetylcholine receptor α subunit (the main autoantigen in MG) was induced by CGRP and CT in PTEC and LT-TEC, respectively. Our data suggest that the neuroendocrine peptides VIP, CGRP, and CT may exert functional roles during MG and malignant transformation of the human thymus.
Context-Dependent Immunogenicity of an S206G-Substituted H-2Db-Restricted Simian Virus 40 Large T Antigen Epitope I Variant
Authors: Mylin Lawrence M.
Published: January 2023

SV40 large tumor Ag (Tag) contains four H-2b-restricted (I, II/III, IV, and V) CTL epitopes. A hierarchy exists among these CTL epitopes. CTL directed against epitopes I, II/III, and IV are readily detected following immunization of H-2b mice with SV40, Tag-transformed syngeneic cells, or a vaccinia recombinant that expresses full-length Tag, while epitope V-specific CTL are not. The mechanisms that define this hierarchy remain unknown. Initial studies have shown that the locations of epitopes I and V within SV40 Tag do not determine the immunological potencies of these epitopes. Like the wild-type Tag, derivatives in which the locations of epitopes I and V were precisely reversed within Tag failed to induce epitope V-specific CTL, but did induce epitope I-specific CTL. The use of an S206G-substituted epitope I variant (GAINNYAQKL) revealed that the S206G variant sequence induced CTL when located within the native epitope I context, but failed to do so when located within the epitope V context of Tag. Mutagenesis of residues adjacent to the S206G-substituted epitope I variant revealed that the identity of the residue flanking the amino terminus of the S206G variant was critical when it resided within the epitope V location, but not within the epitope I location. These results demonstrate that effects imposed by both regional context and adjacent residues can modulate immunogenicity, but that the relative importance of such effects varies in an epitope-dependent manner.
IFN-γ-Mediated Control ofCoxiella burnetiiSurvival in Monocytes: The Role of Cell Apoptosis and TNF
Authors: Dellacasagrande Jérôme , Capo Christian , Raoult Didier , Mege Jean-Louis
Published: January 2023

The treatment of infectious diseases caused by intracellular bacteria, such as Q fever, may benefit from cytokines acting on macrophages. Monocytic THP-1 cells were infected with Coxiella burnetii, the etiological agent of Q fever, and then treated with IFN-γ. While C. burnetii multiplied in untreated monocytes, IFN-γ reduced bacterial viability after 24 h of treatment and reached maximum inhibition after 96 h. IFN-γ also affected the viability of infected cells. Cell death resulted from apoptosis; occurring 24 h after the addition of IFN-γ, it reached a maximum after 48 h and was followed by necrosis. Reactive oxygen intermediates were not required for C. burnetii killing, since monocytes from patients with chronic granulomatous disease were microbicidal in response to IFN-γ. The role of cytokines was also investigated. IFN-γ elicited a moderate release of IL-1β in infected monocytes. Moreover, the IL-1 receptor antagonist did not affect C. burnetii survival, suggesting that IL-1β was not involved in the bacterial killing induced by IFN-γ. TNF was involved in IFN-γ-induced killing of C. burnetii and cell death. IFN-γ induced mRNA expression and sustained secretion of TNF. Neutralizing Abs to TNF as well as Abs directed against TNF receptors I and II, significantly prevented IFN-γ-dependent killing of C. burnetii and cell death. These results suggest that IFN-γ promotes the killing of C. burnetii in monocytes through an apoptotic mechanism mediated in part by TNF.
Bacterial DNA Containing CpG Motifs Stimulates Lymphocyte-Dependent Protection of Mice Against Lethal Infection with Intracellular Bacteria
Authors: Elkins Karen L. , Rhinehart-Jones Tonya R. , Stibitz Scott , Conover Jacqueline S. , Klinman Dennis M.
Published: January 2023

Bacterial DNA containing unmethylated CpG motifs activates mammalian lymphocytes and macrophages to produce cytokines and polyclonal Ig. These include IFN-γ, IL-12, TNF-α, and IL-6, which are important in the control of intracellular bacterial infection. Here, we show that bacterial DNA, as well as synthetic oligonucleotides containing CpG motifs, induce protection against large lethal doses of Francisella tularensis live vaccine strain (LVS) and Listeria monocytogenes. Methylation of DNA at CpG dinucleotides or inversion of the motif abolished this protection. Surprisingly, DNA-mediated protection was highly dependent on lymphocytes, particularly B cells, as well as the production of IFN-γ. Optimal protection was elicited 2–3 days after inoculation with DNA and persisted for up to 2 wk. Further, animals surviving lethal challenge developed pathogen-specific secondary immunity. These findings indicate that host innate immune responses to bacterial DNA may contribute to the induction of protective immunity to bacteria and the subsequent development of memory.
Modulation of Airway Inflammation by Passive Transfer of Allergen-Specific Th1 and Th2 Cells in a Mouse Model of Asthma
Authors: Randolph David A. , Carruthers Cynthia J. L. , Szabo Susanne J. , Murphy Kenneth M. , Chaplin David D.
Published: January 2023

Although evidence is strong that Th cells play a major role in mediating the airway inflammation observed in asthma, the relative contributions of the Th cell subsets, Th1 and Th2, are unclear. It has been suggested that asthma is driven by Th2 predominant responses in the lung, but other data suggest a role for Th1 cells as well. Here we show by intracellular cytokine staining and flow cytometric analysis that in the murine model of OVA-induced airway inflammation, both Th1 and Th2 cells are recruited to the airways. Th1 cells predominate early in the response and Th2 cells predominate late. We further show that increasing the number of Th1 cells by passive transfer of OVA-specific Th1 cells results in increased inflammation. This effect is observed regardless of whether the T cells are transferred before sensitization or after airway inflammation is already in progress. Transfer of Th1 cells also results in increased recruitment of host T cells of both Th1 and Th2 phenotypes. Passive transfer of Th2 cells results in little change in the inflammatory response. These results demonstrate that Ag-specific Th1 cells are not protective in this model of asthma, but rather may potentiate the inflammatory response.
Adjuvanticity of the Cholera Toxin A1-Based Gene Fusion Protein, CTA1-DD, Is Critically Dependent on the ADP-Ribosyltransferase and Ig-Binding Activity
Authors: Ågren Lena C. , Ekman Lena , Löwenadler Björn , Nedrud John G. , Lycke Nils Y.
Published: January 2023

The ADP-ribosylating enterotoxins, cholera toxin (CT) and Escherichia coli heat-labile toxin, are among the most powerful immunogens and adjuvants yet described. An innate problem, however, is their strong toxic effects, largely due to their promiscuous binding to all nucleated cells via their B subunits. Notwithstanding this, their exceptional immunomodulating ability is attracting increasing attention for use in systemic and mucosal vaccines. Whereas others have separated adjuvanticity from toxicity by disrupting the enzymatic activity of the A1 subunit by site-directed mutagenesis, we have constructed a nontoxic molecule that combines the full enzymatic activity of the A1 subunit with a B cell targeting moiety in a gene fusion protein, the CTA1-DD adjuvant. Despite its more selective binding properties, we found comparable adjuvant effects of the novel CTA1-DD adjuvant to that of CT. Here we unequivocally demonstrate, using a panel of mutant CTA1-DD molecules, that the immunomodulating ability of CTA1-DD is dependent on both an intact enzymatic activity and the Ig-binding ability of the DD dimer. Both agents, CT and CTA1-DD, ADP-ribosylate intact B cells. However, contrary to CT, no increase in intracellular cyclic AMP in the targeted cells was detected, suggesting that cyclic AMP may not be important for adjuvanticity. Most remarkably, CTA1-DD achieves similar immunomodulating effects to CT using a ganglioside-GM1 receptor-independent pathway for internalization.