bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Extracellular Degradation of Synthetic Class I Peptides by Human Dendritic Cells
Authors: Amoscato Andrew A. , Prenovitz Damon A. , Lotze Michael T.
Published: December 2022

Dendritic cells (DCs) effectively process exogenous and endogenous Ag and present peptide in the context of both class I and class II molecules. We have demonstrated that peripheral blood DCs efficiently degrade synthetic class I peptides at their cell surface within minutes as determined by analyzing DC supernatants by HPLC. Fragments were verified as bona fide cleavage products by direct sequencing using collision-induced dissociation tandem mass spectrometry. The predominant degradative activities were 1) not secreted but associated with activity at the plasma membrane, 2) ecto-orientated, 3) not induced by peptide-specific interactions, and 4) not associated with nonspecific uptake. Sequence analysis indicated that both N- and C-terminal as well as endoproteolytic events were occurring at the cell surface. The primary exoproteolytic event was identified as CD13 or CD13-like activity through inhibition studies and could be inhibited by ubiquitin and metal-chelating agents. Endoproteolytic events could be inhibited in the presence of DTT, but the precise nature of this enzyme is still undetermined. Compared with the starting monocyte population, DCs cultured in the presence of granulocyte-macrophage CSF/IL-4 exhibited the highest degradative rate (4.3 nmol/min), followed by cultured monocytes (2.9 nmol/min) and freshly isolated monocytes (1.0 nmol/min). In addition to increased enzymatic activity, a change in substrate specificity was noted. Results are discussed with respect to APC loading, and alternatives are offered for circumventing such degradation.
A Nontoxic Adjuvant for Mucosal Immunity to Pneumococcal Surface Protein A
Authors: Yamamoto Masafumi , Briles David E. , Yamamoto Shingo , Ohmura Mari , Kiyono Hiroshi , McGhee Jerry R.
Published: December 2022

In this study, we demonstrated that pneumococcal surface protein A (PspA) nasally administered with a nontoxic A subunit mutant of cholera toxin (mCT) S61F elicited a protective immune response. Immunization with PspA and mCT elicited higher levels of PspA-specific IgG and IgA Abs in serum and of IgG and IgA anti-PspA Ab-forming cells in spleens, cervical lymph nodes (CLN), and lung tissue when compared to nonimmunized mice. Furthermore, significant PspA-specific IgA Abs were induced in saliva and nasal secretions. These responses were dependent on the use of mCT as a mucosal adjuvant. The PspA-specific Ab responses induced by mCT S61F were comparable with those induced by native CT (nCT). Analysis of cytokine responses showed that nasal PspA plus mCT S61F enhanced the induction of PspA-specific CD4+ T cells producing IL-4 but not IFN-γ in CLN at both the protein and mRNA levels. Importantly, significant numbers of mice intranasally immunized with PspA plus mCT S61F were protected from lethal challenge with capsular serotype 3 Streptococcus pneumoniae A66. These results show that intranasal administration of PspA together with mCT S61F is an effective mucosal vaccine against pneumococcal infection and induces CD4+ Th2-type cells, which provide help for both mucosal and systemic Ab responses.
IFN-γ, IL-12, and TNF-α Are Required to Maintain Reduced Liver Pathology in Mice Vaccinated withSchistosoma mansoniEggs and IL-12
Authors: Hoffmann Karl F. , Caspar Pat , Cheever Allen W. , Wynn Thomas A.
Published: December 2022

The development of hepatic fibrosis and portal hypertension is the principal cause of morbidity and mortality in schistosomiasis mansoni. Nevertheless, relatively little is known about the mechanisms that lead to excessive collagen deposition during infection with Schistosoma mansoni. In the murine model, infection leads to significant egg-induced granuloma formation, tissue eosinophilia, and hepatic fibrosis. The pathology has been linked to dominant type 2 cytokine expression, and our recent studies showed that sensitizing animals to egg Ags in combination with IL-12, before infection, led to a highly significant reduction in egg-induced immunopathology. In this study, we demonstrate that in contrast with egg/IL-12-sensitized animals that showed marked decreases in pathology, mice similarly sensitized but depleted of IFN-γ, IL-12, or TNF-α at the time of egg laying developed granulomas that were similar to the non-IL-12-treated control group. Although all three anti-cytokine-treated groups exhibited a dominant type 1 response in lymph node cells restimulated ex vivo, the expression of type 2 cytokine mRNA was markedly restored at the site of granuloma formation, which suggests that all three cytokines are required to maintain the suppressed type 2 pattern. Moreover, egg/IL-12-sensitized mice depleted of IFN-γ or IL-12 displayed a partial reduction in IFN-γ production, suggesting that multiple type 1 cytokines were required to maintain polarized type 1 responses to chronic type 2-inducing stimuli. Together, these data reveal key roles for IFN-γ, IL-12, and TNF-α in the protective effects mediated by this IL-12-based vaccine to prevent pathology.
The Memory B Cell Subset Responsible for the Secretory IgA Response and Protective Humoral Immunity to Rotavirus Expresses the Intestinal Homing Receptor, α4β7
Authors: Williams Marna B. , Rosé Jason R. , Rott Lusijah S. , Franco Manuel A. , Greenberg Harry B. , Butcher Eugene C.
Published: December 2022

Infection of mice with murine rotaviruses induces life-long immunity, characterized by high levels of IgA in the intestine and large numbers of rotavirus (RV)-specific Ab-secreting cells in gut-associated lymphoid tissues. Lymphocyte trafficking into gut-associated lymphoid tissues is mediated by interaction of the α4β7 integrin on lymphocytes with the vascular mucosal addressin cell adhesion molecule-1. To determine whether B cell memory for RV correlates with α4β7 expression, we transferred sorted B220+ phenotypically defined memory (IgD−α4β7high and IgD− α4β7−) and naive (IgD+α4β7+) splenocytes into recombination-activating gene-2 knockout mice (B and T cell-deficient) that were chronically infected with RV. Only mice receiving α4β7high memory (IgD−) B cells produced RV-specific IgA in the stool, cleared the virus, and were immune to reinfection. α4β7high (but not α4β7−) memory B cells from donors boosted as much as 7 mo previously also cleared the virus, indicating that α4β7high memory B cells maintain long term functional immunity to RV. Although only α4β7high memory cells provided mucosal immunity, α4β7− cells from recently boosted donor animals could generate RV-specific serum IgG, but, like naive (IgD+) B cells, were unable to induce viral clearance even 60 days after cell transfer. These data indicate that protective immunity for an intestinal pathogen, RV, resides in memory phenotype B cells expressing the intestinal homing receptor, α4β7.
Virus-Induced Immunoinflammatory Lesions in the Absence of Viral Antigen Recognition
Authors: Gangappa Shivaprakash , Babu John Sam , Thomas Johnson , Daheshia Massoud , Rouse Barry T.
Published: December 2022

Herpetic stromal keratitis (HSK) is a CD4+ T cell-controlled immunopathologic lesion in the eye that results from infection with herpes simplex virus (HSV). Target Ags involved in HSK remain undefined. In this study, we determined if HSK could be induced in animals genetically incapable of generating HSV Ag-specific CD4+ T cells. Mice bearing transgenic TCR specific to OVA peptide 323–339 (DO11.10) were crossed to SCID mice whose offspring (Tg-SCID) possessed CD4+ T cells, >98% of which expressed the OVA peptide-specific TCR. HSV infection of Tg-SCID mice was lethal, and mice failed to generate detectable T cell responses even after repeated immunization with a mutant avirulent virus (AN-1). Immunization with AN-1 virus followed by ocular challenge with HSV resulted in ocular inflammation before encephalitis, in contrast to the protection conferred in the control BALB/c and DO11.10 mice. These results indicate that clinical HSK may not require viral Ag recognition by CD4+ T cells and that T cells of irrelevant specificity can be recruited, activated, and driven into effector function in the HSV-infected cornea. This is suggested to represent a bystander activation effect resulting from the presence of proinflammatory mediators resulting from HSV replication.
Products of Sphingolipid Catabolism Block Activation of the p21-Activated Protein Kinases in Neutrophils
Authors: Lian Jian P. , Huang RiYun , Robinson Dwight , Badwey John A.
Published: December 2022

Neutrophils stimulated with the chemoatttractant FMLP are known to exhibit a rapid and transient activation of two p21-activated protein kinases (Paks) with molecular masses of approximately 63 and 69 kDa. Paks can be detected by their ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297 to 331 of the 47-kDa subunit of the nicotinamide-adenine dinucleotide phosphate-oxidase complex (p47-phox) fixed within a gel. In this study, we demonstrate that N-acetylsphingosine (C2-ceramide) and a variety of sphingoid bases (e.g., d-erythrosphingosine) block activation of the 63- and 69-kDa Paks in neutrophils. The concentrations of these lipids that were effective in blocking Pak activation were similar to those that inhibit a variety of neutrophil responses. Activation of the 63- and 69-kDa Paks was also markedly reduced in neutrophils treated with sphingomyelinase before stimulation. Moreover, we report that addition of C2-ceramide or d-erythrosphingosine to neutrophils after stimulation with FMLP markedly enhances the rate of Pak inactivation. These effects were not mimicked by arachidonate, which is a potent disorganizing agent of neutrophil membranes. These data support and extend the proposal that sphingoid bases may establish a set point in neutrophils for positive stimuli.
Differential Expression of Inflammatory Cytokines Parallels Progression of Central Nervous System Pathology in Two Clinically Distinct Models of Multiple Sclerosis
Authors: Begolka Wendy Smith , Vanderlugt Carol L. , Rahbe Sandra M. , Miller Stephen D.
Published: December 2022

Multiple sclerosis is an immune-mediated demyelinating disease of unknown etiology that presents with either a chronic-progressive or relapsing-remitting clinical course. Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) and relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE) in the SJL/J mouse are both relevant murine CD4+ T cell-mediated demyelinating models that recapitulate the multiple sclerosis disease phenotypes. To determine the cellular and molecular basis for these observed differences in clinical course, we quantitatively analyzed the temporal expression of pro- and antiinflammatory cytokine mRNA expression in the central nervous system (CNS) and the phenotype of the inflammatory mononuclear infiltrates. TMEV-infected SJL/J mice expressed IFN-γ, TNF-α, IL-10, and IL-4 mRNA during the preclinical phase, and their levels continued to increase throughout the duration of the chronic-progressive disease course. These data correlated with the continued presence of both CD4+ T cells and F4/80+ macrophages within the CNS infiltrates. In contrast, SJL/J mice with PLP139–151-induced R-EAE displayed a biphasic pattern of CNS expression for the proinflammatory cytokines, IFN-γ and TNF-α, with expression peaking at the height of the acute phase and relapse(s). This pattern correlated with dynamic changes in the CD4+ T cell and F4/80+ macrophage populations during relapsing-remitting disease progression. Interestingly, IL-4 message was undetectable until disease remission(s), demonstrating its potential role in the intrinsic regulation of ongoing disease, whereas IL-10 was continuously expressed, arguing against a regulatory role in either disease. These data suggest that the kinetics of cytokine expression together with the nature of the persistent inflammatory infiltrates are major contributors to the differences in clinical course between TMEV-IDD and R-EAE.
Cutting Edge: Bystander Apoptosis Triggers Dendritic Cell Maturation and Antigen-Presenting Function
Authors: Rovere Patrizia , Vallinoto Cristina , Bondanza Attilio , Crosti Maria Cristina , Rescigno Maria , Ricciardi-Castagnoli Paola , Rugarli Claudio , Manfredi Angelo A.
Published: December 2022

Physiologic cell death via apoptosis occurs without inflammation or autoimmunity. Here, we investigated the outcome of the interaction of apoptotic cells with dendritic cells (DCs), which are potent professional APCs. DCs internalized apoptotic cells and processed them for presentation to both MHC class I- and class II-restricted T cells with an efficiency that was dependent upon the number of apoptotic cells. The latter event was accompanied by the autocrine/paracrine secretion of IL-1β and TNF-α, with eventual DC maturation. High numbers of apoptotic cells, mimicking a failure of their in vivo clearance, are therefore sufficient to trigger DC maturation and the presentation of intracellular Ags from apoptotic cells, even in the absence of exogenous “danger” signals.
The Rate of Dissociation Between Antibody and Antigen Determines the Efficiency of Antibody-Mediated Antigen Presentation to T Cells
Authors: Guermonprez Pierre , England Patrick , Bedouelle Hugues , Leclerc Claude
Published: December 2022

We analyzed the role of Ab affinity on Ab-mediated Ag uptake and presentation to T cells. Hen egg white lysozyme (HEL) was captured by bifunctional hybrid proteins (Fv-MalE) in which the variable fragment (Fv) of the anti-HEL mAb D1.3 was covalently linked to the Escherichia coli MalE protein. These complexes were targeted via two anti-MalE mAbs to an APC expressing a receptor for the Ab constant region. The combination of Fv-MalE and anti-MalE mAbs increased, specifically, HEL presentation. With this experimental system, we evaluated the impact of six different mutations, affecting the Fv-MalE complementarity determining regions, on the increase of HEL presentation by the corresponding hybrids. These mutations increase the dissociation rate constant (koff), and, thus, the dissociation constant of the HEL/Fv-MalE interaction, up to 650-fold, as compared with the wt Fv-MalE. Increasing the koff from 7 × 10−4 s−1 up to 300 × 10−4 s−1 did not interfere with the enhancement of HEL presentation. A mutant with a koff of 600 × 10−4 s−1 had a reduced enhancement ability, and mutants with koff higher than 5700 × 10−4 s−1 did not enhance HEL presentation at all. These results show that affinity determines the efficiency of Ab-mediated Ag presentation to T cells. One consequence is that affinity maturation in specific B lymphocytes can drastically enhance their ability to collaborate with T cells in an MHC-restricted way. This may contribute to the selection of high affinity B cell clones.
Recombinant Rat Surfactant-Associated Protein D Inhibits Human T Lymphocyte Proliferation and IL-2 Production
Authors: Borron Paul J. , Crouch Erika C. , Lewis James F. , Wright Jo Rae , Possmayer Fred , Fraher Laurence J.
Published: December 2022

Components of the airspace-lining material may contribute to the local regulation of immune function within the lung. We report here that recombinant rat pulmonary surfactant-associated protein D (SP-D) inhibits the lectin- and anti-CD3-stimulated proliferation of human PBMCs. Inhibition was associated with a decreased production of IL-2, and the addition of human rIL-2 blocked the inhibitory action of SP-D. These effects were not inhibited by maltose, indicating that the inhibitory activity was not dependent upon the lectin activity of SP-D. Studies employing mutant SP-D lacking N-linked sugars or defective in multimerization further indicated that inhibition was not dependent upon cellular interactions with the N-linked oligosaccharide on SP-D or the oligomerization of trimeric SP-D subunits. Although a peptide containing an inverted DGR showed similar IL-2-dependent effects on anti-CD3-stimulated proliferation, deletion of the conserved DGRDGR sequence near the amino-terminal end of the collagen domain did not decrease the suppressive activity of SP-D. We hypothesize that SP-D can dampen lymphocyte responses to exogenous stimuli and protect the lung against collateral immune-mediated damage.
Regulation of Mouse CD72 Gene Expression During B Lymphocyte Development
Authors: Ying Han , Healy James I. , Goodnow Christopher C. , Parnes Jane R.
Published: December 2022

CD72 is a 45-kDa transmembrane glycoprotein that is predominantly expressed on cells of the B lineage except plasma cells. Previously, we identified the 255-bp minimal mouse CD72 promoter capable of tissue-specific and developmental stage-specific expression. DNase I footprinting analysis of the 255-bp CD72 promoter revealed three protected elements, footprint (FP) I, FP II, and FP III. FP II, which extends from nucleotide −189 to −169 of the mouse CD72 promoter, exhibited both tissue-specific and developmental stage-specific activity that was reflective of the activity of the CD72 gene in vivo. In this report, we show that FP II is specifically recognized by the transcription factor B cell-specific activator protein (BSAP). Mutations eliminating the binding of BSAP in reporter constructs also eliminated the increase of reporter activity in B cells. In addition, cotransfections with reporter constructs plus different amounts of expression plasmids for BSAP showed that CD72 promoter activity was up-regulated by BSAP in plasmacytoma cells and T cells in a dose-dependent manner. Moreover, the expression level of CD72 decreased 10-fold on normal plasma cells. Compared with the presence of BSAP binding in mature B cells, the binding of BSAP was undetectable in those plasma cells. This study strongly suggests that BSAP-FP II interaction plays a critical role in determining the cell-type specificity of the CD72 promoter. The absence of positive factors such as BSAP accounts for at least part of the loss of mouse CD72 expression in plasma cells and thus might be common for the down-regulation of many molecules at the plasma cell stage.
FcεRI-Mediated Induction of TNF-α Gene Expression in the RBL-2H3 Mast Cell Line: Regulation by a Novel NF-κB-Like Nuclear Binding Complex
Authors: Pelletier Claire , Varin-Blank Nadine , Rivera Juan , Iannascoli Bruno , Marchand Françoise , David Bernard , Weyer Anne , Blank Ulrich
Published: December 2022

Using rat basophilic leukemia (RBL-2H3) cells as a model, we investigated how aggregation of the high affinity receptor for IgE (FcεRI) regulates TNF-α gene expression. Antigenic stimulation of RBL-2H3 cells led to an increase in newly synthesized TNF-α mRNA that was dependent on continuous receptor aggregation and did not require de novo protein synthesis. Kinetic analysis showed that maximal levels were achieved at 60 min and waned by 180 min of stimulation. Concomitant with the transcriptional activation of the TNF-α gene, the rapid appearance and disappearance of a previously uncharacterized nuclear NF-κB DNA binding activity, comprised of two distinct protein complexes, were observed. These protein complexes bound to NF-κB sites within the TNF-α gene and contained novel proteins (three species of Mr between 90,000–110,000) distinct from the classical proteins in NF-κB complexes. The induced NF-κB binding activity required continuous receptor stimulation and induced NF-κB-dependent reporter gene expression. Consistent with a role for the novel NF-κB nuclear binding activity in TNF-α gene expression, deletion of several 5′ κB elements in the TNF-α promoter abolished all measurable FcεRI-dependent induction of a reporter construct. Pharmacologic agents that inhibited the NF-κB binding activity also inhibited TNF-α mRNA expression. Our results demonstrate that a novel NF-κB-like nuclear binding activity plays an important role in regulation of the rapid and transient transcriptional activation of the TNF-α gene via FcεRI.
Defective Function of Langerhans Cells in Tumor-Bearing Animals Is the Result of Defective Maturation from Hemopoietic Progenitors
Authors: Ishida Tadao , Oyama Tsunehiro , Carbone David P. , Gabrilovich Dmitry I.
Published: December 2022

Langerhans cells (LC), the APCs in the skin, serve as a model for investigation of dendritic cell (DC) function in tissues. DC play a crucial role in the generation of antitumor immune responses. In this study, we investigated the effect of the presence of tumor in vivo on the ability of LC to take up Ag, migrate to draining lymph nodes, and stimulate primary T cell responses. In two animal models, these functions were substantially inhibited. This effect was not restricted to LC located in the skin near a tumor but was also seen at sites distant from the tumor. The duration of tumor exposure, and not its ultimate size, were found to be important, suggesting that tumors could be inhibiting the maturation of LC rather than directly inhibiting their function. Model experiments with radiation chimeras supported this hypothesis. To investigate the potential role of vascular endothelial growth factor (VEGF) in these effects we used anti-VEGF-neutralizing Ab to treat animals bearing tumors. Treatment with the Ab at a dose of 10 μg i.p. per mouse, twice a week for 4 wk, significantly improved the number and function of LC as measured by their ability to migrate to lymph nodes and stimulate primary T cell responses, even at doses that do not affect the growth of these established poorly immunogenic tumors. Thus, inhibition of VEGF signaling may improve DC function in tumor-bearing hosts and possibly serve to improve the efficacy of cancer immunotherapy.
Rapid and Delayed p42/p44 Mitogen-Activated Protein Kinase Activation by Nitric Oxide: The Role of Cyclic GMP and Tyrosine Phosphatase Inhibition
Authors: Callsen Dagmar , Pfeilschifter Josef , Brüne Bernhard
Published: December 2022

The exposure of rat mesangial cells to cytokines promoted activation of the p42/p44 mitogen-activated protein kinase (MAPK). We identified a rapid and delayed phase of MAPK activation with distinctive activity increases at 5 to 15 min and 15 to 24 h. Rapid and late MAPK activation were attenuated by the redox-modulating agent N-acetylcysteine. Specifically, late-phase activation coincided with endogenous nitric oxide (NO) generation and in turn was suppressed by the NO synthase-blocking compounds diphenyliodonium or nitroarginine methyl ester. By using NO-liberating agents such as S-nitrosoglutathione and 3-morpholinosydnonimine, we investigated intermediary signaling elements of NO in promoting MAPK activation. Early and transient activation at 5 min was suppressed by the soluble guanylyl cyclase-blocking agent 1H-(1,2,4)-oxdiazolo-(4,3-α)-6-bromoquinoxazin-1-one (NS 2028) and, moreover, was mimicked by the lipophilic cyclic GMP (cGMP) analogue 8-bromo-cGMP. In contrast, NO-mediated activation achieved within hours was unrelated to cGMP signaling. Late and persistent MAPK activation, induced by NO donors or endogenously generated NO, was found in association with inhibition of phosphatase activity. In vitro dephosphorylation of activated and immunoprecipitated p42/p44 by cytosolic phosphatases was sensitive to the readdition of NO and was found to be inhibited in cytosol of S-nitrosoglutathione-stimulated cells. Also, cells that had been exposed to cytokines for 24 h revealed a blocked phosphatase activity, which was successfully attenuated by the NO synthase inhibitor nitroarginine methyl ester and, therefore, was NO mediated. Conclusively, NO affects p42/p44 MAPK in rat mesangial cells twofold: rapid activation is cGMP mediated, whereas late activation is transmitted via inhibition of tyrosine dephosphorylation.
Genetic Immunization Generates Cellular and Humoral Immune Responses Against the Nonstructural Proteins of the Hepatitis C Virus in a Murine Model
Authors: Encke Jens , zu Putlitz Jasper , Geissler Michael , Wands Jack R.
Published: December 2022

Exposure to hepatitis C virus (HCV) is associated with a high prevalence of persistent viral infection and the development of chronic liver disease and hepatocellular carcinoma. Recovery from acute infection may depend upon the generation of broad-based cellular immune responses to viral structural and nonstructural proteins. We used the DNA-based immunization approach in BALB/c mice to determine whether the HCV nonstructural proteins NS3, NS4, and NS5 will induce Ab responses, CD4+ Th cell proliferation, and cytokine release in response to stimulation by recombinant proteins as well as generate CD8+ CTL activity both in vitro and in vivo. We found that the nonstructural proteins were particularly good immunogens and produced cellular immune responses when administered as a DNA construct. Indeed, a tumor model was established following inoculation of syngenic SP2/0 cells stably transfected with NS5. We observed protection against tumor formation and growth only in mice immunized with the NS5-encoding DNA construct, establishing the generation of significant CTL activity in vivo by this technique. The results indicate that genetic immunization may define the cellular immune response of the host to HCV nonstructural proteins and is a promising approach for vaccine development.
IL-12 Is an Effective Adjuvant for Induction of Mucosal Immunity
Authors: Boyaka Prosper N. , Marinaro Mariarosaria , Jackson Raymond J. , Menon Satish , Kiyono Hiroshi , Jirillo Emilio , McGhee Jerry R.
Published: December 2022

We addressed the effects of two cytokines, IL-6 and IL-12, derived from APCs, for the development of mucosal IgA Ab responses following their nasal delivery with the protein vaccine tetanus toxoid (TT). Mice treated nasally with IL-6 and TT showed higher TT-specific serum IgG (mainly IgG1 and IgG2b) Ab responses than did control mice, but exhibited no IgE and negligible secretory IgA (S-IgA) Ab responses. In contrast, IL-12 administered nasally with TT not only induced sharp increases in TT-specific serum IgG (mainly IgG1 and IgG2b) and IgA, but also elevated mucosal S-IgA Ab responses. Coadministration of IL-6 and IL-12 with TT did not enhance the mucosal or serum Ab responses over those seen with IL-12 alone. TT-specific CD4+ T cells from mice given TT with IL-6 or IL-12 produced higher levels of IFN-γ, IL-6, and IL-10 than did those from control mice, but only negligible levels of IL-4 and IL-5. In summary, both intranasal IL-6 and IL-12 induced serum Abs that protected mice from systemic challenge with TT, whereas only IL-12 induced mucosal S-IgA Ab responses. The significance of IL-12-induced Th1-type responses for regulation of both mucosal and systemic immunity is discussed.
Follicular Exclusion and Rapid Elimination of Hen Egg Lysozyme Autoantigen-Binding B Cells Are Dependent on Competitor B Cells, But Not on T Cells
Authors: Schmidt Kerstin N. , Cyster Jason G.
Published: December 2022

In mice with a diverse B cell repertoire, hen egg lysozyme (HEL) autoantigen-binding B cells are excluded from follicles and eliminated in 3 days. To explore the roles of competitor B cells and of T cells in this mechanism of self-tolerance, HEL-specific B cells were transferred into mice containing HEL and deficient in endogenous B cells (μMT), T cells (TCR−/−), or B and T cells (RAG1−/−). Previous studies suggested a dual requirement for B cell receptor (BCR) engagement and competition in HEL autoantigen-binding B cell elimination, but interpretation of these experiments has been confounded by the possible failure to independently regulate autoantigen concentration and competitor B cell frequency. In experiments in this study, we have fixed one variable, HEL concentration, while varying the second, the presence or absence of other B cells. By this approach, we find that follicular exclusion and rapid elimination of autoreactive B cells require BCR engagement plus competition with other B cells, rather than BCR engagement alone. We also find, by transfers into T cell-deficient mice, that T cells are not required for this peripheral tolerance mechanism. Unexpectedly, in mice lacking both T cells and competitor B cells (RAG1−/−), transferred HEL-binding cells survive less well than in mice just lacking competitor B cells. These results suggest T cells can enhance autoreactive B cell survival. Enhanced survival of autoreactive B cells, due to the presence of T cells and the lack of competitor B cells, might contribute to the elevated frequency of autoimmunity in B cell-deficient individuals.
Clathrin-Coated Pit-Associated Proteins Are Required for Alveolar Macrophage Phagocytosis
Authors: Perry Douglas G. , Daugherty Gena L. , Martin William J.
Published: December 2022

During phagocytosis, phagocytic receptors and membrane material must be inserted in the pseudopod membrane as it extends over the phagocytic target. This may require a clathrin-mediated recycling mechanism similar to that postulated for leading edge formation during cell migration. To investigate this possibility, liposomes were used to deliver to intact rat alveolar macrophages (AMs): 1) Abs to clathrin, clathrin adaptor AP-2, and hsc70, and 2) amantadine. Phagocytosis was assayed by fluorometric and colorimetric techniques. Liposome-delivered Abs to clathrin and AP-2 inhibited AM phagocytosis of zymosan-coated, fluorescent liposomes from 16.3 ± 0.3 to 5.8 ± 0.3, and 10.1 ± 0.9 to 4.8 ± 0.2 liposomes/cell (p < 0.01). Similarly, liposome-delivered Ab to clathrin also inhibited AM phagocytosis of IgG-opsonized RBCs from 11.7 ± 1.7 to 3.8 ± 0.7 RBCs/cell (p < 0.01). Amantadine, which blocks the budding of clathrin-coated vesicles, inhibited phagocytosis from 13.8 ± 0.8 to 5.7 ± 0.6 (p < 0.01). Ab blockade of hsc70, which catalyzes clathrin turnover, also inhibited phagocytosis from 9.1 ± 0.5 to 4.3 ± 0.2 (p < 0.01). These findings suggest that clathrin-mediated receptor/membrane recycling is required for phagocytosis.
Activation-Induced Resistance of Human Macrophages to HIV-1 Infection In Vitro
Authors: Zybarth Gabriele , Reiling Norbert , Schmidtmayerova Helena , Sherry Barbara , Bukrinsky Michael
Published: December 2022

Cells of the monocyte/macrophage lineage are the first targets of HIV-1 in patients and also serve as reservoirs for the virus during the course of infection. We investigated the effects of cell activation on early events of HIV-1 infection of monocyte-derived macrophages. Addition of LPS, a potent stimulator of macrophages, at the time of infection stimulated entry of HIV-1 into monocyte-derived macrophages, as judged by accumulation of early products of RT, but inhibited the synthesis of late RT products and strongly repressed nuclear import of the viral DNA, resulting in protection from infection. This effect was mediated by the CD14 receptor and involved activation of the p38 mitogen-activated protein kinase pathway. Disruption of this signaling pathway using a specific inhibitor of the p38 mitogen-activated protein kinase (SB203580) restored HIV-1 infection in the presence of LPS. These results suggest a novel view of the role of macrophage activation in anti-HIV responses of the immune system.
Interaction Between α5β1 Integrin and Secreted Fibronectin Is Involved in Macrophage Differentiation of Human HL-60 Myeloid Leukemia Cells
Authors: Laouar Amale , Collart Frank R. , Chubb Cynthia B. H. , Xie Bei , Huberman Eliezer
Published: December 2022

We examined the role of fibronectin (FN) and FN-binding integrins in macrophage differentiation. Increased FN and α5β1 integrin gene expression was observed in phorbol 12-myristate 13-acetate PMA-treated HL-60 cells and PMA- or macrophage-CSF-treated blood monocytes before the manifestation of macrophage markers. After treatment of HL-60 cells and monocytes, newly synthesized FN was released and deposited on the dishes. An HL-60 cell variant, HL-525, which is deficient in the protein kinase Cβ (PKC-β) and resistant to PMA-induced differentiation, failed to express FN after PMA treatment. Transfecting HL-525 cells with a PKC-β expression plasmid restored PMA-induced FN gene expression and macrophage differentiation. Untreated HL-525 cells (which have a high level of the α5β1 integrin) incubated on FN differentiated into macrophages. The percentage of cells having a macrophage phenotype induced by PMA in HL-60 cells, by FN in HL-525 cells, or by either PMA or macrophage-CSF in monocytes was reduced in the presence of mAbs to FN and α5β1 integrin. The integrin-signaling nonreceptor tyrosine kinase, p72Syk, was activated in PMA-treated HL-60 and FN-treated HL-525 cells. We suggest that macrophage differentiation involves the activation of PKC-β and expression of extracellular matrix proteins such as FN and the corresponding integrins, α5β1 integrin in particular. The stimulated cells, through the integrins, attach to substrates by binding to the deposited FN. This attachment, in turn, may through integrin signaling activate nonreceptor tyrosine kinases, including p72Syk, and later lead to expression of other genes involved in evoking the macrophage phenotype.
Role of the 75-kDa TNF Receptor in TNF-Induced Activation of Neutrophil Respiratory Burst
Authors: Dri Pietro , Haas Elvira , Cramer Rita , Menegazzi Renzo , Gasparini Chiara , Martinelli Roberta , Scheurich Peter , Patriarca Pierluigi
Published: December 2022

The exclusive role of the 55-kDa TNF receptor (TNF-R55) as the signaling receptor in TNF-induced activation of respiratory burst by human polymorphonuclear leukocytes residing on biologic surfaces has been inferred from results obtained with receptor-specific monoclonal and polyclonal Abs. In this work, we confirm this assumption by a more direct approach, i.e., by using receptor-specific TNF mutants (p55TNF and p75TNF) and, as a novel contribution, we show that cooperation of the 75-kDa TNF receptor (TNF-R75) is required for a full blown response to the cytokine. This conclusion stems from three sets of data: 1) none of the TNF-R55-specific agonists used, i.e., mAbs or p55TNF, induced a respiratory burst comparable with that induced by TNF; 2) selective down-modulation of TNF-R75 resulted in a diminished response to TNF but not to TNF-R55-specific agonists or to the chemotactic peptide FMLP; and 3) mAbs that either block or stabilize binding of TNF to TNF-R75 inhibited the response to the cytokine, suggesting that cooperation requires not only TNF binding to the receptor but also an appropriate dissociability from it. The inhibitory effect of the Abs increased as the cytokine concentrations decreased, indicating that cooperation by TNF-R75 becomes more relevant at low TNF doses. Such a cooperation does not seem to rely on the activation of a TNF-R75-linked signaling pathway independent of TNF-R55, since the response to p55TNF and p75TNF given in combination was not higher than the response to p55TNF alone. The possible mechanisms of cooperation are discussed.
Peripheral Tolerance to Class I Mismatched Renal Allografts in Miniature Swine: Donor Antigen-Activated Peripheral Blood Lymphocytes from Tolerant Swine Inhibit Antidonor CTL Reactivity
Authors: Ierino Francesco L. , Yamada Kazuhiko , Hatch Tevis , Rembert James , Sachs David H.
Published: December 2022

Studies utilizing partially inbred miniature swine have demonstrated that a short course of cyclosporin A leads to indefinite survival of two haplotype class I mismatched renal allografts. In the present study, we have examined peripheral regulatory mechanisms that may be involved in maintenance of tolerance by coculturing PBL from long-term tolerant animals with naive recipient-matched PBL in cell-mediated lympholysis assays. We show that PBL from tolerant animals, primed in vitro with donor Ag, suppress antidonor CTL reactivity by naive recipient-matched PBL. Suppression was not observed when PBL from naive animals, primed with donor-matched PBL, were cocultured with PBL from a second naive animal, nor did PBL from either tolerant or naive recipient-matched control animals, primed with third-party Ag, suppress the generation of anti-third-party CTL by a second naive animal. The suppression was cell dose-dependent, radiation-sensitive, required cell-to-cell contact not reversed by the provision of exogenous IL-2, and associated with lower levels of IL-2R expression on the suppressive effector group (particularly the CD8 single positive cells) when compared with the control effector group. These data indicate an association between the presence of peripheral regulatory cells demonstrable in vitro and the maintenance of tolerance to renal allografts.
Activated Eosinophils Are the Major Source of Th2-Associated Cytokines in the Schistosome Granuloma
Authors: Rumbley Catherine A. , Sugaya Hiroko , Zekavat S. Ali , El Refaei Mohamed , Perrin Peter J. , Phillips S. Michael
Published: December 2022

Eosinophils are a numerically dominant cell population within the schistosome granuloma. These granuloma eosinophils can produce a variety of cytokines, including IL-2, IL-4, IL-5, and IFN-γ. Therefore, eosinophils may play a key role in the determination of the unique cytokine microenvironment within the granuloma milieu. These studies investigated the potential role of eosinophils in the regulation of granuloma immunopathology. We have characterized spleen- and granuloma-derived eosinophils based on cellular activation and cytokine production during the development of murine schistosomiasis. Based on the criteria of hypodensity and CD69 expression, granuloma eosinophils were highly activated and very homogeneous at 7 and 11 wk postinfection. Splenic eosinophils were also activated at 7 wk postinfection, but were much more heterogeneous than their granuloma counterparts. By 11 wk postinfection, few hypodense splenic eosinophils were observed. Eosinophils represented the majority of cytokine-producing cells in the granuloma and were a dominant source of IL-4. Eosinophils also produced IL-2, IL-5, and IFN-γ, using the criteria of mRNA in situ hybridization and intracellular cytokine staining by FACS. Granuloma eosinophil activation and cytokine production were greatest at the time of maximum granuloma formation, i.e., 10–12 wk after initial cercarial exposure. Therefore, locally activated eosinophils, not Th2 lymphocytes, produce the majority of Th2 cytokines in the granuloma milieu and may be important determinators of immunopathology in schistosomiasis.
IL-10 Gene Knockout Mice Show Enhanced Th1-Like Protective Immunity and Absent Granuloma Formation FollowingChlamydia trachomatisLung Infection
Authors: Yang Xi , Gartner John , Zhu Lihua , Wang Shuhe , Brunham Robert C.
Published: December 2022

We previously reported that higher IL-10 production is correlated with lower IFN-γ production, weaker delayed hypersensitivity (DTH), and slower organism clearance following chlamydial infection in mice. To assess more directly the role of IL-10, we examined protective immunity and pathological reaction in C57BL/6 IL-10 gene knockout (KO) and wild-type mice. The results showed that in the absence of endogenous IL-10, mice had significantly accelerated chlamydial clearance and developed significantly stronger DTH responses, which could be inhibited by local delivery of rIL-10. Consistent with the enhancement of DTH responses, IL-10 KO mice showed stronger and more persistent CD4 T cell-dependent IFN-γ production and significant elevation of IL-12 and TNF-α production. Additionally, wild-type, but not IL-10 KO, mice showed granuloma formation that was correlated with higher levels of Th2 cytokine (IL-5) production at the later stages of infection. Moreover, chlamydial infection, unlike parasitic protozoan infection, did not induce significant acute toxicity in IL-10 KO mice, which may be due to the low (undetectable) levels of systemic release of proinflammatory cytokines. These results suggest that IL-10 inhibits the priming and expansion of Th1-like T cell responses and that IL-10 plays a role in the fibrotic reaction seen with chlamydial infection.
A Comparison of C3a and C5a-Mediated Stable Adhesion of Rolling Eosinophils in Postcapillary Venules and Transendothelial Migration In Vitro and In Vivo
Authors: DiScipio Richard G. , Daffern Pamela J. , Jagels Mark A. , Broide David H. , Sriramarao P.
Published: December 2022

The comparative ability of the complement anaphylatoxins C3a and C5a to mediate leukocyte adhesion and transendothelial migration in vivo and in vitro was investigated. Superfusion of IL-1β-stimulated rabbit mesentery with C3a resulted in a rapid and stable adhesion of rolling eosinophils, but not neutrophils, to postcapillary venules. However, C3a failed to evoke subsequent transmigration of the adherent eosinophils. In contrast, C5a induced both the rapid activation-dependent firm adhesion and transmigration of eosinophils and neutrophils through venular endothelium. C3a induced selective shedding of L-selectin and an increase in αMβ2 integrin expression on eosinophils but not neutrophils, while C5a induced shedding of L-selectin and up-regulation of αMβ2 integrin on both eosinophils and neutrophils. Both C3a- and C5a-dependent adhesion to venular endothelium was blocked by ex vivo treatment of eosinophils with anti-α4 and anti-β2 integrin mAbs. In vitro, both C3a (but not C3adesArg) and C5a (including C5adesArg)-dependent transmigration of eosinophils across IL-1β-stimulated endothelial monolayer was mediated by α4β1 and αMβ2 integrins. Overall these studies suggest that C3a is eosinophil-specific chemotactic mediator that influences selectively eosinophil adhesion but not transmigration in vivo. C5a in contrast is a complete activator of integrin-dependent adhesion as well as transmigration of eosinophils and neutrophils.
IFN-γ Induces Endothelial Cells to Proliferate and to Invade the Extracellular Matrix in Response to the HIV-1 Tat Protein: Implications for AIDS-Kaposi’s Sarcoma Pathogenesis
Authors: Fiorelli Valeria , Barillari Giovanni , Toschi Elena , Sgadari Cecilia , Monini Paolo , Stürzl Michael , Ensoli Barbara
Published: December 2022

Previous studies indicated that the Tat protein of HIV functions as a progression factor in Kaposi’s sarcoma (KS), an angioproliferative disease common and aggressive in HIV-1-infected individuals (AIDS-KS). In particular, Tat that is released by infected cells stimulates the growth and invasion of spindle cells of endothelial origin derived from KS lesions (KS cells). Other work suggested that inflammatory cytokines may act as initiating factors in KS since they induce normal endothelial cells to acquire the same phenotype and functional features of KS cells, including the responsiveness to Tat. In this study, we show that among the inflammatory cytokines increased in AIDS-KS lesions, IFN-γ alone is sufficient to induce endothelial cells to proliferate and to invade the extracellular matrix in response to Tat. This is because IFN-γ up-regulates the expression and activity of the receptors for Tat identified as the integrins α5β1 and αvβ3. These results suggest that, by triggering Tat effects, IFN-γ plays a major role in AIDS-KS pathogenesis.
CTLA-4 (CD152) Inhibits the Specific Lysis Mediated by Human Cytolytic T Lymphocytes in a Clonally Distributed Fashion
Authors: Saverino Daniele , Tenca Claudya , Zarcone Daniela , Merlo Andrea , Megiovanni Anna M. , Valle Maria T. , Manca Fabrizio , Grossi Carlo E. , Ciccone Ermanno
Published: December 2022

Since the functional outcome of effector T lymphocytes depends on a balance between activatory and inhibitory receptors, we studied the ability of CTLA-4 (CD152) to inhibit the cytolytic function of CTL. In 22 TCRαβ+CD3+8+ CTL clones, activation induced by anti-CD3, anti-CD28, or anti-CD2 mAb was inhibited by anti-CD152 mAb in a redirected killing assay. In eight clones inhibition was >40%, in 10 it ranged between 20–40%, and in four it was <20%. This suggests the existence of a clonal heterogeneity as well as for the ability of CTLA-4 to inhibit CD3/TCR-, CD28-, or CD2-mediated CTL activation. To support further this contention, we used an experimental model based upon Ag-specific CTL. Eight Ag-specific T cell clones that lyse autologous EBV-infected B lymphocytes, but are unable to lyse allogeneic EBV-infected B cell lines, were used in a cytolytic assay in which anti-CD152 mAb or soluble recombinant receptor (i.e., CTLA-4 Ig) were included. In this system, at variance from the redirected killing assay, cross-linking of surface molecules by mAb does not occur. Thus, addition of anti-CD152 mAb or of CTLA-4 Ig and anti-CD80/CD86 mAb to the assay should result in a blockade of receptor/ligand interactions. As a consequence, inhibition of a negative signal, such as that delivered via CD152, should enhance lysis. A >40% increment of target cell lysis was achieved in three of eight clones studied. Since it is not equally shared by all CTL clones, this feature also appears to be clonally distributed.
Thymic Stromal Lymphopoietin: A Cytokine That Promotes the Development of IgM+ B Cells In Vitro and Signals Via a Novel Mechanism
Authors: Levin Steven D. , Koelling Ray M. , Friend Sherree L. , Isaksen Deborah E. , Ziegler Steven F. , Perlmutter Roger M. , Farr Andrew G.
Published: December 2022

A novel cytokine from a thymic stromal cell line (thymic stromal lymphopoietin (TSLP)) promotes the development of B220+/IgM+ immature B cells when added to fetal liver cultures, long term bone marrow cultures, or bone marrow cells plated in semisolid medium. Because the activities of TSLP overlap with those of IL-7 in some in vitro assays, we compared the signaling mechanisms employed by TSLP and IL-7. Proliferation of a factor-dependent pre-B cell line (NAG8/7) in response to either TSLP or IL-7 was inhibited by anti-IL-7Rα mAbs, suggesting that the functional TSLP receptor complex uses IL-7Rα. In contrast, three different Abs to the common cytokine receptor γ-chain had no effect on the response of these cells to TSLP, indicating that the functional TSLP receptor complex does not use the common cytokine receptor γ-chain. Both cytokines induced activation of Stat5, but only IL-7 induced activation of the Janus family kinases Jak1 and Jak3. In fact, TSLP failed to activate any of the four known Janus family kinases, suggesting that Stat5 phosphorylation is mediated by a novel mechanism. Taken together, these data support the idea that TSLP can make unique contributions to B lymphopoiesis and indicate that it does so by mechanisms distinct from IL-7.
T Cell Responses to Heat-Shock Protein 60: Differential Responses by CD4+ T Cell Subsets According to Their Expression of CD45 Isotypes
Authors: Ramage Judith M. , Young Joyce L. , Goodall Jane C. , Hill Gaston J. S.
Published: December 2022

We demonstrate that human T lymphocytes proliferate in vitro to highly purified human heat-shock protein 60 (Hu.hsp60). The response to this self Ag was confined to the CD45RA+RO− T cell subset, with minimal responses by adult CD45RA−RO+ T cells. Experiments using keyhole limpet hemocyanin as a prototypic novel Ag, or tetanus toxoid as a recall Ag, were consistent with the notion that CD45RA+RO− and CD45RA−RO+ T cell subsets can be designated as naive and memory cells, respectively; thus, responses to Hu.hsp60 were confined to the putative naive subset. In contrast, both CD45RA+RO− and CD45RA−RO+ T cell populations proliferated to bacterial hsp60 from Mycobacterium leprae, Escherichia coli, or Chlamydia trachomatis. However, only CD45RA−RO+ (memory) T cells responded to a mycobacterial hsp60-derived peptide previously defined as a major bacteria-specific epitope. Experiments with cord blood T cells, which are CD45RA+RO− and can be considered truly naive, showed that the peptide could elicit responses from naive T cells in vitro; cord blood cells also responded to Hu.hsp60. Since bacterial hsp60 Ags contain both conserved and nonconserved epitopes, we speculate that in vivo challenge with bacterial hsp60 will activate T cells capable of seeing either type of epitope, but only those that see nonconserved epitopes maintain the CD45RA−RO+ memory phenotype. However, T cells recognizing conserved epitopes, while not apparently being recruited to the memory pool, may nevertheless play a role in immunoregulation, particularly in the context of inflammation, when expression of Hu.hsp60 is increased.