bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibitory activity of IL-1 receptor antagonist depends on the balance between binding capacity for IL-1 receptor type 1 and IL-1 receptor type II.
Authors: Ruggiero P , Bossù P , Macchia G , Del Grosso E , Sabbatini V , Bertini R , Colagrande A , Bizzarri C , Maurizi G , Di Cioccio V , D'Andrea G , Di Giulio A , Frigerio F , Grifantini R , Grandi G , Tagliabue A , Boraschi D
Published: December 2022

A series of mutants of human IL-1 receptor antagonist (IL-1ra) has been designed by comparison of IL-1ra and IL-1beta structures in order to increase receptor antagonist capacity. Upon in vitro and in vivo assay of IL-1 antagonism, the IL-1ra mutants DoB 0039 (N91-->R), DoB 0040 (T109-->A) and DoB 0041 (N91/T109-->R/A) could inhibit IL-1beta effects more efficiently than wild-type IL-1ra, with DoB 0041 being the most active. Analysis of the receptor-binding capacity of the IL-1ra mutants showed that all three mutants could inhibit binding of IL-1alpha or IL-1beta to IL-1RI-bearing cells more efficiently than wild-type IL-1ra. Conversely, binding of IL-1beta to IL-1RII-bearing cells could be inhibited by DoB 0041 much less efficiently than by wild-type IL-1ra. It is known that the two types of IL-1 receptors (IL-1RI and IL-1RII) play different roles in the regulation of IL-1 activity, with IL-1RI being solely responsible for cell triggering upon IL-1 binding, whereas IL-1RII acts as a scavenger of IL-1 and can thus be considered as a natural IL-1 inhibitor. Thus, the enhanced inhibitory capacity of DoB 0041 as compared with wild-type IL-1ra is explained in terms of better binding to the activating receptor IL-1RI and poorer interaction with the inhibitory receptor IL-1RII.
Genetically engineered nontoxic vaccine adjuvant that combines B cell targeting with immunomodulation by cholera toxin A1 subunit.
Authors: Agren L C , Ekman L , Löwenadler B , Lycke N Y
Published: December 2022

Cholera toxin (CT) is an exceptionally potent adjuvant but, unfortunately, also very toxic. Here we present a powerful new approach to separate toxicity from adjuvanticity by constructing a fusion protein that combines the enzymatically active cholera toxin A1 subunit (CTA1) with targeting to B cells. The CTA1 was genetically linked at its C-terminal end to two Ig-binding domains, DD, of staphylococcal protein A and produced in Escherichia coli. The highly purified, monomeric CTA1-DD fusion protein, with a molecular mass of 37 kDa, was found to exhibit strong ADP-ribosyltransferase activity and bound, via the DD moiety, to both Fc and Fab fragments and to all IgG subclasses--IgE, IgA, and IgM. After i.v. injection of the fusion protein, FACS analysis revealed binding of CTA1-DD to splenic IgM+ B cells, but not CD3+ T cells, indicating cell-specific targeting in vivo. Strikingly, we found that the adjuvant ability of CTA1-DD to enhance systemic IgG as well as mucosal IgA responses to the unrelated Ags, OVA, or keyhole limpet hemocyanin, administered i.v or intranasally, was comparable to that of intact CT. In addition, the enhancing effect on specific IgG1, IgG2a, and IgG2b responses mimicked that of CT and suggested involvement of both Th1 and Th2 CD4+ T cell activity. The CTA1-DD, as well as CT, up-regulated expression of the CD80 and CD86 molecules on the targeted B cells, indicating that enhanced T cell costimulation may be responsible for the adjuvant effect. Contrary to CT, however, CTA1-DD was completely nontoxic. Thus, the CTA1-DD adjuvant should find general applicability in systemic and mucosal vaccines, and the strategy used may also be explored for other regimens requiring targeted immunomodulation.
Attenuation of proinflammatory response by recombinant human IL-10 in human endotoxemia: effect of timing of recombinant human IL-10 administration.
Authors: Pajkrt D , Camoglio L , Tiel-van Buul M C , de Bruin K , Cutler D L , Affrime M B , Rikken G , van der Poll T , ten Cate J W , van Deventer S J
Published: December 2022

To determine the effects of IL-10 on cytokine and granulocyte responses during endotoxemia, two groups of eight healthy male volunteers were challenged with endotoxin (4 ng/kg) on two occasions, once in combination with placebo injection, and once in conjunction with i.v. administered recombinant human IL-10 (rhIL-10) (25 microg/kg). In group 1, rhIL-10 was administered 2 min before endotoxin challenge; in group 2, the intervention was delayed for 1 h after endotoxin administration. rhIL-10 pretreatment reduced the LPS-induced rises in temperature and release of TNF, IL-6, IL-8, and IL-1 receptor antagonist. Endotoxin-induced granulocyte accumulation in lungs, as determined by dynamic granuloscintigrams, was prevented by rhIL-10 pretreatment, whereas granulocyte recruitment in liver and spleen was only modestly reduced. In addition, granulocyte degranulation, as measured by plasma elastase/alpha1-antitrypsin complexes, was blunted significantly by rhIL-10 pretreatment. Post-treatment with rhIL-10 did not influence LPS-induced temperature responses, cytokine release, or granulocyte degranulation. Both rhIL-10 pretreatment and post-treatment reduced LPS-induced cortisol levels. These results indicate that pretreatment with rhIL-10 reduces endotoxin-induced febrile responses, cytokine responses, and granulocyte accumulation in lungs, while in this acute model post-treatment with rhIL-10 exerts limited anti-inflammatory effects.
Enhancement of cell-mediated immunity against HIV-1 induced by coinnoculation of plasmid-encoded HIV-1 antigen with plasmid expressing IL-12.
Authors: Tsuji T , Hamajima K , Fukushima J , Xin K Q , Ishii N , Aoki I , Ishigatsubo Y , Tani K , Kawamoto S , Nitta Y , Miyazaki J , Koff W C , Okubo T , Okuda K
Published: December 2022

Previous investigations have demonstrated that CTL may play an important role in suppressing the disease progression of HIV infection. In this study, we inoculated mice with IL-12 expression plasmid together with plasmid-encoding HIV-1 envelope to enhance CTL activity by activating a Th1-type response. The results of delayed-type hypersensitivity using the footpad swelling response and of CTL activity clearly showed that HIV-1-specific cell-mediated immunity was enhanced by inoculation of the IL-12 expression plasmid. Quantitation of cytokine in the sera of IL-12-inoculated mice revealed that IFN-gamma significantly increased. The enhanced cell-mediated immunity responses were abrogated by combined administration of the IL-12 expression plasmid and neutralizing anti-IFN-gamma Ab. Together, these results suggest that enhanced virus-specific cell-mediated immunity occurred via an endogenously produced IFN-gamma by inoculation of IL-12 expression plasmid.
Peptide antagonists inhibit proliferation and the production of IL-4 and/or IFN-gamma in T helper 1, T helper 2, and T helper 0 clones bearing the same TCR.
Authors: Dittel B N , Sant'Angelo D B , Janeway C A
Published: December 2022

Engagement of a TCR by its peptide Ag bound by MHC class II molecules leads to T cell activation, resulting in proliferation and cytokine production. This agonist response can be antagonized by analogue peptides containing single amino acid substitutions. We used T cell clones isolated from a mouse transgenic for the rearranged TCR alpha- and beta-chains of the D10.G4.1 (D10) Th2 clone specific for hen egg conalbumin peptide 134-146 (CA 134-146) to characterize antagonist peptides for the D10.TCR. The D10.TCR CD4 T cell clones proliferated in a dose-dependent manner to CA 134-146, and this proliferation was accompanied by secretion of IL-4 and/or IFN-gamma with Th1, Th2, and Th0 patterns. Proliferation of the clones was inhibited completely by CA 134-146 analogue peptides containing a substitution of the glutamic acid at position 8 with alanine (E8A) or threonine (E8T). The E8A and E8T peptides also antagonized the production of mRNA and subsequent cytokine secretion of IFN-gamma and/or IL-4. Our results, showing that antagonist peptides can inhibit both T cell proliferation and cytokine production in Th1, Th2, and Th0 clones all bearing the same TCR, demonstrate that the TCR:peptide interaction determines the outcome regardless of the phenotype of the clone. Thus, antagonism by peptides acts through the TCR.
Fas ligand is positioned in mouse uterus and placenta to prevent trafficking of activated leukocytes between the mother and the conceptus.
Authors: Hunt J S , Vassmer D , Ferguson T A , Miller L
Published: December 2022

Despite intimate juxtaposition of maternal and fetal tissues during mammalian pregnancy, reciprocal migration of cells is limited. To evaluate the postulate that cell traffic is restricted by expression of Fas ligand (FasL) in the uterus and placenta, FasL mRNA was identified by using reverse transcription-PCR, and FasL protein was identified by Western blotting and immunohistology. FasL mRNA and protein were detected at all stages tested (gestation days (g.d.) 6-18). At g.d. 6 to 10, immunoreactive FasL was prominent in glandular epithelial cells and decidual cells. Between g.d. 12 and 14, expression shifted to placental trophoblast cells bordering maternal blood spaces and fetal placental endothelial cells. Thus, FasL is appropriately positioned, first in the uterus and then in the placenta, to deter trafficking of activated Fas+ immune cells between the mother and the fetus. To test whether the absence of functional FasL affects pregnancy, uteroplacental units from homozygous matings of gld mice, a mutant strain lacking functional FasL, were examined. Extensive leukocytic infiltrates and necrosis at the decidual-placental interface were observed from day 10 onward, resorption sites were common, and small litters were delivered by gld mice. These observations are consistent with the idea that FasL at the maternal-fetal interface protects the placenta against a maternal leukocytic influx that reduces fertility.
Developmentally ordered V-J recombination in mouse T cell receptor gamma locus is not perturbed by targeted deletion of the Vgamma4 gene.
Authors: Sunaga S , Maki K , Komagata Y , Miyazaki J , Ikuta K
Published: December 2022

Mouse TCR gamma genes in the gamma1 cluster are arranged in the order of Vgamma5, Vgamma2, Vgamma4, Vgamma3, Jgamma1, and Cgamma1 on the chromosome. During thymic ontogeny, each Vgamma gene recombines with the Jgamma1 gene in the order of proximity to Jgamma1. To explore the mechanism of the ordered recombination, we generated Vgamma4-deficient mice by gene targeting and the Cre/loxP system, by deleting the 4.8-kb DNA region between 3' of the Vgamma2 and 3' of the Vgamma4. In semiquantitative PCR analysis, Vgamma2-Jgamma1 recombination was detected frequently in adult thymus, while Vgamma3-Jgamma1 recombination preferentially occurred in fetal thymus of the mutant mice. There was no difference in the frequency of V-J recombinations between control and mutant mice. Southern blot analysis also revealed that recombination of the Vgamma2 gene occurred as frequently as in control mice. In addition, there was no difference in the levels of germline transcripts of Vgamma2 and Vgamma3 genes between control and mutant mice. Therefore, regulation of the Vgamma-Jgamma recombination was not affected by deletion of the Vgamma4 gene. These results suggest that the ordered recombination is controlled by regulatory elements near each Vgamma gene.
Programmed cell death of Mycobacterium avium serovar 4-infected human macrophages prevents the mycobacteria from spreading and induces mycobacterial growth inhibition by freshly added, uninfected macrophages.
Authors: Fratazzi C , Arbeit R D , Carini C , Remold H G
Published: December 2022

Mycobacterium avium, an opportunistic pathogen in AIDS patients, replicates in human macrophages (Mphi) and induces programmed cell death (PCD). In this study we examine the effect of freshly added, uninfected Mphi on M. avium growth in apoptotic Mphi cultures. Incubation of uninfected autologous Mphi with apoptotic Mphi infected with M. avium for 6 h results in 90% inhibition of bacterial growth. The uninfected Mphi adhere to M. avium-infected apoptotic, but not to nonapoptotic M. avium-infected Mphi, suggesting a specific interaction between apoptotic and nonapoptotic Mphi. PCD of the host Mphi also prevents the release of intracellular components and the spread of the mycobacterial infection. Once the apoptotic infected Mphi reach the necrotic stage, mycobacteria and other intracellular material are released; the latter suffice to support extracellular mycobacterial replication. Necrosis of M. avium-infected Mphi is significantly augmented by the transglutaminase inhibitors dansyl-cadaverine and cystamine, indicating that apoptosis of Mphi is dependent on the extent of cross-linking of cell proteins by transglutaminases. Consequently, transglutaminase inhibitors accelerate the release of mycobacteria and intracellular components from the infected Mphi into the medium. These findings indicate that PCD of M. avium-infected Mphi is an important defense mechanism, preventing the spread of infection by sequestering the mycobacteria and by contributing to their demise by activation of newly recruited uninfected Mphi.
The lipid A moiety of Porphyromonas gingivalis lipopolysaccharide specifically mediates the activation of C3H/HeJ mice.
Authors: Tanamoto K , Azumi S , Haishima Y , Kumada H , Umemoto T
Published: December 2022

The lipid A preparation isolated from Porphyromonas gingivalis was found to induce splenocyte mitogenicity and TNF-alpha release from peritoneal macrophages in LPS-unresponsive C3H/HeJ mice to the same extent as in LPS-responsive mice. In order to clarify whether the activation of C3H/HeJ mice was specifically caused by the lipid A and not by contaminating protein, two strategies were employed. The lipid A fraction from P. gingivalis was subjected to either hydrochloric acid or alkaline treatment to eliminate either glycosylated phosphate or O-acylated fatty acids from the lipid A structure, and the biologic activities of the derivatives were compared in both LPS-responsive and unresponsive C3H/HeJ mice. De-1-O-phosphorylated P. gingivalis lipid A showed partial loss, and de-O-acylated lipid A complete loss of splenocyte mitogenic and TNF-alpha-inductive activities from peritoneal macrophages in both LPS-responsive and unresponsive mice. The relative activities of the intact and treated lipid A compounds in splenocyte mitogenicity and TNF-alpha-inductive activity in macrophages were similar to the relative activities of these preparations in Limulus gelation activities. The LPS-specific antagonist, succinylated lipid A precursor, inhibited P. gingivalis lipid A-mediated splenocyte mitogenicity and TNF-alpha induction in macrophages in a similar manner in LPS-responsive and unresponsive mice. These results strongly suggest that the activation of LPS-unresponsive C3H/HeJ mice by P. gingivalis lipid A was specifically mediated by the lipid A portion and not by contaminating protein. The characteristic action of P. gingivalis lipid A on LPS-unresponsive C3H/HeJ mice was thought to reflect the unique chemical properties of this compound.
Macrophage triggering with cecropin A and melittin-derived peptides induces type II nitric oxide synthase expression.
Authors: Velasco M , Díaz-Guerra M J , Díaz-Achirica P , Andreu D , Rivas L , Boscá L
Published: December 2022

Triggering of RAW 264.7 cells with a cecropin A-melittin hybrid peptide (CA(1-8)M(1-18)) promoted a rapid rise in the intracellular calcium concentration that was followed, after a lag period of 6 h, by nitric oxide synthesis through the expression of the cytokine-inducible form of nitric oxide synthase (type II NOS or iNOS). The maximal effect was obtained at peptide concentrations in the 2 to 5-microM range. Simultaneous incubation with the peptide and LPS abrogated the nitric oxide synthesis elicited after LPS treatment of the cells. CA(1-8)M(1-18) induced a rapid activation of nuclear factor kappaB as evidenced by the presence of p50/p65 heterodimers of the nuclear factor kappaB/c-Rel family in the nuclei of activated cells. This peptide also activated the reporter activity of cells transfected with a plasmid harboring a 1-kb fragment corresponding to the 5'-flanking region of the murine iNOS gene. CA(1-8)M(1-18) promoted apoptotic cell death at concentrations below 1 to 2 microM, whereas higher concentrations altered the plasma membrane integrity. These results suggest the involvement of multiple intracellular signaling pathways in the mechanism by which this peptide elicits macrophage triggering.
Interaction of CTLA-4 with the clathrin-associated protein AP50 results in ligand-independent endocytosis that limits cell surface expression.
Authors: Chuang E , Alegre M L , Duckett C S , Noel P J , Vander Heiden M G , Thompson C B
Published: December 2022

CTLA-4 is a lymphocyte cell surface receptor expressed by activated T cells that functions to down-regulate T cell responses induced by TCR and CD28 stimulation. Since CTLA-4 competes with CD28 for binding to the common ligands B7-1 and B7-2, the level of CTLA-4 surface expression is likely to play an important role in its ability to inhibit CD28-dependent T cell activation. The factors that regulate these levels are poorly understood. Recent studies have revealed that following T cell activation, the majority of CTLA-4 is localized intracellularly rather than on the cell surface, and surface CTLA-4 is rapidly reinternalized. In this study, we investigate the molecular mechanism underlying the rapid clearance of CTLA-4 from the cell surface. The data demonstrate that cell surface CTLA-4 is endocytosed into clathrin-coated vesicles even in the absence of ligand. The targeting of CTLA-4 to clathrin-coated vesicles is mediated by the clathrin-associated adaptor complex AP-2. The cytoplasmic domain of CTLA-4 was found to specifically bind to AP50, the medium chain subunit of AP-2 in both yeast two-hybrid and coimmunoprecipitation assays. The interaction requires the peptide sequence 199-GVYVKM-204 in the cytoplasmic tail of CTLA-4. Mutation of the CTLA-4 amino acid residue Y201 abrogates the interaction with AP50, resulting in the accumulation of CTLA-4 at the cell surface. Together these data suggest that the interaction of CTLA-4 with AP50 plays an important role in regulating the cell surface expression of CTLA-4.
Direct MHC class I complementary DNA transfer to thymus induces donor-specific unresponsiveness, which involves multiple immunologic mechanisms.
Authors: Knechtle S J , Wang J , Graeb C , Zhai Y , Hong X , Fechner J H , Geissler E K
Published: December 2022

Our purposes were 1) to determine whether direct transfer of cDNA encoding allogeneic MHC class I Ag to the rat thymus would be capable of inducing donor-specific unresponsiveness and 2) to study the immunologic mechanism of this effect. Plasmid DNA encoding donor strain (ACI-RT1.Aa) MHC class I Ag was directly injected into Lewis (RT1(l)) rat recipient thymus 7 to 10 days before ACI liver transplantation. A single dose of anti-lymphocyte serum was given i.p. on the day of thymic injection. Rats injected intrathymically with plasmid DNA and treated with anti-lymphocyte serum demonstrated prolonged survival in 9 of 13 rats (>100 days). PCR was used to demonstrate that RT1.Aa cDNA was expressed in thymus transiently and later appeared in spleen. CTL limiting dilution assays showed that CTL precursor frequency was decreased in tolerant liver recipients. To test the hypothesis of clonal deletion vs anergy, CTL limiting dilution assays cultures were restimulated with donor cells and IL-2 to reverse anergy. Restimulation caused CTL precursor frequency to return to near normal in only one of five tolerant rats, suggesting clonal deletion or a dense anergic state. Passive transfer of splenocytes from tolerant rats to naive recipients prolonged cardiac allograft survival, suggesting that suppressor-type cells may also contribute to thymic tolerance in our model. In summary, our data suggest that donor MHC class I Ag expressed in thymus by direct DNA injection, followed by liver allografting, results in donor-specific unresponsiveness. The mechanism of this effect is complex, involving multiple immunologic mechanisms.
In vivo mechanism by which leflunomide controls lymphoproliferative and autoimmune disease in MRL/MpJ-lpr/lpr mice.
Authors: Xu X , Blinder L , Shen J , Gong H , Finnegan A , Williams J W , Chong A S
Published: December 2022

Two activities have been identified for the immunosuppressive metabolite of leflunomide, A77 1726: inhibition of dihydroorotate dehydrogenase (DHO-DHase), an enzyme involved in the biosynthesis of pyrimidine nucleotides (PyN); and inhibition of protein tyrosine kinases. The in vitro potency of A77 1726 as a DHO-DHase inhibitor is reported to be 10- to 500-fold greater than as a tyrosine kinase inhibitor. These observations suggested that the immunosuppressive efficacy of leflunomide in vivo is related to inhibition of DHO-DHase. However, observations that patients with disorders in the PyN synthetic pathway are not overtly immunodeficient militate against this hypothesis. We investigated the effects of leflunomide in vivo and report that amelioration of lymphoproliferative and autoimmune diseases in MRL/MpJ-lpr/lpr (lpr/lpr) mice by leflunomide is not accompanied by reduced PyN concentrations in lymph node cells. Our hypothesis that lymphocytes could salvage serum uridine to counter the effects of reduced PyN synthesis in vivo was supported by in vitro studies. Finally, we observed that amelioration of disease correlated with a reduction of tyrosine phosphorylated proteins in lymph node cells of lpr/lpr mice. These observations suggest that the primary mechanism by which leflunomide prevents autoimmune and lymphoproliferative diseases in lpr/lpr mice is not depletion of PyN, but correlates with reduced tyrosine phosphorylation concentrations in lymph node cells.
Antigen specificity of dual reactive T hybridomas determines the requirement for CD40 ligand-CD40 interactions.
Authors: Ozaki M E , Karlsson L , Peterson P A , Webb S R
Published: December 2022

CD40 ligand (CD40L) expression on T cells is known to play a crucial role in B cell responses. Some evidence also supports a role for CD40L-CD40 interactions in T cell responses, at least in vivo. Whether the T cell requirement for these interactions is an invariable finding, however, is less clear. Here, we provide evidence that the Ag specificity of T cells influences the requirement for CD40L. T cell hybridomas with dual reactivity for two different Ags, allo-H2-Ap and Mls(a) superantigens, display a differential requirement for CD40L expression. Whereas the response to splenic APC expressing Mls(a) Ags requires CD40L expression, the response to alloantigen-bearing APC does not. The requirement for CD40L expression for the Mls(a) response appears to reflect a strong dependence of this response on ICAM-1 (intercellular adhesion molecule-1) and the ability of CD40-mediated signals to regulate ICAM-1 expression. These findings demonstrate that CD40L-CD40-mediated cross-talk is important for some but not all T cell responses and is influenced by both the type of Ag recognized and the type of APC.
Production of IL-18 (IFN-gamma-inducing factor) messenger RNA and functional protein by murine keratinocytes.
Authors: Stoll S , Müller G , Kurimoto M , Saloga J , Tanimoto T , Yamauchi H , Okamura H , Knop J , Enk A H
Published: December 2022

Recently, the novel cytokine IL-18 (IFN-gamma-inducing factor) has been described as a growth and differentiation factor for Th1 cells. Epidermal keratinocytes (KC) are known to direct T cell education by production of cytokines. Therefore, expression of IL-18 was sought in KC. Epidermal RNA was analyzed following stimulation with contact sensitizers or controls for IL-18 mRNA expression by semiquantitative reverse transcription-PCR. Constitutive expression of IL-18 mRNA was low in untreated epidermal cells (EC), but early up-regulation of IL-18 mRNA signals was detected following application of a contact allergen in vivo. The peak strength of IL-18 signals was observed within 4 to 6 h following stimulation with an allergen. Application of an irritant (benzalconiumchloride) or solvents did not result in increased signal strength. To determine the cellular origin of IL-18 mRNA in EC, depletion experiments were performed. IL-18 signals were not affected by depletion with anti-CD3 (T cells) or anti-MHC class II mAb-coupled beads identifying KC as a major source of IL-18. These results were confirmed by analysis of mRNA derived from the KC cell line PAM 212. Strong IL-18 signals could be detected by reverse transcription-PCR. To delineate whether IL-18 protein was produced by EC/KC, a sandwich ELISA was used to assay for IL-18 production. Supernatants from allergen-stimulated EC and KC showed production of IL-18 protein. To confirm that IL-18 protein was functional, EC or KC supernatants were tested for their ability to induce IFN-gamma production. Significant amounts of IFN-gamma were detected in supernatants of allergen-treated cells. In aggregate, our data indicate that murine KC are a source of both IL-18 mRNA and functional protein.
NKR-P1A stimulation of arachidonate-generating enzymes in rat NK cells is associated with granule release and cytotoxic activity.
Authors: Grazia Cifone M , Roncaioli P , Cironi L , Festuccia C , Meccia A , D'Aló S , Botti D , Santoni A
Published: December 2022

NKR-P1A protein has been implicated in the triggering of NK-mediated natural killing contributing to target cell recognition by NK cells. The aim of the present work was to assess whether NKR-P1A receptor triggering also induced arachidonic acid (AA) generation and to determine the possible role of this event on granule release and cytotoxicity. We demonstrated that activation of fresh peripheral blood rat NK cells by cross-linking with the anti-NKR-P1A 3.2.3 mAb induced calcium-dependent AA release, which is due to the activation of cytosolic phospholipase A2 (cPLA2), secretory phospholipase A2 (sPLA2), and diacylglycerol/monoacylglycerol lipase. We also documented the presence of a type II sPLA2 activity in the supernatant fluids from NKR-P1A-activated rat NK cells, suggesting that AA and lysophospholipids could be mobilized from the outside of the cell. The involvement of AA-generating enzymes in NKR-P1A-induced cytotoxic functions was also investigated. Treatment of effector cells with arachidonyl trifluoromethylketone, a cPLA2 inhibitor; p-bromophenacylbromide, a sPLA2 inhibitor; or RHC80267, a diacylglycerol lipase inhibitor, led to a partial inhibition of the redirected lysis against P815 target cells as well the granule content release induced by NKR-P1A cross-linking. A complete abolishment of these events was observed when the cells were simultaneously incubated with all three inhibitors. Taken together, our results support a crucial role for the arachidonate-generating enzymes in the induction of lytic activity of NK cells directly or by leading to generation of additional mediators that can play a role in the context of NK cell activation and cytotoxic functions.
IL-12 treatment attenuates T helper cell type 2 and B cell responses but does not improve vaccine-enhanced lung illness.
Authors: Hussell T , Khan U , Openshaw P
Published: December 2022

In humans and mice, sensitization to respiratory syncytial virus (RSV) Ags can result in severe inflammatory lung disease during subsequent infection with RSV. Although specific antiviral T cells are thought to be responsible for this augmentation of disease, the precise roles of different functional subsets are unknown, and no protective nonpathogenic subset has been defined. BALB/c mice sensitized to the major surface glycoprotein of RSV (G) expressed by recombinant vaccinia virus develop Th2-driven lung eosinophilia after intranasal challenge with RSV. In an attempt to manipulate the outcome of vaccination, we treated mice with IL-12 at various times during vaccination and challenge. IL-12 treatment reduced the vaccine-induced lung eosinophilia during RSV challenge, but increased the total lymphoid cell infiltration into the alveolar space. Analysis of intracellular cytokines by flow cytometry showed that IFN-gamma production during challenge was increased, and IL-4 and IL-5 levels were reduced by IL-12 treatment. In control treated mice, 40 to 50% of the lung lymphoid cells were B cells. Treatment with IL-12 reduced this figure to approximately 1.5%. Although IL-12 treatment reduced lung eosinophilia, illness (as assessed by weight loss) was not eliminated and, in some experiments, was increased. The present study shows that reversing Th2-associated pathology with IL-12 does not necessarily benefit the host.
Differential expression of transcription directed by a discrete NF-AT binding element from the IL-4 promoter in naive and effector CD4 T cells.
Authors: Aune T M , Flavell R A
Published: December 2022

Acquiring the ability to selectively produce IFN-gamma or IL-4 is a fundamental property of the immune system and enables T cell subsets (Th1, Th2) to deliver their effector functions. To create an experimental system to examine regulation of critical promoter elements within the IL-4 gene, we have prepared and analyzed transgenic mice expressing the luciferase gene under the control of the distal NF-AT binding site from the IL-4 promoter. This site is immediately adjacent to an AP-1 site, and this NF-AT/AP-1 composite site is termed either P1 or PubB. The distal NF-AT site we have used to prepare these reporter transgenic mice lacks the AP-1 binding site but contains the NF-AT binding site (P1(NF-AT)) These transgenic mice were also intercrossed with transgenic mice expressing a single MHC class II-restricted TCR. Expression of transcriptional activity under the control of P1(NF-AT) was observed only in effector T cells, and not naive T cells, after stimulation with Ag or polyclonal stimuli. By contrast, gel mobility shift assays showed that nuclear extracts from both naive and effector T cells contained NF-AT, which could effectively bind to the P1(NF-AT) element. IL-4-stimulated Th2 differentiation did not increase the TCR responsiveness of the P1(NF-AT) element by more than 2-fold but increased production of IL-4 protein by more than 10-fold. These data suggest that factors interacting with the P1(NF-AT) element regulate transcriptional activity in a naive/effector T cell-specific manner but not in a Th1/Th2-specific manner. Th1/Th2-specific regulation of the composite P1 element may result from regulation of transacting factors that bind to the AP-1 portion of this element.
Effect of the inducible nitric oxide synthase inhibitors aminoguanidine and L-N6-(1-iminoethyl)lysine on zymosan-induced plasma extravasation in rat skin.
Authors: Ridger V C , Pettipher E R , Bryant C E , Brain S D
Published: December 2022

The effect of nitric oxide synthase (NOS) inhibitors on plasma extravasation in a rat model of zymosan-induced inflammation has been investigated. Plasma extravasation was determined in response to intradermal test agents over 0 to 45 min or 0 to 4 h by the accumulation of i.v. injected 125I-labeled human serum albumin. Zymosan (1-100 microg/site) produced a dose- and time-dependent plasma extravasation. N(G)-nitro-L-arginine methyl ester (30-300 nmol/site), but not aminoguanidine (AG; 10-300 nmol/site) or L-N6-(1-iminoethyl)lysine (L-NIL; 10-300 nmol/site), significantly (p < 0.01) inhibited zymosan-induced (10 microg/site) plasma extravasation over 0 to 45 min. However, both AG and L-NIL produced significant (p < 0.05) inhibition over 0 to 4 h. The inhibition produced by AG was reversed by i.v. L-arginine or by coinjection of the vasodilator, calcitonin gene-related peptide. Zymosan (10-100 microg/site) induced an increase in dermal blood flow (laser-Doppler flowmetry) and this was inhibited by AG. Neutrophils were depleted selectively with antiserum, but this did not affect plasma extravasation except at the highest dose of zymosan (100 microg/site). Furthermore, zymosan-induced edema was not modified at either time point by pretreatment with the cyclooxygenase inhibitor indomethacin (30 micromol/kg, s.c., -30 min). In conclusion, in this model of dermal inflammation, it is suggested that inducible NOS inhibitors selectively remove an inducible NOS component that, at least in part, acts to increase microvascular blood flow and thus the edema formation observed during 0 to 4 h. There is no evidence of a contributory role for neutrophils or cyclooxygenase products in this model.
Transgenic monocyte chemoattractant protein-1 (MCP-1) in pancreatic islets produces monocyte-rich insulitis without diabetes: abrogation by a second transgene expressing systemic MCP-1.
Authors: Grewal I S , Rutledge B J , Fiorillo J A , Gu L , Gladue R P , Flavell R A , Rollins B J
Published: December 2022

Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine that attracts monocytes and T lymphocytes in vitro; however, its in vivo functions are poorly understood. To address this question, we constructed transgenic mice expressing MCP-1 controlled by an insulin promoter. These mice developed a chronic insulitic infiltrate composed of F4/80+ monocytes with minor populations of CD4+, CD8+, and B220+ cells. Despite persistent transgene expression, the insulitis never progressed, and blood glucose levels remained normal. Thus, MCP-1 alone is sufficient to elicit a monocytic infiltrate, but not to activate elicited cells. These results differ from those obtained with another transgenic model using the mouse mammary tumor virus long terminal repeat, in which mice expressed substantial MCP-1 in several organs but had no infiltrates. However, mice expressing both transgenes had minimal insulitis, indicating that high systemic levels of MCP-1 prevented monocytes from responding to local MCP-1. Thus, the ability of MCP-1 to elicit monocytic infiltration depends on its being expressed at low levels in an anatomically restricted area.
Differential regulation of indoleamine 2,3-dioxygenase expression by nitric oxide and inflammatory mediators in IFN-gamma-activated murine macrophages and microglial cells.
Authors: Alberati-Giani D , Malherbe P , Ricciardi-Castagnoli P , Köhler C , Denis-Donini S , Cesura A M
Published: December 2022

Induction of indoleamine 2,3-dioxygenase (IDO) and nitric oxide synthase (NOS) is involved in the immunomodulatory roles of IFN-gamma and evidence suggests that these pathways are functionally cross-regulated. We report here that nitric oxide (NO) negatively modulates the expression of IDO activity in IFN-gamma-primed macrophages, but not in microglial cells from mouse. In MT2 macrophages, the induction of IDO activity by IFN-gamma was further increased by the presence of NOS inhibitors, whereas culturing of IFN-gamma-activated MT2 cells with NO generators produced a marked reduction of IDO activity expression. Conversely, neither NOS inhibitors nor exogenous NO affected the induction of the enzyme activity in N11 microglial cells after IFN-gamma activation. LPS and picolinic acid, two costimulatory agents that up-regulate inducible NOS in activated cells, regulated IDO induction differently in the two cell lines. LPS and picolinic acid caused a significant decrease of IDO activity in IFN-gamma-activated MT2 cells. This effect, however, did not appear to be mediated by the ability of LPS and picolinic acid to stimulate NO production. In N11 cells, LPS further stimulated the enzyme activity and picolinic acid had no effect. Northern blot analysis revealed that, in MT2 macrophages, NOS inhibitors increased the levels of IDO mRNA, while a reduction was observed with picolinic acid. No changes in IDO mRNA levels were detected in N11 cells. Consistent with the functional heterogeneity of phagocytes, the reported results indicate the existence of marked differences in the regulation of IDO expression between murine macrophages and microglial cells.
Role of macrophages and macrophage-derived cytokines in the pathogenesis of Kilham rat virus-induced autoimmune diabetes in diabetes-resistant BioBreeding rats.
Authors: Chung Y H , Jun H S , Kang Y , Hirasawa K , Lee B R , Van Rooijen N , Yoon J W
Published: December 2022

The diabetes-resistant BioBreeding (DR-BB) rat, derived from diabetes-prone forebears, does not normally develop spontaneous insulitis or diabetes, but when infected with Kilham rat virus (KRV) this animal develops autoimmune diabetes similar to the diabetes-prone BioBreeding (DP-BB) rat. In this study, we attempted to determine whether macrophages and macrophage-derived cytokines play a role in the development of KRV-induced diabetes in DR-BB rats. Seventy-eight percent of DR-BB rats treated with KRV and poly(I:C) develop diabetes, whereas depletion of macrophages with liposome-encapsulated dichloromethylene diphosphonate (lip-Cl2MDP) in KRV and poly(I:C)-treated DR-BB rats results in the near-complete prevention of insulitis and diabetes. Measurement of the macrophage-derived cytokines IL-12, TNF-alpha, and IL-1beta revealed a selective increase of their expression, after KRV infection, in the splenic lymphocytes and the pancreatic islets. Measurement of CD4+ T cell-derived cytokines revealed that IL-2 and IFN-gamma cytokine gene expression closely correlates with an elevation of IL-12, but IL-4 and IL-10 do not change. Depletion of macrophages before the isolation of splenic lymphocytes from DR-BB rats treated with KRV and poly(I:C) resulted in the loss of ability to transfer diabetes to young DP-BB rats. On the basis of these observations, we conclude that macrophages and macrophage-derived cytokines play a critical role in the cascade of events leading to the destruction of pancreatic beta cells, culminating in the development of insulin-dependent diabetes mellitus.
American Association of Immunologists Presidential Address: doing it together: a perspective on the process of experimental science.
Authors: Knight K L
Published: December 2022

We are witnessing profound changes in how experimental science is done and the rate at which scientists are discovering nature's secrets. While we are experiencing this phenomenal change, we are also at risk, because along with these changes comes the potential for altering the values by which we do science. I share with everyone in our field an abiding concern that scientists of the next generation embrace the values that have guided my generation and the generations of scientists that have preceded me. Because relationships in science are key to passing on these values, I have focused the first part of this address on the importance that relationships have played in guiding my scientific process. In the second part; Looking Toward Our Future, I discuss how the mentoring relationship can serve as an antidote to pressures that threaten our scientific values.
The effect of NK cell activation by tumor cells on antigen-specific antibody responses.
Authors: Koh C Y , Yuan D
Published: December 2022

In addition to mediating direct cytotoxicity, NK cells can exert regulatory effects on specific immune responses. For example, injection of poly (I:C) can alter specific Ab responses, which is attributable to the production of IFN-gamma by NK cells. To test whether direct activation of NK cells can exert the same effect, we have injected, at the same time as Ag challenge, BCL1-C11 tumor cells, which are highly effective inducers of IFN-gamma production by NK cells. The results show a specific enhancement of the IgG2a response, which does not occur with a tumor (70Z/3) that does not induce IFN-gamma production. This enhancement is NK cell and IL-12 dependent. However, BCL1-C11 cells cannot directly induce IL-12 production in peritoneal exudate cells (PECs). On the other hand, PECs from tumor-treated mice produce IL-12 in response to LPS, suggesting that they are primed in vivo. Furthermore, the IL-12 production is NK cell and IFN-gamma dependent. These results indicate that if tumor cells can directly activate NK cells to produce IFN-gamma, this cytokine initiates an amplification loop by activating macrophages to produce IL-12, which in turn activates NK cells further, resulting in the alteration of the isotype distribution of specific Ab responses. Production of the appropriate Ab isotype should enhance Ab-dependent cellular cytotoxicity against targets mediated by NK cells, implicating their role(s) in the specific immune response as well as the initial nonspecific phase.
Induction of parasite antigen-specific antibody responses in unsensitized human B cells is dependent on the presence of cytokines after T cell priming.
Authors: Garraud O , Perler F B , Bradley J E , Nutman T B
Published: December 2022

Using two recombinant filarial protein Ags and keyhole limpet hemocyanin, we sensitized T cells from uninfected, nonatopic individuals in such a manner that they were able to provide help for the selective induction of an Ag-specific Ab response. IL-2 and IL-4 were shown to be critical for sensitizing the T cells; once sensitized, these T cells could provide the necessary signals for B cells to produce Ag-specific Abs, provided that IL-4 (or IL-2) was supplied exogenously. Primary exposure of T cells to IFN-gamma, but not to IL-12, prevented the Ag-sensitized T cells from helping B cells to produce specific Abs, apart from the IgG2 isotype. These data suggest that Ab-producing B cells of a defined Ag specificity and isotype can be generated differentially after in vitro priming of human T cells by Ag, providing regulatory cytokines are also present.
Appropriate developmental expression of human CD8 beta in transgenic mice.
Authors: Kieffer L J , Yan L , Hanke J H , Kavathas P B
Published: December 2022

The human CD8 glycoprotein is expressed either as an alpha beta heterodimer or as an alpha alpha homodimer on thymocytes, mature T cells, and subpopulations of intestinal intraepithelial lymphocytes (IELs). The homodimeric form of CD8 is exclusively expressed on TCR gamma delta IELs and on subsets of NK cells and TCR alpha beta IELs. To understand the molecular mechanisms by which these genes are regulated, we created transgenic mice with a 95-kb human genomic DNA fragment containing the entire CD8 beta gene as well as a cluster of tissue-specific DNase I-hypersensitive sites 7 to 10 kb upstream of the gene. These sites were present in CD8 alpha beta+- but not CD8 alpha beta- T cell lines nor in a B cell line. We found that transgenic mice had correct developmental expression of human CD8 beta on thymocytes and mature CD8+ cells and no expression on mature CD4+ T cells or B cells. Interestingly, the percentage of mouse CD8 alpha+ cells that were human CD8 beta+ varied, depending on the founder line, from 4 to 88%, whereas the percentage among siblings was similar, indicative of a variegated phenotype resulting from site of integration effects. Expression was also observed on intestinal IELs, but only on those expressing the TCR alpha beta receptor and not the TCR gamma delta cells, which exclusively express CD8 alpha alpha. Of the TCR alpha beta+ cells, the transgene was expressed in both the CD8 alpha alpha and alpha beta subpopulations. These results indicate that this 95-kb fragment affords developmentally correct expression of the human CD8 beta gene on thymus-derived T cells in transgenic animals. Therefore, CD8 lineage-specific regulatory sequences must be located within the fragment.
HLA-DP2: self peptide sequences and binding properties.
Authors: Chicz R M , Graziano D F , Trucco M , Strominger J L , Gorga J C
Published: December 2022

Although self peptides bound to HLA-DQ and, especially, HLA-DR allotypes have been described in some detail, few ligands that bind to HLA-DP have been identified. Toward this aim, naturally processed peptides were isolated from immunoaffinity-purified HLA-DP2 molecules expressed in cultured B lymphocytes. The size distribution of the peptide repertoire is generally similar to those reported for self peptides bound to HLA-DR and HLA-DQ molecules. Twelve peptides representing individual sequences including two nested sets were sequenced by mass spectrometry and/or N-terminal Edman analysis. Source proteins included MHC molecules and other integral membrane proteins as well as secretory and serum proteins. No dominant amino acid markers suggestive of particular enzymatic processing events were detected. Peptide specificity and affinity were examined in binding assays using synthetic peptides and purified HLA-DP and HLA-DR molecules. Anchor residues were tentatively assigned using alanine-substituted analogues of two self peptides. Some structural features of HLA-DP2 that may relate to peptide binding are considered.
Natural killer cell cytokine production, not cytotoxicity, contributes to resistance against blood-stage Plasmodium chabaudi AS infection.
Authors: Mohan K , Moulin P , Stevenson M M
Published: December 2022

Our recent study showed that IL-12 treatment of susceptible A/J mice induces Th1-mediated, protective immunity against lethal blood-stage Plasmodium chabaudi AS infection. To further understand the mechanism of this protection, we examined NK cell cytotoxic (NKCC) and cytokine secretory functions in untreated and IL-12-treated A/J mice, along with resistant C57BL/6 (B6) mice. Normal A/J mice receiving six daily doses of 0.1 microg IL-12 exhibited significant increases in NKCC in total spleen cell populations. Defective NKCC evident in vitro in enriched NK cells from infected A/J mice was corrected by addition of 10 ng/ml IL-12 and was comparable with that seen in B6 mice. In vivo and in vitro analyses revealed that enriched NK cells from day 6 infected A/J mice were defective not only in NKCC, but also in IFN-gamma, and to a certain extent, TNF-alpha secretion, which could also be corrected by IL-12 treatment. Depletion of NK cells from resistant B6 mice resulted in a more severe course of infection, while NK cell-depleted, IL-12-treated A/J mice had significantly higher parasitemia, as well as 100% mortality, suggesting the importance of NK cells in IL-12-mediated protection. NKCC-defective bg/bg mice produced optimum IFN-gamma and TNF-alpha and recovered from infection similar to bg/+ controls; in vivo depletion of these cytokines resulted in significantly higher parasitemia early in infection. Based on these results, we conclude that IFN-gamma, and possibly TNF-alpha, secretion by NK cells during early infection plays a major role in protective immunity to blood-stage malaria.
Formyl peptide receptors are coupled to multiple mitogen-activated protein kinase cascades by distinct signal transduction pathways: role in activation of reduced nicotinamide adenine dinucleotide oxidase.
Authors: Rane M J , Carrithers S L , Arthur J M , Klein J B , McLeish K R
Published: December 2022

Formyl peptide receptor activation of three mitogen-activated protein kinase (MAPK) cascades, extracellular signal-regulated kinases (ERKs), N-terminal kinases (JNKs), and p38 MAPK was examined in differentiated HL-60 granulocytes. FMLP stimulated a concentration- and time-dependent increase in ERK, JNK, and p38 MAPK activities, all of which were dependent on a pertussis toxin-sensitive G protein. Pharmacologic inhibitors were used to examine the roles of tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C. FMLP-stimulated ERK activity was dependent on tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C; p38 MAPK activation was dependent on phosphatidylinositol 3-kinase and phospholipase C; while JNK activation was independent of all of these signaling components. The mitogen-activated protein kinase/ERK kinase inhibitor PD098059 reduced ERK activation by 90%, while an inhibitor of p38 MAPK, SB203580, inhibited p38 MAPK activation by 80%. Both PD098059 and SB203580 inhibited FMLP-stimulated superoxide release, as did inhibitors directed against protein kinase C, tyrosine kinases, and phosphatidylinositol 3-kinase. We conclude that formyl peptide receptors are coupled to three MAPK cascades by Gi proteins. ERKs, p38 MAPK, and JNKs are each activated by distinct proximal signal transduction pathways. Activation of p38 MAPK is necessary for FMLP stimulation of respiratory burst activity; however, a second signal that may involve ERK is also required for this activity.