bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-12-induced protection against blood-stage Plasmodium chabaudi AS requires IFN-gamma and TNF-alpha and occurs via a nitric oxide-dependent mechanism.
Authors: Stevenson M M , Tam M F , Wolf S F , Sher A
Published: December 2022

The effects of IL-12 administration on the development of protective immunity to blood-stage Plasmodium chabaudi AS were analyzed. Treatment of susceptible A/J mice on the day of infection and for 5 days postinfection with various doses 0.025-0.3 microgram) of rIL-12 significantly decreased the peak parasitemia level, but only treatment with 0.1 microgram resulted in increased survival. Treatment of resistant B6 mice with 0.1 microgram of rIL-12 using the same regimen also significantly decreased the peak parasitemia level, but 40% of the animals died. Treatment of these mice with anti-IL-12 mAb resulted in a more severe course of infection, but survival was not significantly altered. The mechanism of IL-12-induced resistance was examined in A/J mice during infection. Compared with spleen cells from untreated mice, cells from IL-12-treated mice produced significantly higher levels of IFN-gamma spontaneously as well as in response to Con A or Ag stimulation on day 7 postinfection. Significantly higher levels of INF-gamma and TNF-alpha were found in the sera of IL-12-treated mice, which correlated with high levels of the nitric oxide (NO) metabolite, NO3-. Furthermore, CD4+T cell depletion was found to abrogate IL-12-induced resistance. Administration of neutralizing mAb against IFN-gamma or TNF-alpha to IL-12-treated mice showed that simultaneous depletion of both cytokines resulted in 100% mortality. The role of NO was investigated by administration of aminoguanidine, a selective inhibitor of cytokine-inducible nitric oxide synthase, to IL-12-treated mice. Significantly increased mortality was observed following treatment twice daily with 9 mg of aminoguanidine, but there was no effect on parasitemia. In conclusion, these results demonstrate that IL-12 regulates the development of resistance to P. chabaudi AS via a CD4+ Th1 response, which involves the cytokines IFN-gamma and TNF-alpha, and is in part NO dependent. Therefore, IL-12, given in the appropriate dose, may be useful in the induction of protective immunity to blood-state malaria.
Comparative studies of cytotoxicity and the release of TNF-alpha, nitric oxide, and eicosanoids of liver macrophages treated with lipopolysaccharide and liposome-encapsulated MTP-PE.
Authors: Dieter P , Ambs P , Fitzke E , Hidaka H , Hoffmann R , Schwende H
Published: December 2022

LPS and liposome-encapsulated MTP-PE induce liver macrophages cytotoxicity against tumor target cells and a release of TNF-alpha, nitric oxide, and eicosanoids but not a generation of superoxide anions. Neither agent elicits a formation of inositol phosphates, a change in intracellular free calcium, or a translation of protein kinase C-beta. Inhibition or down-regulation of protein kinase C does not inhibit the release of TNF-alpha and nitric oxide but inhibits the formation of prostanoids. In contrast to LPS, liposome -encapsulated MTP-PE induces an elevation of diacylglycerol mass and an enhanced expression of protein kinase C-delta. LPS, but not liposome-encapsulated MTP-PE, elicits an enhanced expression of cytosolic phospholipase A2 and a predominant formation of PGE2. Both agents elicit different responses when given to cells pretreated with one of the immunomodulators, with dexamethasone, or with PGE2. In contrast, to liposome-encapsulated MTP-PE, LPS induces only cytotoxicity when added to liver macrophages simultaneously or a maximum of 2 h before the addition of tumor target cells. The observed differences might reflect partly differences in the potencies of LPS and some liposome-encapsulated MTP-PE as immunomodulators.
Murine mast cells express two types of C1q receptors that are involved in the induction of chemotaxis and chemokinesis.
Authors: Ghebrehiwet B , Kew R R , Gruber B L , Marchese M J , Peerschke E I , Reid K B
Published: December 2022

Although previous studies have shown that different cells and cell lines of murine origin bind human C1q, suggesting that they display cell surface receptors for C1q, no information is available to indicate whether mouse or human mast cells express C1q receptors. This paper presents the first evidence to show that murine mast cells express specific receptors for C1q. Western blot analysis of cell membrane proteins prepared from a bone marrow-derived mouse cell line using two monospecific polyclonal Abs, one directed against the 60-kDa C1q receptor (C1q-R) that binds to the collagen-like stalk of C1q (cC1q-R) and the other directed against the 33-kDa molecule that binds to the globular "heads" of C1q (gC1q-R), show that both of these receptors are present on these cells. In addition, C1q can induce mast cell migration in a specific and dose-dependent manner. Interestingly, the C1q-induced migratory response was found to be biphasic; the first response peaked at a C1q concentration of 0.1 nM, whereas the second phase peaked at approximately 40 nM. Checkerboard analysis of the mast cell migratory response to C1q showed that the first phase was primarily due to chemotaxis and the second phase was attributable to chemokinesis. Preincubation of C1q with Abs specific for the collagen-like tail of the molecule abolished both its chemotactic and chemokinetic response, whereas heat inactivation of C1q (56 degrees C, 1 h) resulted in 85% abrogation of the chemotactic phase and 42% reduction in the chemokinetic phase. The observed mast cell migratory responses were mediated by cell surface C1q-R(s), as inclusion of a mixture of anti-C1q-R and anti-gC1q-R Abs with the cells inhibited their migratory response toward C1q. However, incubation of cells with various doses of C1q did not result in histamine release. Furthermore, engagement of mast cell C1q-Rs by the ligand C1q induced an antiproliferative response, as coculturing of mast cells with C1q resulted in a specific and dose-dependent decrease in DNA synthesis. These data suggest that C1q-Rs may play a significant role in mast cell function and regulation by providing an important signal through which mast cells can be recruited to inflammatory sites of increased C1q concentration.
Heterogeneous T cell responses to beta-lactam-modified self-structures are observed in penicillin-allergic individuals.
Authors: Brander C , Mauri-Hellweg D , Bettens F , Rolli H , Goldman M , Pichler W J
Published: December 2022

To investigate the role of T cells in drug allergy, we stimulated PBMC from penicillin-allergic patients with reactive penicillin G itself or penicillin G coupled with human serum albumin (BPO-HSA). T cell clones specific for penicillin G or BPO-HSA were established and their phenotype and reactivity to both forms of the beta-lactam were analyzed. T cell clones stimulated by penicillin G were CD4 and CD8 positive, whereas BPO-HSA stimulated the growth of CD4+ T cells. The penicillin G-specific clones were HLA class I or class II restricted and processing was not required as fixed APC could still present penicillin G. In contrast, BPO-HSA has to undergo processing to stimulate BPO-HSA-specific T cell clones. In addition to classical APC, activated MHC class II expressing T cells could also restimulate the penicillin G-specific clones, indicating that various cell types might serve as APC. Penicillin G and BPO-HSA-specific T cell clones produced a heterogeneous cytokine pattern as most clones produced high amounts of IL-2, IFN-gamma, TFN-alpha, and rather variable levels of IL-4 and IL-5. Since no Ag processing was required, penicillin G may stimulate T cells by binding directly to MHC molecules on the cell surface or to their embedded peptide. Alternatively, it may bind to soluble proteins like HSA, which are processed and subsequently presented in an immunogenic form. These different modes of presentation, which elicit a variety of immunological reactivities, may explain the great heterogeneity of the clinical pictures seen in penicillin allergy.
A proteoglycan (aggrecan)-specific T cell hybridoma induces arthritis in BALB/c mice.
Authors: Buzás E I , Brennan F R , Mikecz K , Garzó M , Negroiu G , Holló K , Cs-Szabó G , Pintye E , Glant T T
Published: December 2022

Aggrecan, the high buoyant density cartilage proteoglycan (PG), has been shown to induce progressive polyarthritis and ankylosing spondylitis in genetically susceptible BALB/c mice. To further characterize the nature of the autopathogenic effector T cells operating in these mice and to determine the region(s) of the PG molecule recognized by these T cells, we generated PG-specific T cell hybridomas from arthritic mice. One of the PG-specific T cell hybridomas (5/4E8), when injected into naive irradiated BALB/c mice, was capable of inducing clinical and histopathologic signs of arthritis. Massive swelling and redness of the paws dominated the clinical picture. A reactive synovial cell proliferation, the accumulation of hybridoma and inflammatory cells in the enlarged joint space, the loss of PG from the superficial layer of the articular cartilage, and the erosion of articular surface were identical histopathologic signs to those found either in primary or adoptive transfer of PG-induced arthritis. The PG-specific and arthritogenic T cell hybridoma (5/4E8) expressed TCR-alpha beta + (V beta 4), CD4+, and CD8- phenotypes and belonged to the Th1 subset, as the cells secreted IL-2 and IFN-gamma, but not IL-4 upon PG stimulation, and the response was MHC class II (I-Ad)-restricted. These observations provide direct evidence that PG-specific Th cells play crucial roles in autoimmune arthritic processes.
Unexpected presence of polyreactive catalytic antibodies in IgG from unimmunized donors and decreased levels in rheumatoid arthritis.
Authors: Kalaga R , Li L , O'Dell J R , Paul S
Published: December 2022

Polyclonal IgG from healthy humans and unimmunized mice was screened for peptide-methylcoumarinamide (peptide-MCA) hydrolyzing activity. The activity was detected in every IgG sample examined. Contaminant enzymes were precluded as an explanation, as the activity tracked exactly with the 150-kDa IgG peak separated by gel filtration in denaturing solvent (6 M guanidine hydrochloride) and with the 50-kDa Fab fragment peak produced by papain-digestion of the IgG. Patients with rheumatoid arthritis displayed a 3.2-fold reduced peptide-MCA hydrolyzing activity (mean) compared to healthy subjects. Control osteoarthritis patients showed no diminution in activity. A progressive decrease in the activity by 7.4-fold of the pre-immune levels was observed in mice over the course of hyperimmunization with SRBC, indicating that exogenous Ag challenge, like rheumatoid arthritis, is associated with decreased catalytic activity. Apparent Km values of the IgG for Pro-Phe-Arg-MCA were 0.39 to 0.53 mM, values approximately 3-orders of magnitude greater than observed previously for Ag-specific catalysis by Abs. The only common structural feature in peptide-MCA conjugates utilized by the Abs as substrates was the presence of Arg-MCA and Lys-MCA bonds. The IgG hydrolyzed Pro-Phe-Arg-Phe at the Arg-Phe peptide bond, showing that the activity is relevant to cleavage of peptide bonds in natural Ags. The universal occurrence of this polyreactive catalytic activity in unimmunized donors and its diminution in an autoimmune disease and nonspecific Ag challenge suggest that it may possess an important, but as yet unidentified, biologic role.
Molecular genetic analysis of X-linked hypogammaglobulinemia and isolated growth hormone deficiency.
Authors: Stewart D M , Notarangelo L D , Kurman C C , Staudt L M , Nelson D L
Published: December 2022

In 1980 the clinical syndrome of X-linked hypogammaglobulinemia and isolated growth hormone deficiency (XLA/GHD) was described. XLA/GHD patients have reduced serum levels of Ig and normal cell-mediated immunity, and thus resemble patients with Bruton's X-linked agammaglobulinemia (XLA). However, XLA/GHD patients also have isolated GHD. Mutations and deletions in the Bruton's tyrosine kinase gene (BTK) are responsible for Bruton's XLA. We investigated BTK gene expression in an XLA/GHD patient from the family originally described by Northern analysis, cDNA sequencing, and Western analysis of protein production using mAb to BTK. BTK mRNA was normal in size and abundance, and the mRNA sequence was normal over the coding region, except for a single silent mutation. BTK protein was present in normal amounts in PBMC of this patient. Thus, at the molecular level, XLA/GHD is a different disease entity from Bruton's XLA. These results suggest that undescribed genes critical for B cell development and growth hormone production exist on the X chromosome.
Ig domains 1 and 2 of murine CD22 constitute the ligand-binding domain and bind multiple sialylated ligands expressed on B and T cells.
Authors: Law C L , Aruffo A , Chandran K A , Doty R T , Clark E A
Published: December 2022

Baby hamster kidney cells transfected with murine CD22 (mCD22) mediate adhesion to B- and T-lineage cells. To further characterize mCD22-mediated cell adhesion, we generated a panel of recombinant globulins (Rg) consisting of different extracellular Ig-like (Ig) domains of mCD22. FACS analysis using these mCD22.Rgs revealed that ligands for mCD22 are expressed on both B and T cell lines and also normal B and T cells. In B-lineage cells, the expression of mCD22 ligands began on sIgM- pre-B cells in bone marrow. The ligand-binding site of mCD22 for ligands was mapped to Ig domains 1 and 2: mCD22.Rgs containing Ig domains 1 and 2 bound target cells and immunoprecipitated sets of glycoproteins similar to Rgs containing Ig domains 1 to 3 or all 7 CD22 Ig domains, whereas Rgs containing Ig domains 2 to 3 or 3 to 7 did not bind either B or T cells. Furthermore, B cells apparently expressed higher levels of mCD22 ligands than that of T cells, suggesting a potential competition for CD22 binding between ligands expressed on the same B cell and those expressed on another B cell or T cells. Immunoprecipitation experiments using the mCD22.Rgs identified mCD22 itself and the B cell-specific isoform of mCD45RA (B220) as two of the mCD22 ligands expressed on B cells. Thus, mCD22 may potentially regulate B cell activation through interactions with itself or mCD45RA/B220.
Ig VH hypermutation is absent in the germinal centers of aged mice.
Authors: Miller C , Kelsoe G
Published: December 2022

After injection with immunogenic conjugates of the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP), two distinct B cell populations can be identified in the spleen during the primary response. One of these populations is specialized for Ab production; the other, the germinal centers (GCs), has been identified as the site of Ig somatic hypermutation. Ag-driven selection of GC B cells bearing mutated receptors with higher affinity leads to the affinity maturation of serum Ab and increased protective humoral immunity. Microdissection of GC B cell populations specific for NP and sequencing of the recovered Ig heavy chain variable region genes revealed that the somatic hypermutation process is absent in the GCs of aged C57BL/6 mice. However, selection for Ag appears to occur in the absence of hypermutation in the form of competition between unmutated clones of Ag-activated B lymphocytes. Thus, affinity maturation in these animals is limited to the affinities of Ab encoded by the germline.
Human B cell activation. Effect of T cell cytokines on the physicochemical binding requirements for achieving cell cycle progression via the membrane IgM signaling pathway.
Authors: Mongini P K , Highet P F , Inman J K
Published: December 2022

Given the range of mIg-binding affinities expressed by Ag-specific B cells, the ligand:receptor affinity threshold for achieving full B cell activation via the mIgM-mediated signaling pathway is quite high. Several recombinant, or semi-purified, cytokines were found to reduce the very high mIgM:ligand affinity threshold for induction of human B cell S phase entry by bivalent, affinity-diverse anti-IgM mAbs without notably affecting the lower affinity threshold for G1-related RNA synthesis. Two-stage culture experiments suggested that one major means by which IL-4, IL-2, and low m.w. B cell growth factor lower the affinity threshold for S phase entry is an indirect one, i.e., rescue of B cells whose mIg engagements with Ag are of sufficient affinity for achieving G1 entry, but of insufficient affinity for initiating the late-phase mIgM-mediated signals needed for the G1-->S phase transition. IL-4 had additional effects in early G1. In contrast to the above cytokines, IFN-gamma, did not function as an independent cell cycle progression factor, but rather required the concomitant presence of mIgM-cross-linking ligand for enhancement. A greater potential of multivalent anti-IgM-dextran conjugates to trigger S phase entry in the absence of cytokines was found to reflect a greater potential for initiating mIgM signals during the late phase in B cell activation. The results indicate that progression of mIgM receptor-activated B cells past a G1-->S phase restriction point is dependent upon continued signal transduction via either the mIgM receptor and/or a cytokine receptor signaling pathway. When mIgM-engaging ligands are ineffective at initiating late-phase signals, due to limited size and binding site valency and/or affinity, ancillary signal transduction through cytokine receptors becomes most relevant.
Distinct phenotypes of antigen-selected CD8 T cells emerge at different stages of an in vivo immune response.
Authors: Walker P R , Ohteki T , Lopez J A , MacDonald H R , Maryanski J L
Published: December 2022

We have previously described a unique system for identifying Ag-selected CD8 T cells during an in vivo response in normal mice. In this system, lymphocytes isolated from DBA/2 mice injected i.p. with HLA-CW3 transfected syngeneic (H-2d) P815 cells show a remarkable expansion of CD8 cells that utilize TCR expressing the V beta 10 gene segment and additional structural features characteristic of Kd-restricted CW3-specific CTL clones. We have now taken advantage of this system to characterize the surface phenotype of CD8 cells selected by Ag in vivo. We observed several distinct phenotypes at different stages of the response. At the peak of the response, Ag-selected cells were low in CD62L and CD45RB expression but displayed high levels of CD44. In addition, there was a partial down-regulation of CD8 and TCR. Cells of this phenotype were present in lymphoid tissues for several mo after immunization. Much later in the response, Ag-selected cells expressed higher levels of CD8 and TCR. Moreover, a distinct subset of these long-term immune cells emerged that now expressed CD62L and CD45RB. Analysis of CD8 cells from different tissues also revealed certain differences, particularly in TCR and co-receptor levels from liver-derived cells compared with circulating cells at the peak of the response. Our findings suggest that the function of Ag-selected CD8 cells may be regulated over time and according to location by subtle changes in cell-surface phenotype.
Radiation-induced apoptosis is differentially regulated in primary B cells from normal mice and mice with the CBA/N X-linked immunodeficiency.
Authors: Woodland R T , Schmidt M R , Riggs J E , Korsmeyer S J , Lussier A M , Gravel K A
Published: December 2022

Normal B cells responsive to thymus independent-type 1 Ags (TI-1) are resistant to low doses of ionizing radiation in vivo (200-300 cGy), compared with TI-1 responsive B cells of mice with the CBA/N X-linked immunodeficiency (xid). This difference in radiosensitivity is an intrinsic B cell property; normal B cells adoptively transferred into xid mice remain TI-1-responsive after irradiation in situ. Because irradiation induces programmed cell death (PCD) in lymphocytes, we determined whether PCD were regulated differently in normal and xid B cells. B cells isolated immediately after irradiation from normal or xid donors when cultured without stimulators became apoptotic with the same kinetics and to the same extent, showing that apoptosis was induced equally in both populations. Apoptosis could be suppressed and mitogenesis could be induced frequently, however, if irradiated B cells were cultured with B cell activators. When activators using separate signal transduction pathways were compared, a hierarchy of efficiency at effecting apoptosis rescue was observed, and activators used singly without effect could synergize to protect. xid B cells were more resistant to rescue than normal B cells unless PMA was used as a stimulant. Although the mechanism of activator-induced rescue was not established, selective overexpression of a bcl-2 transgene rendered xid B cells radioresistant. The data suggest that a signal(s) delivered to irradiated B cells in the in vivo microenvironment suppresses apoptosis and that xid B cells and a radiosensitive subpopulation of normal B cells are refractory to this signal(s).
Down-regulation of CD8 on mature antigen-reactive T cells as a mechanism of peripheral tolerance.
Authors: Zhang L , Fung-Leung W , Miller R G
Published: December 2022

Previously we have shown that intravenous injection of male B6 lymphocytes containing CD8+ cells into B6 female anti-HY TCR transgenic mice results at 6 wk in the disappearance of the majority of male Ag-reactive T cells (TghighCD8+) from the periphery. Here we investigate the process in more detail. B6 female anti-HY TCR transgenic mice were intravenously injected with viable lymphocytes from male B6 normal, CD4-/-, CD8-/- or CD8-/- carrying a CD8 transgene lacking its cytoplasmic tail ("CD8 tail-less") mice. The fate of TghighCD8+ cells was followed in vivo. There was always a large (at least twofold) expansion of these cells in the periphery 4 days after encountering male Ag. Their subsequent fate differed, however, depending on whether or not the injected male lymphocytes contained normal CD8-expressing cells. If the injected male lymphocytes contained normal CD8 cells, at 6 wk there was a large drop in the number of TghighCD8+ cells associated with a rise in the number of TghighCD8- cells. If the injected male lymphocytes lacked CD8 cells or expressed only tail-less CD8, TghighCD8+ cell numbers returned to normal by 6 wk, while TghighCD8- cell numbers remained unchanged. The TghighCD8- cells, although carrying high levels of the male specific TCR, did not react to male Ag. In addition, their ability to respond in anti-CD3-induced activation, which does not require CD8 as a co-receptor, was significantly impaired. Our data suggest that down-regulation of CD8 on the Ag-reactive T cells accounts, at least partially, for the disappearance of HY-reactive T cells from the periphery. Further, some step in the process requires signaling through the cytoplasmic domain of CD8 on the injected Ag-bearing cells.
Polymorphism at position nine of the MHC class I heavy chain affects the stability of association with beta 2-microglobulin and presentation of a viral peptide.
Authors: Ribaudo R K , Margulies D H
Published: December 2022

To identify residues that control the interactions between MHC-heavy chains and (beta 2m) sequence comparisons were made between murine class I MHC molecules with high (H-2Dd, H-2Kb) and low (H-2Ld, H-2Db) affinities for beta 2m. A single residue at position 9 was evaluated for its contribution to the stability of the complex. Mutagenesis of the glutamic acid at position 9 of H-2Ld to valine, as is found in H-2Dd and H-2Kb, resulted in both qualitative and quantitative effects on inter-chain interactions, intracellular transport, peptide binding, and peptide presentation. In in vitro translation and assembly studies, the E9V mutation resulted in a more stable association of beta 2m with the heavy chain after immunoprecipitation with the alpha 2 domain-specific Ab 30-5-7 in the presence of an H-2Ld-restricted peptide. E9V variant expressed in transfected L cells had similar surface expression compared with H-2Ld despite exhibiting a slower rate of maturation. However, cells expressing E9V were unable to present peptide Ag to a specific T cell hybridoma. H-2LdE9V in E-3 cells, which are defective in TAP-dependent peptide transport, was expressed at higher levels than H-2Ld and was stabilized more efficiently by the addition of exogenous human beta 2m. Thus, amino acid position 9 not only plays an important role in the interaction of the MHC-1 molecule with the beta 2m, it also qualitatively and quantitatively influences peptide binding and Ag presentation.
Lethal shock is inducible by lipopolysaccharide but not by superantigen in mice with retrovirus-induced immunodeficiency syndrome.
Authors: Aoki Y , Hiromatsu K , Arai T , Usami J , Makino M , Ishida H , Yoshikai Y
Published: December 2022

The retrovirus-induced murine AIDS (MAIDS) shares many features with human AIDS. Here, we examined the susceptibility of mice with MAIDS to staphylococcal enterotoxin-triggered shock. Following sensitization with D-galactosamine (D-Gal), mice with MAIDS were resistant to the otherwise lethal effect of superantigen staphylococcal enterotoxin A (SEA). Peak IL-2 levels in these mice after D-Gal/SEA challenge were 10-fold higher than those in uninfected controls, and concurrently, IL-10 levels rose markedly with reduction of circulating IL-1 and IFN-gamma. Treatment with neutralizing anti-IL-10 mAb before D-Gal/SEA challenge led to increased IFN-gamma levels in mice with MAIDS, and resulted in a dose-dependent mortality. In contrast, mice with MAIDS were more susceptible to the toxicity of bacterial endotoxin LPS than were uninfected controls. Administration of 100 micrograms LPS alone induced 50% lethality in mice infected with MAIDS virus 8 wk previously but not in uninfected controls. Administration of 10 micrograms LPS caused acute shock in D-Gal-sensitized mice with MAIDS. Peak TNF-alpha levels in these mice after LPS challenge were increased more than 10-fold, whereas IL-10 levels were one-third of those after SEA challenge. Moreover, serum IFN-gamma was undetectable in uninfected controls and rose to 1063 +/- 483 pg/ml in mice with MAIDS 4 h after LPS challenge. These results suggest that aberrant profiles of cytokine production are crucial in determining fatal outcome in these two types of septic shock in MAIDS.
Metabolic requirements for induction of contact hypersensitivity to immunotoxic polyaromatic hydrocarbons.
Authors: Anderson C , Hehr A , Robbins R , Hasan R , Athar M , Mukhtar H , Elmets C A
Published: December 2022

Experiments were performed to define the metabolic requirements for induction of contact hypersensitivity to polyaromatic hydrocarbons (PAHs), environmental xenobiotics that are both immunotoxic and carcinogenic. Evidence that conversion of the parent compound to a reactive metabolite was necessary for the development of contact hypersensitivity included the fact 1) that contact hypersensitivity to the polyaromatic hydrocarbon dimethylbenz(a)anthracene (DMBA) only occurred in strains of mice that could metabolize the compound, 2) that among the PAHs, only those that could induce aryl hydrocarbon hydroxylase, the rate-limiting enzyme in the PAH metabolic pathway, were immunogenic, and 3) that inhibitors of PAH metabolism reduced DMBA contact hypersensitivity. Cells from the XS52 Langerhans cell-like dendritic cell line were able to metabolize the PAH benzo(a)pyrene to its diol, quinone, and phenol metabolites. GM-CSF augmented benzo(a)pyrene metabolism in XS52 cells. Finally, in vivo depletion of CD8+, but not CD4+, T cell populations inhibited contact hypersensitivity to DMBA. The implications of these experiments are that at least for some contact allergens, the metabolic status of the host is a key determinant of individual susceptibility to the development of allergic contact dermatitis, and the metabolic pathway of an individual hapten may have ramifications for the T cell subpopulation-CD4 or CD8-that is activated.
Selective inhibition of E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 expression by inhibitors of I kappa B-alpha phosphorylation.
Authors: Chen C C , Rosenbloom C L , Anderson D C , Manning A M
Published: December 2022

The promoters of the E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) genes contain recognition sequences for the inducible nuclear transcription factor kappa B (NF-kappa B). We demonstrate that the appearance of NF-kappa B DNA-binding activity in the nucleus of TNF-alpha-stimulated HUVEC is associated with the rapid phosphorylation and subsequent degradation of I kappa B-alpha, the cytoplasmic inhibitor of NF-kappa B. Serine protease inhibitors prevented the TNF-alpha-induced accumulation of phosphorylated I kappa B-alpha, and prevented I kappa B-alpha degradation and the appearance of NF-kappa B DNA-binding activity. These inhibitors had no direct effect upon the ability of NF-kappa B to bind its cognate recognition sequences, nor upon the DNA-binding activities of other transcription factors. Inhibition of I kappa B-alpha proteolysis resulted in the inhibition of the cytokine- and LPS-induced transcriptional up-regulation and cell-surface expression of E-selectin, VCAM-1, and ICAM-1. In contrast, the TNF-alpha-induced expression of plasminogen activator inhibitor-1 and the constitutive expression of ICAM-2 were unaffected. These inhibitors also had no effect on cellular viability or rates of RNA or protein synthesis. Inhibitors of other proteases and various protein kinases had no effect on the cytokine-induced expression of these endothelial adhesion molecules. These findings indicate that it is possible, using a single pharmacologic agent, to selectively inhibit the expression of the E-selectin, VCAM-1, and ICAM-1 genes without affecting the constitutive or inducible expression of other genes. Pharmacologic inhibition of I kappa B-alpha proteolysis represents a novel approach to the development of anti-inflammatory therapeutics.
Contribution of IL-1, CD14, and CD13 in the increased IL-6 production induced by in vitro monocyte-synoviocyte interactions.
Authors: Chomarat P , Rissoan M C , Pin J J , Banchereau J , Miossec P
Published: December 2022

Rheumatoid synovitis is characterized by an infiltration of mononuclear cells and by the proliferation of synoviocytes. Monocytes and synoviocytes are major producers of cytokines, growth factors, and enzymes that contribute to the rheumatoid arthritis (RA) process. Since they are in close contact in vivo, we engaged in an in vitro study of the functional consequences of their interactions. Coculture of unstimulated elutriated normal blood monocytes over RA synoviocytes resulted in a synergistic increase of the production of IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), and IL-8, when compared with their respective production in culture alone. In contrast, cytokines such as IL-10, IL-1 beta, IL-1 alpha, and TNF-alpha could not be detected. The IL-6 production in coculture was further increased by the addition of IL-1 beta, GM-CSF, IFN-gamma, or TNF-alpha, but was inhibited by the addition of IL-10, IL-4, IL-13, or IL-1Ra, an effect reverted by the addition of IL-1 beta. Moreover, an inhibition was also observed with anti-CD14 mAb and newly raised mAbs directed against RA synoviocytes. Under reducing conditions, the mAb SY12 precipitated a 150-kDa surface membrane protein, identified as amino-peptidase N (CD13/AP-N). Collectively, these results indicate that 1) monocytes and synoviocytes interact with each other to produce proinflammatory cytokines, 2) pro- and antiinflammatory cytokines have opposite effects on IL-6 production, and 3) molecules such as IL-1, CD14, and CD13 are involved.
Anti-CD3:anti-IL-2 receptor-bispecific mAb-mediated immunomodulation. Low systemic toxicity, differential effect on lymphoid tissue, and inhibition of cell-mediated hypersensitivity.
Authors: MacLean J A , Su Z , Colvin R B , Wong J T
Published: December 2022

An anti-CD3:anti-CD25 (CD3,25) bispecific mAb was developed with the objective of combining the advantages of the parent anti-CD3 and anti-CD25 mAbs. The in vivo effects of the CD3,25 were examined in comparison to the parent Abs. The CD3,25 was well tolerated in vivo, in contrast to the parent anti-CD3 mAb, which induced systemic toxicity in recipient animals. Anti-CD3 mAb induced cell death, lymphoblast formation, and T cell activation in peripheral lymphoid organs; these were observed to a lesser extent in CD3,25-treated animals. In the thymus, anti-CD3 caused a progressive depletion of the CD4+ CD8+ "double positive" thymocytes, which was not seen in CD3,25-treated animals. This finding suggests that monovalent CD3 binding is insufficient to induce thymocyte apoptosis. Animals treated with a combination of anti-CD3 and anti-CD25 mAbs demonstrated changes in the lymphoid organs that were similar to anti-CD3-treated mice. This finding demonstrates that the effect of the CD3,25 is different than the sum of the parent Abs and suggests that the bispecific nature of the CD3,25 results in a reagent with unique immunomodulatory properties. The functional efficacy of the CD3,25 was assessed in a murine model of delayed-type hypersensitivity. The CD3,25 was as effective as the anti-CD3 mAb in inhibiting the delayed-type hypersensitivity reaction and was more effective than the parent anti-CD25 mAb. These data demonstrate that appropriately designed bispecific mAbs can be used as effective immunosuppressive agents with low systemic toxicity.
Peptide-selected T cell lines from myasthenia gravis patients and controls recognize epitopes that are not processed from whole acetylcholine receptor.
Authors: Matsuo H , Batocchi A P , Hawke S , Nicolle M , Jacobson L , Vincent A , Newsom-Davis J , Willcox N
Published: December 2022

To study pathogenic T helper cells in myasthenia gravis (MG) reacting against the acetylcholine receptor (AChR), we have previously selected five CD4+ T cell lines/clones from MG patients (or healthy controls) against full-length recombinant human AChR alpha subunit (alpha 1-437); these can all recognize AChR solubilized from human muscle. Recently, T cells selected with pooled AChR subunit synthetic peptides have shown greater heterogeneity than above. Hoping to validate that, we have characterized three MG and six control T cell lines selected with pooled peptides (averaging 33 residues long) covering the alpha subunit sequence; recurring responses to three particular peptides each showed preferred HLA class II restrictions--p75-115/DR4, p138-167/DR4, and p309-344/DR3 (or DR52a). However, none of three lines from MG patients recognized p138-167--even one from a previous responder to this epitope in full-length alpha 1-437; otherwise they resembled those from controls. Moreover, no peptide-selected line responded significantly to whole AChR, alpha 1-437, or even to shorter polypeptides sharing one terminus with the peptide, suggesting specificity for epitopes not naturally processed by APCs from blood. Of 20 sublines maintained with individual peptides, at least 10 responded to independently synthesized overlapping sequences, but four others depended on contaminants in the original peptides. A single line did recognize one longer polypeptide, but only after tryptic digestion; the processing of this cryptic epitope was evidently the limiting factor here rather than its concentration or the T cell sensitivity. Therefore, while synthetic peptides are essential for mapping epitopes, assessment of the pathogenic MG T cell repertoire requires full-length Ag processed naturally.
Activation of telomerase in human lymphocytes and hematopoietic progenitor cells.
Authors: Hiyama K , Hirai Y , Kyoizumi S , Akiyama M , Hiyama E , Piatyszek M A , Shay J W , Ishioka S , Yamakido M
Published: December 2022

This is the first report describing up-regulation of telomerase activity in human normal cells. Telomerase, a ribonucleoprotein enzyme, has been thought to be involved in maintaining telomere length stability in germline and most cancer cells, but not in normal cells. However, in the present study, we demonstrate that telomerase activity is detectable at low levels in normal human T and B cells, increases by in vitro mitogenic stimulation, increases in hematopoietic progenitor cells upon their proliferation and differentiation, and decreases with aging. Understanding the regulation of telomerase activity in normal cells may provide important insights not only into the mechanisms of normal cellular senescence but also into the mechanisms of telomerase activity deregulation as part of cancer development.
Opposing effects of TGF-beta 2 on the Th1 cell development of naive CD4+ T cells isolated from different mouse strains.
Authors: Hoehn P , Goedert S , Germann T , Koelsch S , Jin S , Palm N , Ruede E , Schmitt E
Published: December 2022

The development of naive dense CD4+ T cells from different mouse strains toward Th1 cells, as monitored by measuring secondary IFN-gamma production, was affected by TGF-beta 2 in a differential way. Th1 cell development of naive CD4+ T cells from strains C57Bl/6, BALB/c, and NMRI primed by immobilized anti-CD3 mAb was strongly inhibited in the presence of TGF-beta 2. Even when the Th1 cell-inducer IL-12 was added, the same effect of TGF-beta 2 was observed. In contrast, Th1 development was substantially promoted by TGF-beta 2 with T cells from C3H/He and CBA/J mice. Further analyses using CD4+ T cells from (C57Bl/6xCBA/J)F1 hybrids or DBA/1 mice showed that Th1 development was inhibited by TGF-beta 2 if the T cells were activated by anti-CD3 mAb, but it was enhanced upon costimulation with anti-CD28 mAb. Determination of primary IL-2 production revealed that T cells from (C57Bl/6xCBA/J)F1 and DBA/1 mice produced low amounts of IL-2 following stimulation by anti-CD3 mAb alone and comparatively high amounts after coactivation by anti-CD28 mAb. In the presence of TGF-beta 2, the production of IL-2 was completely suppressed if such T cells were activated solely by anti-CD3 mAb, but it was only partially inhibited after costimulation by anti-CD28 mAb. Furthermore, TGF-beta 2-promoted Th1 development of such T cells was strongly inhibited after neutralization of endogenously produced IL-2 and completely restored by the addition of human IL-2. Thus, our results indicate that the TGF-beta 2-mediated stimulation of Th1 cell development requires the presence of relatively high concentrations of IL-2. Therefore, the opposing effect of TGF-beta 2 on the Th1 cell development of naive CD4+ T cells from different mouse strains appears to be the result of the variable potency of the respective CD4+ T cells to produce IL-2 in the presence of TGF-beta 2.
T cell-dependent secretion of IL-1 beta by a dendritic cell line (XS52) derived from murine epidermis.
Authors: Kitajima T , Ariizumi K , Mohamadazadeh M , Edelbaum D , Bergstresser P R , Takashima A
Published: December 2022

IL-1 beta has been reported to play an essential role in the induction of T cell-mediated immune responses in skin, and Langerhans cells are considered to be the primary source of IL-1 beta in epidermis. We have established recently a long-term dendritic cell line (XS52) from mouse epidermis. This line resembles resident epidermal Langerhans cells in many respects, including the potent capacity to present a protein Ag (KLH) to a CD4+ Th1 clone (HDK-1) and the expression of IL-1 beta mRNA. We sought to determine whether XS52 cells secrete IL-1 beta upon Ag-dependent interaction with T cells and, if so, to elucidate the mechanism. Despite constitutive expression of mRNA for both IL-1 beta and the IL-1 beta-converting enzyme (ICE), XS52 cells secreted no detectable IL-1 beta spontaneously. When they were cultured with HDK-1 T cells and KLH, relatively large amounts of IL-1 beta (17.5-kDa form) were detected in the culture supernatant. IL-1 beta was secreted by LPS-stimulated XS52 cells, but not by LPS- or Con A-stimulated HDK-1 cells, suggesting that IL-1 beta is secreted primarily by XS52 cells in the coculture system. Incubation with HDK-1 cells alone or with KLH alone caused no IL-1 beta secretion, indicating the requirement for both T cells and Ag. IL-1 beta secretion was associated with a striking up-regulation of IL-1 beta mRNA and a modest up-regulation of ICE mRNA and enzymatic activity. IL-1 beta secretion was blocked by Ac-YVAD-CHO (a peptide inhibitor of ICE), CTLA4-Ig fusion protein, or anti-Ia mAb. IL-1 beta secretion was triggered in a T cell-independent manner by either CTLA4-Ig or anti-Ia mAb in immobilized forms. Thus, the XS52 dendritic cell line secretes, by an ICE-dependent mechanism, biologically relevant amounts of IL-1 beta upon Ag-dependent interaction with T cells, with both Ia molecules and B7-related molecules playing essential roles.
Intermediate steps in thymic positive selection. Generation of CD4-8+ T cells in culture from CD4+8+, CD4int8+, and CD4+8int thymocytes with up-regulated levels of TCR-CD3.
Authors: Kydd R , Lundberg K , Vremec D , Harris A W , Shortman K
Published: December 2022

Minor thymus subpopulations representing possible intermediates in thymic positive selection were isolated by cell sorting from bcl-2 transgenic mice, and cultured 1 to 4 days in simple medium to assess their ability to spontaneously develop the surface phenotype of mature T cells. Recovery of cells was in the 60 to 80% range, and no cell proliferation occurred. Only cells originally expressing high, near mature T cell levels of CD3 developed further in culture by down-regulation of CD4 or CD8. The main mature cell product was CD4-8+, regardless of whether the starting phenotype of the CD3high intermediates was CD4+8+, CD4int8+, or CD4+8int; only an intermediate subpopulation expressing the highest levels of CD4 (CD4high8int) produced a dominance of CD4+8- mature progeny. Partial down-regulation of CD8 was therefore not a good indicator of CD4+ T lineage commitment. These and previous results indicate that maturation to the CD8+ T lineage involves a rapid up-regulation of the TCR-CD3 complex, but a relatively slow down-regulation of CD4; it may also involve a partial, transient reduction in surface CD8. In contrast, maturation to the CD4+ T lineage involves a relatively rapid down-regulation of CD8, with maintenance of high levels of CD4. There appears to be a marked asymmetry in the developmental steps leading from CD4+8+ thymocytes to the CD8+ or to the CD4+ T cell lineage.
Binding of FimD on Bordetella pertussis to very late antigen-5 on monocytes activates complement receptor type 3 via protein tyrosine kinases.
Authors: Hazenbos W L , van den Berg B M , Geuijen C W , Mooi F R , van Furth R
Published: December 2022

Nonopsonized Bordetella pertussis bind to human monocytes by means of the virulence factors filamentous hemagglutinin (FHA), pertactin, and the minor fimbrial subunit FimD. Receptors on monocytes that mediate binding of B. pertussis to these cells include complement receptor type 3 (CR3), which binds to FHA of B. pertussis, and very late antigen-5 (VLA-5), which binds to an, as yet, unknown ligand on these bacteria. In the present study, the possibility that FimD acts as a ligand for VLA-5 was investigated. Soluble fibronectin, which is the natural ligand for VLA-5, or mAbs against VLA-5 inhibited binding to monocytes of B. pertussis strains that express FimD but not of mutant strains that lack FimD. Beads that were coated with the fusion protein maltose-binding protein-FimD bound to adherent monocytes, and this binding was inhibited by soluble fibronectin or mAb against the alpha- or beta-chain of VLA-5, while soluble collagen or mAb against VLA-4, VLA-6, CR3, or HLA class II had no effect. Down-modulation of VLA-5 on the apical surface of monocytes by plating the cells onto surfaces precoated with anti-VLA-5 mAb also inhibited binding of beads coated with maltose-binding protein-FimD to monocytes, while precoating of the surfaces with mAb against VLA-6 or CR3 had no effect. These results indicate that VLA-5 on monocytes serves as a receptor for FimD on B. pertussis. Binding of C3bi-coated erythrocytes to monocytes, which is a measure of the binding activity of CR3, was enhanced when monocytes were adhered onto plates precoated with purified fimbriae of B. pertussis, while precoating with fimbriae lacking FimD had no effect. Precoating of the plates with FimD-containing fimbriae also enhanced binding of B. pertussis, which express FHA, but not of strains that lack FHA, to monocytes. The enhanced binding of C3bi-coated erythrocytes and B. pertussis to monocytes could be markedly inhibited by tyrphostin-47, a protein tyrosine kinase inhibitor. These results demonstrate that interaction of FimD of B. pertussis with VLA-5 on monocytes activates CR3, which requires protein tyrosine kinases and results in enhanced binding of B. pertussis to the latter receptor via FHA.
A nonlethal rat parvovirus infection suppresses rat T lymphocyte effector functions.
Authors: McKisic M D , Paturzo F X , Gaertner D J , Jacoby R O , Smith A L
Published: December 2022

Inoculation of the UMass strain of rat virus (RV-UMass) into adult immunocompetent rats results in a prolonged subclinical infection that is resolved in 4 to 8 wk. Co-labeling studies, using in situ hybridization (ISH) and immunohistochemistry (IHC), confirmed that RV-UMass was lymphocytotropic and capable of infecting CD4+ and CD8+ T cells as well as B cells. ISH studies also revealed that virus replication was restricted in unstimulated cells but was productive in concanavalin A-stimulated lymphocytes. A corollary of productive infection of lymphocytes was the suppression of lymphocyte functions. Although RV-UMass did not appear to induce phenotypic changes during the course of infection, cells from infected rats had diminished proliferation and cytolytic responses. Both peripheral and mesenteric lymph node cells exhibited only partial recovery of their proliferative and cytolytic capacities one month after infection. Furthermore, RV-UMass-infected tissue culture maintained alloreactive CD4+ T cells in vitro, and a nonlethal infection of this T cell line inhibited Ag- and IL-2-induced proliferation. Because parvoviruses are widespread among laboratory rodents, these findings emphasize the importance of identifying and excluding parvovirus infection in rodents and in cultures of rat T lymphocytes.
T lymphocytes from volunteers immunized with irradiated Plasmodium falciparum sporozoites recognize liver and blood stage malaria antigens.
Authors: Krzych U , Lyon J A , Jareed T , Schneider I , Hollingdale M R , Gordon D M , Ballou W R
Published: December 2022

The model of protective immunity induced by immunization with irradiated plasmodia sporozoites (SPZ) has become the prototype for a promising vaccine strategy based on Ab and CTL responses directed against pre-erythrocytic stage Ags, in particular the circumsporozoite protein (CSP) and sporozoite surface protein 2 (SSP2). However, results from recently conducted vaccine studies suggest that T cell responses directed against additional specificities might also be required for protection. We have tested this hypothesis by examining human T lymphocytes from irradiated Plasmodium falciparum SPZ-immune volunteers for proliferative reactivities to parasitized red blood cells (pRBC) and recombinant proteins and synthetic peptides representing certain liver and blood stage Ags. In this work, we report that although SPZ-induced protective immunity is stage-specific, SPZ-immune lymphocytes recognized determinants associated with erythrocytic and liver stage parasites. Thus, protective immunity induced by irradiated SPZ may depend upon responses against pre-erythrocytic Ags in addition to CSP and SSP2.