bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced production of nitric oxide by rat alveolar macrophages after inhalation of a pulmonary irritant is associated with increased expression of nitric oxide synthase.
Authors: Pendino K J , Laskin J D , Shuler R L , Punjabi C J , Laskin D L
Published: December 2022

Alveolar macrophages represent the first line of defense of the lung against inhaled environmental agents. These cells release a variety of inflammatory mediators including reactive oxygen and nitrogen intermediates that have been implicated in host defense and in tissue injury. In the present studies we characterized production of these mediators by lung phagocytes after exposure of rats to an inhaled pulmonary irritant. Freshly isolated alveolar macrophages from control rats were found to produce nitric oxide as well as hydrogen peroxide and superoxide anion in response to in vitro treatment with inflammatory mediators such as IFN-gamma or LPS and phorbol esters, respectively. Production of nitric oxide by lung phagocytes was enhanced in the presence of superoxide dismutase. Western blot analysis revealed that production of nitric oxide after treatment of the cells with IFN-gamma and LPS was a result of increased expression of inducible nitric oxide synthase. After brief exposure of rats to ozone (O3, 1 to 2 ppm, 3 h), a pulmonary irritant and inflammatory agent that is rapidly converted to molecular oxygen, lung phagocytes produced significantly increased amounts of nitric oxide when compared with control animals. These cells were also sensitized to produce more nitric oxide in response to in vitro treatment with IFN-gamma and LPS. This was due, at least in part, to increased expression of inducible nitric oxide synthase by the cells, which was evident in protein blots and in immunohistochemically stained sections of lung tissue. In further studies we found that O3 inhalation also caused enhanced production of hydrogen peroxide, but an apparent decrease in release of superoxide anion by lung phagocytes. Taken together, these data demonstrate that acute irritant exposure modifies production of reactive oxygen and nitrogen intermediates by lung phagocytes. These alterations may be important in the pathophysiologic response of the lungs to irritants.
Close correlation between Daudi and mycobacterial antigen recognition by human gamma delta T cells and expression of V9JPC1 gamma/V2DJC delta-encoded T cell receptors.
Authors: Davodeau F , Peyrat M A , Hallet M M , Gaschet J , Houde I , Vivien R , Vie H , Bonneville M
Published: December 2022

Recent studies have demonstrated that a large fraction of human gamma delta PBL recognize Ag of prokaryotic and eukaryotic origins, respectively found in hydrosoluble mycobacterial extracts and on the Daudi Burkitt's lymphoma cells. The structural basis of the recognition of these Ag have been presently studied in detail, through analysis of a large panel of thymus- and peripheral blood-derived gamma delta T-cell clones. Our results suggest that Daudi and mycobacteria-reactive gamma delta subsets are strictly overlapping and hence that gamma delta T-cell responses against these two Ag are closely related. Daudi cells and mycobacteria were recognized by V gamma 9+V delta 2+, but not by V gamma 9+V delta 2-, V gamma 9-V delta 2+, or V gamma 9-V delta 2- PBL clones. However, not all V gamma 9+V delta 2+ clones were reactive and, in particular: 1) the proportion of Ag-reactive lymphocytes was much lower among thymus- than PBL-derived clones (respectively 24/36 vs 72/73); 2) none of the V gamma 9+V delta 2+ clones expressing a V9J2C2 gamma chain (n = 4) were reactive to Daudi or mycobacteria, indicating that expression of a disulfide-linked TCR is probably a prerequisite for recognition of these Ag; and 3) among V gamma 9+V delta 2+ clones bearing disulfide-linked TCR, almost all (50/53) clones expressing a V9JPC1 gamma chain were reactive, whereas a large fraction (6/10) of those expressing a V9J1C1 gamma chain were weakly or nonreactive. Together, these observations suggest that germline residues specific to V gamma 9, V delta 2, and J gamma P elements directly contribute to recognition of Daudi and mycobacterial Ag. Furthermore, these findings may provide an explanation for coordinate use of these gene elements by a large fraction of gamma delta PBL, through peripheral selection events mediated by ligands identical or structurally related to the above Ag.
Identification of T cell peptides from C gamma 2ab involved in IgG2ab T cell-induced allotypic suppression. Relationship with the MHC haplotype of the suppressor T cell-donor Igha mice.
Authors: Majlessi L , Roth C , Benaroch P , Claverie J M , Kourilsky P , Bordenave G
Published: December 2022

Using the algorithm of the peptidic self-model we selected, from the constant part of the IgG2ab polypeptidic heavy chain, four peptides susceptible to being recognized by the T cells of Igha mice that prevent IgG2ab expression. Among these four peptides, two were capable of enhancing in vivo this T cell activity of Igha mice almost as well as the whole IgG2ab allotype. One of these peptides, C gamma 2ab-248-263, located in the CH3 domain of IgG2ab, was effective in H-2d mice, while the other one, C gamma 2ab-103-118, located in the hinge region of IgG2ab, was efficient in H-2b mice. This demonstrates for the first time that this suppression involves the TCR of the effector cells and peptides derived from the allotype presented in the context of MHC molecules of the target cells, the presentation specificity being dependent on the MHC haplotype. The availability of these peptides will allow us to further understand the role of class I and/or class II MHC molecules in this suppression.
T cell receptor V alpha-V beta combinatorial selection in the expressed T cell repertoire.
Authors: Vacchio M S , Granger L , Kanagawa O , Malissen B , Tomonari K , Sharrow S O , Hodes R J
Published: December 2022

This study has evaluated whether preferential pairing occurs between TCR alpha- and beta-chains expressing specific V alpha and V beta gene products in the mature peripheral T cell population, as a result of either thymic selection or of structural constraints on chain pairing. The association of specific V alpha products with specific V beta products on individual T cells was found, in multiple instances, to be highly selective. Moreover, patterns of preferential V alpha-V beta association were highly strain-specific and were independently expressed in CD4+ and CD8+ T cell subsets. Although these findings do not exclude the possibility that structural constraints may limit V alpha-V beta pairing in other instances, they indicate that the observed instances of skewed expression are not caused by structural constraints in chain pairing. Rather, they suggest that strain-specific selective events alter the expressed V alpha V beta repertoire as a result of recognition of self or environmental Ag during T cell repertoire selection.
Chemoattraction of neutrophils by substance P and transforming growth factor-beta 1 is inadequately explained by current models of lipid remodeling.
Authors: Haines K A , Kolasinski S L , Cronstein B N , Reibman J , Gold L I , Weissmann G
Published: December 2022

"Classical" chemoattractants, such as FMLP, C5a, or leukotriene B4, not only elicit directed motility but also activate neutrophils (degranulation, release of active oxygen species). Signal transduction after ligation of receptors for these classical chemoattractants is mediated by pertussis toxin (PT)-sensitive, heterotrimeric G proteins and the early production of lipid messengers via phospholipases. In contrast, we have previously shown that substance P (SP) and transforming growth factor-beta 1 (TGF-beta 1) are "pure" chemoattractants in that they elicit chemotaxis without activating neutrophils. Paradoxically, pure chemoattractants also activate G proteins (plasmalemmal GTPase activity) without eliciting increments in cytosolic calcium ([Ca]i) and thus inositol trisphosphate. We therefore determined lipid remodeling and signal transduction in response to pure chemoattractants. Increments in plasmalemmal GTPase activated by SP (0.1 microM) and TGF-beta 1 (40 fM), like that after FMLP, were PT-sensitive (SP = 6.6 +/- 2 pm/mg/min vs SP + PT = 1.1 +/- 0.9 over basal activity; TGF-beta 1 = 4.3 +/- 1.6 vs TGF-beta 1 + PT = 2.3 +/- 0.9). In parallel, treatment of PMN with PT (1 microgram/ml, 30 min) inhibited chemotaxis (under agarose) after FMLP (2175 +/- 176 (SEM) microns vs 726 +/- 267) and SP (411 +/- 99 microns vs 103 +/- 62 microns) and TGF-beta 1 (40 fM, 375 +/- 53 microns vs 83 +/- 47). However, G proteins coupled to receptors for SP and TGF-beta 1, unlike FMLP, did not appear to be linked to phospholipases in that neither increments in diacylglycerol were detected after receptor ligation (FMLP = 152 +/- 22% resting levels; SP = 101 +/- 5%; TGF-beta 1 = 105 +/- 4%) nor was alkylacylglycerol increased by exposure to SP or TGF-beta 1 (SP = 92 +/- 4%; TGF-beta 1 = 101 +/- 8%; FMLP = 226 +/- 40%). Moreover, polymorphonuclear leukocytes failed to generate phosphatidates (PA) of either species after SP (DA-PA = 79 +/- 9% resting at 60 s; EA-PA = 103 +/- 4%) or TGF-beta 1 (DA-PA = 101 +/- 5%; EA-PA = 98 +/- 9%) in contrast to FMLP (DA-PA = 155 +/- 22%; EA-PA = 149 +/- 16%). The data clearly contravene the current dogma that all chemoattractants use inositol trisphosphate and diglycerides as intracellular signals and suggest the presence of a unique subset of PT-sensitive G proteins, not coupled to "classical" phospholipases, transduce chemoattraction.
Identification by microsequencing of lipopolysaccharide-induced proteins secreted by mouse macrophages.
Authors: Meheus L A , Fransen L M , Raymackers J G , Blockx H A , Van Beeumen J J , Van Bun S M , Van de Voorde A
Published: December 2022

The conditioned medium of the murine macrophage PU5.1.8 was analyzed by two-dimensional gel electrophoresis in order to detect LPS-induced proteins. Spots of interest were identified by microsequencing of internal peptides generated by limited in situ acid hydrolysis. In total conditioned medium, several monokines (TNF-alpha and macrophage inflammatory protein-1 alpha and 1 beta) were identified as LPS-induced spots. Because minor spots could be masked by the complexity of the 2-D pattern, conditioned medium was successively fractionated by zinc precipitation and affinity chromatography (Procion red and Con A agarose). Zinc supernatant fraction, Procion red flow-through, and Con A eluate fractions were further analyzed by 2-D gel electrophoresis for the presence of LPS-induced spots. In these fractions serum amyloid A3, lipocalin 24p3, cathepsin B, and plasminogen activator inhibitor-I were characterized as LPS-induced proteins secreted by macrophages. Lipocalin 24p3 protein was retrieved for the first time. In addition to these proteins that follow a classical secretory pathway, several cellular proteins (mainly ribosomal proteins) were retrieved as LPS-induced proteins in the conditioned medium. Control experiments argue against the obvious explanation that the latter observation is caused solely by cellular leakage.
Susceptibility of human trophoblast to killing by human complement and the role of the complement regulatory proteins.
Authors: Tedesco F , Narchi G , Radillo O , Meri S , Ferrone S , Betterle C
Published: December 2022

The susceptibility of trophoblast to cytolysis by human complement was investigated using cells purified to over 90% from first trimester placentae. Two assay systems were employed to measure the killing of trophoblasts, an antibody-dependent complement-mediated cytolysis and the reactive lysis. The antibody obtained from a patient with Addison's disease reacted specifically with syncytiotrophoblasts and induced a dose-dependent killing of the cells not exceeding 50% even in the presence of excess antibody and complement. The percentage of cells killed by the terminal complement complex in the reactive lysis system was somewhat higher, reaching values of 60%. Immunofluorescence analysis revealed the presence of CD46 and CD59 on all syncytiotrophoblasts, whereas CD55 was only detected on approximately 30% of the cells. Inhibition of CD46 and CD59 resulted in increased susceptibility of syncytiotrophoblasts to complement lysis. The protective function of CD55 could not be evaluated because of its reduced expression on isolated trophoblasts. These results suggest that syncytiotrophoblasts may be killed by complement and that membrane regulators to some extent protect these cells from complement damage.
Evidence for selective accumulation of V beta 8+ T lymphocytes in experimental autoimmune uveoretinitis induced with two different retinal antigens.
Authors: Egwuagu C E , Mahdi R M , Nussenblatt R B , Gery I , Caspi R R
Published: December 2022

Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease that serves as a model of human intraocular inflammatory disease (uveitis) and is initiated in susceptible animals by immunization with retinal Ag, such as interphotoreceptor retinoid binding protein (IRBP) and S-Ag (SAg). Previous studies of TCR usage by uveitogenic T cells suggested a possible connection between pathogenicity of T lymphocytes and usage of V beta 8 family genes. Here, we have analyzed the T cell repertoire at the autoimmune site by examining V beta gene expression in the retinas of rats with SAg- or IRBP-induced uveitis. Our data show the following: 1) T cell response to SAg or IRBP is clonally heterogeneous: at least 15 of the 20 known rat TCR families were detected in the retinas of rats with EAU. 2) V beta 8+T cells were selectively increased in retina during the early stage of EAU, and the pattern of V beta 8 subfamily member utilization in the retina was Ag-dependent: V beta 8.2+ and V beta 8.3+ T cells were found in the retina of rats immunized with IRBP but only V beta 8.2+ cells in SAg-induced EAU. V beta 8.1 cells were not detected in any of the samples. 3) Kinetic change in the proportions of V beta 8.2+ and V beta 8.3+ T cells was observed in IRBP-EAU: on the day of clinical onset of disease, V beta 8.3+ cells were the only member of the V beta 8 family detected, but 24 h later, V beta 8.2+ T cells appeared and became the dominant V beta 8 clonotype in the retina. 4) The pattern of V beta 8 family member usage by T cells in uveitogenic lines specific to the corresponding Ag was similar to their utilization in the uveitic response in the retina. DNA sequence analysis of 75 V beta 8 cDNA clones from uveitogenic T cell lines revealed that, of 20 V beta 8 clones isolated from SAg-specific T cell lines, all were V beta 8.2 TCR, whereas among 55 V beta 8 clones from IRBP-specific lines, 36 were V beta 8.2 and 19 were V beta 8.3. In similarity to the retina, no V beta 8.1+ T cells were detected from these T cell lines. Taken together, our data suggest a bias towards usage of V beta 8+ cells in EAU and, depending on the autoantigen mediating the disease, particular V beta 8 subfamily member(s) were preferentially utilized or excluded from the autoimmune response.
Tumor-specific cytotoxic T cell clones from patients with breast and pancreatic adenocarcinoma recognize EBV-immortalized B cells transfected with polymorphic epithelial mucin complementary DNA.
Authors: Jerome K R , Domenech N , Finn O J
Published: December 2022

Human breast and pancreatic adenocarcinomas are tumors of ductal epithelial cell origin and as such produce and express on their surface polymorphic epithelial cell mucin encoded by the MUC 1 gene. We have previously reported that tumor-specific cytotoxic T cells derived from patients bearing such tumors recognize specific epitopes on the mucin polypeptide core. This recognition was not MHC-restricted because of the highly repetitive sequence of the polypeptide core, which allows simultaneous recognition of many identical epitopes, and cross-linking and aggregation of TCR on mucin-specific T cells. Those studies were performed with limited numbers of tumor cells or allogeneic tumor cell lines. A renewable source of autologous cells presenting this Ag was necessary to further explore mucin-specific immunity. We report here successful establishment and functional analysis of mucin-specific CTL lines and clones derived from breast and pancreatic cancer patients, using either autologous or allogeneic mucin-transfected B cells as Ag. Our results demonstrate that transfection of autologous or allogeneic B cells with mucin confers upon them tumor Ag-presenting ability as well as susceptibility to lysis by mucin-specific CTL. Transfection of APC with this or any other human tumor Ag that may be molecularly defined in the future provides a unique and powerful tool with which to examine the ability of a tumor-associated Ag to stimulate T cell responses.
Increasing the immunogenicity of protein antigens through the genetic insertion of VQGEESNDK sequence of human IL-1 beta into their sequence.
Authors: Beckers W , Villa L , Gonfloni S , Castagnoli L , Newton S M , Cesareni G , Ghiara P
Published: December 2022

The immunogenicity of two recombinant protein Ag containing the immunostimulatory sequence of human IL-1 beta 163-171 (VQGEESNDK) genetically engineered into their structure has been evaluated. The IL-1 beta sequence was inserted into the loop between alpha helices D and E of recombinant human ferritin H chain and into the hypervariable region of recombinant flagellin from Salmonella muenchen. The chimeric proteins were injected into mice and the level of humoral immune response developed against the native proteins was assessed by measuring the number of Ag-specific plaque forming cells/spleen or as the level of serum IgG response. The response was compared to that of mice receiving injections with wild-type protein Ag not containing the VQGEESNDK sequence or with hybrid constructs containing unrelated foreign peptide sequences of the same length. A significantly higher immune response was observed in mice immunized with chimeric constructs containing the human IL-1 beta 163-171 sequence. These data suggest that the insertion of the VQGEESNDK sequence may prove useful to increase the immune response against poorly immunogenic recombinant proteins.
Priming immunization determines T helper cytokine mRNA expression patterns in lungs of mice challenged with respiratory syncytial virus.
Authors: Graham B S , Henderson G S , Tang Y W , Lu X , Neuzil K M , Colley D G
Published: December 2022

Defining the mechanism for the vaccine-enhanced illness associated with respiratory syncytial virus (RSV) is critical for advancing RSV vaccine development. Previous studies in which infants were vaccinated with formalin-inactivated alum-precipitated whole virus did not protect from RSV infection, and those infected had a high incidence of severe illness. In contrast, previous clinical trials evaluating live attenuated RSV showed no associated vaccine-enhanced illness. We have used a mouse model to explore the immunopathogenesis of RSV infection. In this study cytokine mRNA expression was examined using 32P-labeled oligonucleotide probes in Northern blot analyses of polyA RNA extracted from lungs of mice primed with various vaccine preparations then challenged nasally with live RSV. We have shown that upon challenge, priming of mice with inactivated virus or subunit F glycoprotein induced a pattern of cytokine mRNA expression suggesting a dominant Th2-like lymphocyte response (relative increase in IL-4 mRNA expression). In contrast, challenge of mice primed with live RSV by parenteral or mucosal routes induced a Th1-like pattern of cytokine mRNA expression (relative decrease in IL-4 mRNA expression compared to IFN-gamma mRNA expression). Thus, the formulation and route of delivery of vaccine products can influence the pattern of cytokine expression in lung upon RSV challenge.
Pentoxifylline attenuates neutrophil activation in experimental endotoxemia in chimpanzees.
Authors: van Leenen D , van der Poll T , Levi M , ten Cate H , van Deventer S J , Hack C E , Aarden L A , ten Cate J W
Published: December 2022

Costimulation of neutrophils and cytokines may play an important role in organ injury in sepsis. Pentoxifylline inhibits various neutrophil functions in vitro, and attenuates endotoxin-induced production of TNF in both in vitro and in vivo models. To assess the effect of pentoxifylline on neutrophil activation in endotoxemia, nine adult chimpanzees (Pan troglodytes) were i.v. injected with saline (n = 2), Escherichia coli endotoxin (4 ng/kg; n = 4), or E. coli endotoxin (4 ng/kg) in combination with pentoxifylline (500 mg/3 h, starting 30 min before the endotoxin injection; n = 3). Serial blood samples were obtained for measurements of leukocyte counts and the granulocytic proteinases elastase complexed with alpha 1-antitrypsin and lactoferrin, and cytokines during the next 5 h. No changes were observed in the saline-treated chimpanzees. Endotoxin induced a marked leukocytosis and neutrophilia, which were slightly reduced by pentoxifylline. In contrast, pentoxifylline almost completely prevented endotoxin-induced neutrophil degranulation: peak elastase-alpha 1-antitrypsin was 164 +/- 21 ng/ml (mean +/- SE) after endotoxin alone, vs 71 +/- 7 ng/ml after endotoxin with pentoxifylline (t = 3 h; p < 0.05); peak lactoferrin was 329 +/- 15 and 182 +/- 5 ng/ml, respectively (t = 5 h; p < 0.05). Pentoxifylline also inhibited the endotoxin-induced release of TNF (271 +/- 26 vs 55 +/- 23 pg/ml at t = 1.5 h; p < 0.05) and IL-6 (225 +/- 42 vs 73 +/- 25 pg/ml at t = 2 h; p < 0.05). IL-8 release was not significantly inhibited by pentoxifylline. In none of the animals activation of the C system could be detected. We conclude that pentoxifylline attenuates neutrophil activation in endotoxemia in chimpanzees, probably in part by inhibiting the release of TNF.
Inhibition of Langerhans cell antigen-presenting function by IL-10. A role for IL-10 in induction of tolerance.
Authors: Enk A H , Angeloni V L , Udey M C , Katz S I
Published: December 2022

IL-10 is a product of activated keratinocytes and is released during the induction phase of contact sensitivity. As IL-10 effects have been described as being mediated by APC, we investigated effects of IL-10 on epidermal Langerhans cells (LC), the resident APC in the epidermis. Initial studies failed to demonstrate effects of IL-10 on MHC class II Ag expression by LC or anti-CD3 mAb- or alloantigen-induced LC-dependent T cell proliferation. However, production of IFN-gamma and IL-2, (but not IL-6) was markedly reduced in these assays. When the soluble-protein Ag specific T cell clones AE7 (Th1) and D10.G4 (Th2) were substituted for unprimed T cells, differential effects of IL-10 on T-cell proliferation were observed. Whereas IL-10-pretreated and untreated LC supported Th2 cell proliferation equally well, IL-10-pretreated LC were essentially unable to induce Th1 cell proliferation in response to native protein or peptide Ag. The inhibitory influence of IL-10 on Th1 cells was observed when fresh or 1 day cultured LC were used; 2- or 3-day cultured LC were affected to a much lesser extent by IL-10 pretreatment. Further, coculture experiments using IL-10-pretreated or untreated LC of a different haplotype suggest that IL-10 negatively regulates a costimulatory signal required for induction of Th1 cell proliferation. To assess whether T cells incubated with Ag and IL-10-pretreated LC were responsive to further stimulation, T cells were rescued after 1 day of coculture with IL-10-pretreated LC and restimulated, either immediately or after 1 to 5 days of rest, with untreated LC in the presence of Ag. T cells incubated with IL-10-pretreated LC were found to be anergic, whereas T cells incubated with untreated LC proliferated normally after further stimulation. However, anergic T cells responded vigorously to IL-2. These data indicate that although IL-10-pretreated LC are effective APC for Th2 cells, they fail to induce Th1 cell proliferation and rather induce clonal anergy in these cells.
Regulation of TL antigen expression. Analysis of the T18d promoter region and responses to IFN-gamma.
Authors: Wang I M , Mehta V , Cook R G
Published: December 2022

Type I (alpha/beta) and type II (gamma) IFN enhance MHC class I gene expression through an IFN-responsive element (IRE) present in the 5' flanking region of the class I-a genes. Comparison of the 5' sequences between classical class I-a genes and T region class I-b genes reveals little homology except for presence of a potential IRE. We have found that cell surface expression of thymus leukemia Ag (TL) was up-regulated by IFN-gamma to a greater extent than H-2K,D in all TL+ T cell lines tested. In contrast, IFN-alpha/beta, which significantly increased H-2K and H-2D Ag expression, had only minor effects on TL expression. Resting peripheral T cells, which were considered to be TL- from previous studies, were found to express TL at a low level as determined by flow cytometry, immunoprecipitation, as well as polymerase chain reaction; the level of expression also could be elevated by IFN-gamma. To examine the control of TL gene transcription and its regulation by IFN-gamma, varying lengths of the T18d 5' flanking region were analyzed in chloramphenicol acetyl transferase assays. By deletion analysis, promoter activity and IFN-gamma responsiveness were localized to an 86-bp fragment that contains the IRE. Both responses were localized further to a 32-bp fragment that contained the IRE at its 3' end. RNase protection assays revealed two major transcription initiation sites, one immediately 5' of the IRE and another approximately 60 bp downstream. Furthermore, polymerase chain reaction analysis of mRNA from resting T cells, thymocytes, and T cell tumor lines confirmed the RNase protection data. Thus, transcription of T18d initiates much further upstream than the classical class I genes, can utilize an unusual promoter element, and can be elevated by IFN-gamma.
Macrophage inflammatory protein-1 alpha expression in interstitial lung disease.
Authors: Standiford T J , Rolfe M W , Kunkel S L , Lynch J P , Burdick M D , Gilbert A R , Orringer M B , Whyte R I , Strieter R M
Published: December 2022

Mononuclear phagocyte (M phi) recruitment and activation is a hallmark of a number of chronic inflammatory diseases of the lung, including sarcoidosis and idiopathic pulmonary fibrosis (IPF). We hypothesized that macrophage inflammatory protein-1 (MIP-1 alpha), a peptide with leukocyte activating and chemotactic properties, may play an important role in mediating many of the cellular changes that occur in sarcoidosis and IPF. In initial experiments, we demonstrated that human rMIP-1 alpha exerted chemotactic activities toward both polymorphonuclear leukocytes and monocytes, and these activities were inhibited by treatment with rabbit anti-human MIP-1 alpha antiserum. In support of the potential role of MIP-1 alpha in interstitial lung disease, we detected MIP-1 alpha in the bronchoalveolar lavage fluid of 22/23 patients with sarcoidosis (mean 443 +/- 76 pg/ml) and 9/9 patients with IPF (mean 427 +/- 81 pg/ml), whereas detectable MIP-1 alpha was found in only 1/7 healthy subjects (mean 64 +/- 64 pg/ml). In addition, we found a 2.5- and 1.8-fold increase in monocyte chemotactic activity in BALF obtained from patients with sarcoidosis and IPF respectively, as compared to healthy subjects, and this monocyte chemotactic activity, but not neutrophil chemotactic activity, was reduced by approximately 22% when bronchoalveolar lavage fluid from sarcoidosis and IPF patients were preincubated with rabbit antihuman MIP-1 alpha antibodies. To determine the cellular source(s) of MIP-1 alpha within the lung, we performed immunohistochemical analysis of bronchoalveolar lavage cell pellets, transbronchial biopsies, and open lung biopsies obtained from patients with IPF and sarcoidosis. Substantial expression of cell-associated MIP-1 alpha was detected in M phi, including both alveolar AM phi and interstitial M phi. In addition, interstitial fibroblasts within biopsies obtained from sarcoid and IPF patients also expressed immunoreactive MIP-1 alpha. Minimal to no detectable MIP-1 alpha was expressed in alveolar M phi from healthy subjects or interstitial cells in lung biopsy specimens obtained from patients undergoing thoracotomy for malignancy. Furthermore, pulmonary fibroblasts isolated from patients with IPF produced greater amounts of MIP-1 alpha after challenge with IL-1 beta than did similarly treated pulmonary fibroblasts recovered from patients without fibrotic lung disease. Our findings suggest that MIP-1 alpha is expressed in increased amounts within the airspace and interstitium of patients with sarcoidosis and IPF, and that this cytokine may be an important mediator of both M phi activation and recruitment that characterize these disease states.
Loxoribine (7-allyl-8-oxoguanosine) activates natural killer cells and primes cytolytic precursor cells for activation by IL-2.
Authors: Pope B L , Chourmouzis E , Victorino L , MacIntyre J P , Capetola R J , Lau C Y
Published: December 2022

Guanine ribonucleosides that have been substituted at the C8 position with bromine or thiol groups have been shown previously to activate NK cells and to act as sparing agents for IL-2 in the generation of LAK cells. Herein, we examined a disubstituted guanosine, 7-allyl-8-oxoguanosine (loxoribine), for the ability to activate NK cells and to interact with IL-2 in the generation of LAK cells. Loxoribine enhanced the NK activity of murine spleen cells with optimal activity occurring after 10 h of culture at concentrations ranging from 50 to 150 microM. The response was, however, short lived, approaching baseline levels by 24 h of culture. In contrast, if spleen cells were cultured with a suboptimal concentration of IL-2 (10 U/ml) in combination with loxoribine, a prolonged and enhanced cytolytic activity was seen. The enhancement was greatest if the loxoribine and IL-2 were both added to the cultures at the beginning of the incubation period. Analysis of the expression of the alpha-chain of the IL-2 receptor after loxoribine stimulation indicated that gene transcription was enhanced within 4 h, and cell surface expression was observed on NK1.1+ Thy1+ and NK1.1+ Thy1- cell populations within 24 h of loxoribine treatment. The priming of LAK cell precursors by loxoribine did not appear to be mediated by IFN-alpha/beta, because anti-IFN antibodies did not block either the activation of cytolytic cells by IL-2 or the expression of IL-2 receptors after culture with loxoribine. These data suggest that one mechanism by which cytolytic precursor cells are primed by loxoribine to respond to IL-2 faster and with enhanced cytolytic activity may be through the expression of high affinity IL-2 receptors due to the up-regulation of the alpha-chain.
Stimulation of IL-2-activated natural killer cells through the Kp43 surface antigen up-regulates TNF-alpha production involving the LFA-1 integrin.
Authors: Aramburu J , Balboa M A , Rodríguez A , Melero I , Alonso M , Alonso J L , López-Botet M
Published: December 2022

We have previously described that a mAb directed against a surface dimer (Kp43) expressed by NK cells was able to regulate cell proliferation, enhance the cytotoxicity of IL-2-activated NK cells, and activate phospholipase D. In this work we have analyzed the ability of the anti-Kp43 mAb to regulate the production of TNF-alpha. Our results show that the stimulation of IL-2-activated NK cells with soluble anti-Kp43 mAb or its F(ab')2 activated the secretion of the cytokine, inasmuch as this effect is associated with the induction of cellular aggregation. The intensity of the Kp43-mediated stimulation varied among different IL-2-activated NK cell samples. Even in those instances where the anti-Kp43 mAb alone could not detectably enhance TNF-alpha production, it displayed a synergistic effect combined to a soluble anti-CD16 mAb, which on its own did not efficiently activate cytokine production. The anti-Kp43 mAb also cooperated with a phorbol ester, although it did not modify the TNF-alpha production triggered by a Ca2+ ionophore. The anti-Kp43-mediated effect, which required the preactivation of cells with IL-2, was inhibited by cycloheximide and actinomycin D and was associated with an increase in the levels of TNF-alpha-specific mRNA. It is noteworthy that the Kp43-mediated production of TNF-alpha was partially inhibited by anti-CD18, anti-CD11a and anti-ICAM-1 mAb that blocked LFA-1-dependent cellular interactions, which impaired NK cell aggregation and, moreover, was dependent on the presence of extracellular Mg2+, thus suggesting that the leukocyte integrin is involved in the activation process triggered through Kp43.
Deficient antigen presentation by Langerhans cells from athymic (nu/nu) mice. Restoration with thymic transplantation or administration of cytokines.
Authors: Grabbe S , Gallo R L , Lindgren A , Granstein R D
Published: December 2022

Epidermal Langerhans cells (LC) are a unique subtype of I-A+ dendritic cells able to present Ag for CD4-dependent immune responses. To investigate whether cutaneous Ag presentation is regulated by thymic elements or soluble factors produced by thymus-derived cells, we compared LC function in athymic nude mice and euthymic normal controls. Examination of the ability of LC to present alloantigens to T cell-enriched responder populations, and insulin to an insulin-specific T cell hybridoma, demonstrated that this function is deficient in LC from inbred and outbred strains of congenitally athymic (nu/nu) mice compared with euthymic litter mates. Adoptive transfer of thymic tissue from euthymic to athymic mice reconstituted the ability of LC derived from athymic mice to present alloantigens. To investigate whether an altered local cytokine microenvironment was responsible for the diminished LC function in athymic mice, various cytokines were administered in vivo and in vitro before determination of alloantigen presentation by epidermal cells from athymic and euthymic mice. Continuous intraperitoneal infusion of granulocyte-macrophage colony stimulating factor (GM-CSF) or TNF-alpha, but not IL-1 alpha or IL-2, restored alloantigen presenting ability in athymic LC. In vitro preincubation of LC in GM-CSF or TNF-alpha but not in other cytokines tested also reconstituted alloantigen presentation by LC from athymic mice in most, but not all, of the experiments performed. Furthermore, analysis of cytokine production by epidermal cells in athymic and euthymic mice revealed that epidermal cells from athymic mice produce less GM-CSF and more TNF-alpha, but normal amounts of various other cytokines. However, reconstitution of athymic mice with thymic tissue did not result in normalization of GM-CSF or TNF-alpha production by epidermal cells. These data suggest that LC Ag presenting ability is regulated by thymic factors and that adequate function of cutaneous APC in situ may require the continuous presence of sufficient amounts of cytokines including GM-CSF and TNF-alpha.
CD45-negative mutants of a rat natural killer cell line fail to lyse tumor target cells.
Authors: Bell G M , Dethloff G M , Imboden J B
Published: December 2022

To examine the role of CD45 in NK cell activation, we isolated three mutants and one variant of a rat NK cell line, RNK-16. Each of these lacked cell-surface expression of CD45 and did not have detectable transcripts for CD45 on Northern blot analysis. The CD45-negative cells expressed CD2, CD53, and NKR-P1, but mAb-induced perturbations of these molecules did not induce protein tyrosine phosphorylations and increases in the concentration of cytoplasmic-free calcium, as occurred in the wild-type RNK-16. Unlike the wild-type cells, the CD45-negative cells failed to lyse YAC-1 and RL-male-1 tumor targets. The cytolytic activity of the CD45-negative cells could be stimulated pharmacologically by ionomycin and PMA, which, when added to the cytotoxicity assays, induced killing of tumor targets. These studies suggest that CD45 is required for the response of RNK-16 cells to target cells and for signaling through CD2, CD53, and NKR-P1.
Immunogenicity evaluation of a lipidic amino acid-based synthetic peptide vaccine for Chlamydia trachomatis.
Authors: Zhong G , Toth I , Reid R , Brunham R C
Published: December 2022

Lipidic amino acid-based synthetic peptides derived from the variable domains (VD) of Chlamydia trachomatis outer membrane protein 1 were evaluated as potential candidate sequences in a vaccine. A peptide sequence designated P2 from the VD IV of serovar B contained a B cell epitope capable of eliciting antibodies binding to serovar B elementary bodies (EB) and a T helper site capable of presentation by multiple H-2 alleles. Polymerization of the P2 into polylysine to form lipid core peptides (LCP) significantly enhanced immunogenicity compared with P2 monomer alone. The LCP system incorporates lipidic amino acids into the polylysine system and enhances lipophobicity and membrane binding effects of the peptide. A second peptide sequence derived from the VD I of serovar C was cosynthesized with P2 into lipidic polylysine LCP and was designated LCP-H1. Antibodies to this construct reacted at high titer with EB of the three major trachoma causing C. trachomatis serovars A, B, and C. LCP-H1 was immunogenic among four of five murine H-2 alleles. Pepscan analysis showed that the fine specificity of antibodies generated to LCP-H1 were directed to the predetermined neutralizing epitope sequences. An in vitro HAK cell neutralization assay showed that LCP-H1 elicited neutralizing antibodies to serovars A, B, and C, but these were of low titer. Because LCP-H1 antibodies bound to the peptide sequence with 10-100-fold higher titer than to EB, the low neutralization titers most likely result from conformational differences between the synthetic peptide and antigenic sites on the native organism. Modification of LCP-H1 to incorporate a predefined conformation may result in improved antigenic properties.
Evidence for superantigen production by Yersinia pseudotuberculosis.
Authors: Abe J , Takeda T , Watanabe Y , Nakao H , Kobayashi N , Leung D Y , Kohsaka T
Published: December 2022

Yersinia pseudotuberculosis is an enteric pathogen that induces a variety of clinical symptoms, fever, scarlatiniform rash, diarrhea, vomiting, and arthritis. Characteristic histopathologic findings in Y. pseudotuberculosis infection such as lymphoid hyperplasia, typically seen in mesenteric lymph nodes, suggest that the stimulation of a large proportion of T lymphocytes may be involved in the pathogenesis of this infection. In this study, we assessed the mitogenic activity of culture supernatants of the clinical isolates of Y. pseudotuberculosis and investigated the mechanism by which these culture sups activate T cells. The culture sups, as well as partially purified fractions obtained by gel filtration, were found to selectively stimulate T cells bearing V beta 3, V beta 9, V beta 13.1, and V beta 13.2 compared with stimulation by anti-CD3. Furthermore, fibroblasts transfected with different HLA class II molecules, either HLA-DPw9, -DQw6, -DR1, or -DR4 Dw15, were capable of presenting Y. pseudotuberculosis culture supernatants to purified T cells. The T cell response to this sup was not restricted by donor HLA-DR types and was not neutralized by antibodies against the known staphylococcal superantigens, Staphylococcal enterotoxin (SE)A, SEB, SEC2, SED, SEE, and TSST1. These results suggest that Y. pseudotuberculosis produces superantigenic toxins that may mediate some of the systemic illnesses associated with infection by this organism.
Promoter analysis of an interferon-inducible gene associated with macrophage activation.
Authors: Nicolet C M , Paulnock D M
Published: December 2022

We have investigated the regulation of an activation-associated guanylate-binding protein gene (mGBP-1/mag-1) in murine macrophage cell lines in response to the cytokine IFN-gamma. One of the cell lines utilized (RAW 264.7) acquires the ability to kill tumor cells after IFN-gamma and LPS treatment, whereas the other (WEHI-3) does not. We previously have demonstrated that mGBP-1 is induced by IFN-gamma in RAW 264.7 but not WEHI-3 cells. Here we present information concerning the cloning, sequencing, and initial characterization of the upstream region of the mGBP-1 gene as a first step towards understanding the differential control of this gene in RAW 264.7 versus WEHI-3 cells. Genomic fragments encompassing a portion of the mGBP-1 5' flanking region were inserted into vectors containing a luciferase reporter gene. 928 bp of upstream sequence were found to be sufficient for IFN-gamma-mediated induction of luciferase activity in the RAW 264.7 cell line. Furthermore, sequences within 100 bp of the major transcription initiation site conferred strong IFN-gamma responsiveness to the reporter gene. A perfect match to the interferon-stimulated response element (ISRE) was present within this region, and was shown to be essential for interferon-induced expression. An oligonucleotide corresponding to the mGBP-1 ISRE bestowed interferon-inducible expression on a heterologous minimal promoter. Site-specific mutation of the ISRE within the 106-bp upstream region eliminated interferon inducibility of this construct. Taken together, the results indicate the ISRE is necessary and sufficient for IFN-gamma induction of the mGBP-1 gene. Transient transfection assays carried out with the WEHI-3 cell line indicated that all promoter constructs were transcriptionally inactive in these cells, including the ISRE-minimal promoter construct. The inability of the WEHI-3 cell line to utilize an ISRE after IFN-gamma induction may underlie the functional differences exhibited by the two cell lines after IFN-gamma stimulation.
IL-2-induced expression of TTK, a serine, threonine, tyrosine kinase, correlates with cell cycle progression.
Authors: Schmandt R , Hill M , Amendola A , Mills G B , Hogg D
Published: December 2022

We have recently isolated the cDNA for a unique human 97-kDa kinase, TTK, by expression screening of a cDNA expression library using anti-phosphotyrosine antibodies. When expressed in Escherichia coli, TTK can phosphorylate serine, threonine, and tyrosine residues. Thus TTK appears to belong to a newly described family of kinases able to phosphorylate all three hydroxy amino acids. This family of multispecific kinases includes several other kinases involved in cell cycle progression. In support of a possible role in regulating cell cycle progression, TTK message is readily detected in rapidly proliferating tissues in vivo including testes, thymus, bone marrow, and many malignant tumors, but not in benign tissues with a low proliferative rate in vivo. To determine the effect of cell activation and cell cycle progression on TTK expression, we measured TTK mRNA and protein levels as well as kinase activity in freshly isolated T cells or IL-2-expanded T cell blasts activated to proliferate by the addition of a variety of mitogens. TTK mRNA levels, protein levels, and kinase activity were greatly enhanced when either freshly isolated PBL or T cell blasts were activated by cross-linking the TCR complex by mitogenic lectins or by bypassing the TCR with phorbol esters and cation ionophores. Incubation with IL-2 increased TTK expression in PBL blasts, which proliferate in response to IL-2, but not in fresh PBL, which do not proliferate in response to IL-2. TTK expression was blocked by either cyclosporin A or FK520, which inhibit IL-2 production and could be recovered by the addition of exogenous IL-2. Furthermore, TTK expression was prevented by incubation of the cells with rapamycin, which blocks IL-2 signaling. Thus, TTK expression in T cells appears to be a consequence of IL2-induced cell proliferation. Agonist-induced TTK expression was a delayed event occurring 12 to 24 h after activation of PBL blasts and 48 to 72 h after activation of fresh PBL. TTK protein and mRNA expression increased in both fresh PBL and T cell blasts concurrently with passage of cells through S phase as indicated by [3H]TdR incorporation and cell cycle analysis of propidium iodide-stained cells. TTK mRNA and protein levels reached a maximum as cells entered the G2 phase of the cell cycle. These results were confirmed by cell cycle blockade studies with aphidicolin and nocodazole wherein TTK protein levels are not detected in cells in G1 and are readily detectable in cells in the S and G2 phases of the cell cycle.(ABSTRACT TRUNCATED AT 250 WORDS)
beta-Amyloid activates complement by binding to a specific region of the collagen-like domain of the C1q A chain.
Authors: Jiang H , Burdick D , Glabe C G , Cotman C W , Tenner A J
Published: December 2022

beta-amyloid peptides that accumulate within the brain of individuals with Alzheimer's disease bind to C1q and activate the classical C pathway via a specific interaction with a site within the collagen-like domain of C1q (C1q-CLF). Synthetic analogues of beta-amyloid peptides, beta 1-42 and beta 1-40, bound to C1q and were strong activators of C as assessed by both total C consumption and C4 consumption. beta 1-42 was significantly more effective than beta 1-40 in binding to C1q and triggering C activation, whereas beta 1-28 demonstrated little or no binding or C activation. This C-activating capacity seems to be largely correlated with the assembly of the beta 1-42 into low speed sedimentable aggregates and/or macromolecular fibrils. Radiolabeled C1q and C1q-CLF bind specifically to these aggregates or amyloid fibrils. In addition, using synthetic C1q peptides in a solid phase binding assay, the major binding site of beta 1-42 to C1q was localized to the C1q A chain collagen-like residues 14-26, a region previously described as a novel interaction site for Ab-independent activators of C1. C1q A chain peptide 14-26 blocked the ability of beta-amyloid peptides to activate the classical C pathway, providing evidence that this relatively unrecognized mechanism of C activation (via binding to the C1q-CLF) may have crucial physiologic consequences. Finally, these observations provide further support for the hypothesis that C activation and inflammation may be a component in the pathogenesis of AD and suggest possibilities for modulating the progression of AD.
Selective induction of the beta chemokine C10 by IL-4 in mouse macrophages.
Authors: Orlofsky A , Lin E Y , Prystowsky M B
Published: December 2022

The beta chemokines are a family of 8- to 12-kDa leukocyte chemoattractants that are typically produced by activated macrophages or lymphocytes. We examined the expression in primary macrophages of a recently described, and as yet functionally uncharacterized, murine beta chemokine, C10, and contrasted its regulation with that of several other beta chemokines. Although three other beta chemokines, macrophage inflammatory protein-1 alpha (MIP-1 alpha), JE, and RANTES, were all induced by LPS treatment of bone marrow-derived macrophages (BMM) and/or resident peritoneal macrophages (RPM), LPS stimulation of C10 was never observed. Conversely, IL-3 and granulocyte macrophage-CSF (GM-CSF) strongly induced C10 in both macrophage populations, whereas MIP-1 alpha and RANTES showed a weaker induction restricted to BMM. JE was strongly induced but only in BMM. Finally, IL-4 strongly induced C10 in a dose-dependent manner in both BMM and RPM but failed to stimulate any of the other three beta chemokines. The accumulation of C10 protein in culture supernatants paralleled the induction of mRNA, and the combination of IL-4 and GM-CSF led to enhanced protein levels. The expression of the C10 message in response to cytokines was completely blocked by cycloheximide, whereas the other three chemokines were all overexpressed in the presence of this inhibitor. These results demonstrate a sharp divergence between the regulation of C10 expression and that of other chemokines and suggest that this molecule may have distinct functions in host defense.