bluuebunny/crossref_metadata_2025 huggingface dataset:
Autocrine/paracrine involvement of platelet-activating factor and transforming growth factor-beta in the induction of phosphatidylserine recognition by murine macrophages.
Authors: Rose D M , Fadok V A , Riches D W , Clay K L , Henson P M
Published: December 2022

The specific recognition of phosphatidylserine (PS) by macrophages is believed to be one means by which effete and apoptotic cells expressing PS on their outer membrane leaflet are targeted for phagocytosis. The aim of this study was to better understand the autocrine/paracrine factors involved in beta-glucan induction of PS recognition by macrophages. We provide evidence that both platelet-activating factor (PAF) and TGF-beta are involved in beta-glucan induction of PS recognition. This is based on the observations that the PAF receptor antagonist WEB 2086 and Ab against TGF-beta each could partially inhibit beta-glucan-induced PS recognition when used alone and could completely inhibit induction when used in combination. PAF, like TGF-beta, was found to prime macrophages for PS recognition, which could then be triggered by costimulation with a nonspecific phagocytic stimulus, latex particles. We also provide evidence that the priming by PAF and that by TGF-beta can occur independently of each other. This is based on the observations that 1) PAF priming was not blocked by anti-TGF-beta Ab, nor was TGF-beta priming prevented by WEB 2086; and 2) PAF did not increase the steady state level of TGF-beta mRNA, and TGF-beta did not induce PAF synthesis in these cells.
Augmentation or inhibition of IFN-gamma-induced MHC class II expression by lipopolysaccharides. The roles of TNF-alpha and nitric oxide, and the importance of the sequence of signaling.
Authors: Sicher S C , Chung G W , Vazquez M A , Lu C Y
Published: December 2022

MHC class II expression on macrophages is one determinant of Ag presentation and the vigor of CD4+ T cell immunity. We show that LPS may either inhibit or augment IFN-gamma-induced MHC class II on macrophages depending on the sequence of the IFN-gamma and LPS signals. LPS inhibited MHC class II when added simultaneously with IFN-gamma, but augmented class II expression when added after IFN-gamma. Inhibition was due to nitric oxide (NO), which was only produced if LPS was given simultaneously with IFN-gamma. However, even when NO production was inhibited, LPS given simultaneously with IFN-gamma did not augment MHC class II expression. This suggests that LPS delivers different signals when given simultaneously vs after IFN-gamma. LPS augmentation of class II expression was functionally important because it correlated with increased Ag presentation. Augmentation by LPS of IFN-gamma-induced class II expression by macrophages has not been previously reported. We found that TNF-alpha, like LPS, inhibited IFN-gamma-induced class II expression if NO was produced, but augmented it in the absence of NO formation. Studies with a neutralizing anti-TNF-alpha Ab, however, indicate that LPS augmentation of MHC class II did not require TNF-alpha. LPS augmentation involved a different mechanism than IFN-gamma induction of MHC class II. LPS augmentation occurred at a post-transcriptional level, whereas IFN-gamma-induction occurred at the level of gene transcription. LPS augmentation was apparent after 2 h of stimulation by LPS, while IFN-gamma induction of class II expression required more than 8 h.
Matrix metalloproteinases in immunity.
Authors: Goetzl E J , Banda M J , Leppert D
Published: December 2022

Matrix metalloproteinases (MMPs) are a family of zinc-containing endo-proteinases that share structural domains but differ in substrate specificity, cellular sources, and inducibility. Macrophage production and secretion of large quantities of many MMPs, after contact with matrix proteins, is enhanced by surface determinants on activated T cells and suppressed by cytokines from Th1 and Th2 cells. T cells secrete predominantly the gelatinases MMP-2 and -9, after beta 1, integrin- or vascular cell adhesion molecule (VCAM)-1-dependent stimulation by cytokines and inflammatory mediators. MMPs of both T cells and macrophages facilitate secretion of TNF-alpha, by cleavage of the membrane-bound form. T cell MMPs prepare connective tissue matrices for T cell chemotaxis across basement membranes and through tissues. The greater amounts of diverse MMPs from macrophages are capable of degrading connective tissues, which may release stored growth factors. In limited studies of animal models of autoimmunity, specific MMP inhibitors have significantly decreased edema and inflammatory tissue damage, suggesting possible therapeutic benefits.
CD40 ligand is expressed and functional on activated neonatal T cells.
Authors: Splawski J B , Nishioka J , Nishioka Y , Lipsky P E
Published: December 2022

Deficient CD40 ligand (CD40L) expression by neonatal T cells has been proposed to explain the diminished Ig production by newborns. Therefore, we examined the expression and function of CD40L on activated neonatal CD4+ T cells isolated from cord blood. Anti-CD3 activation of neonatal T cells resulted in the expression of CD40L demonstrated with specific mAb or a soluble CD40.G1 construct. The level of expression was comparable to that of adult memory CD4+ T cells. In contrast, PMA and ionomycin induced only minimal CD40L expression by neonatal T cells, whereas adult memory T cells expressed CD40L comparably after stimulation with PMA and ionomycin or anti-CD3. The expression of CD40L was blocked by cyclosporine and prostaglandin E2 (PGE2). IL-2 IL-4 partially reversed the inhibition of CD40L expression by cyclosporine, and IL-2 reversed the inhibition by PGE2. CD40L expressed by neonatal T cells was functionally active, since neonatal T cell-dependent production of IgG4 and IgE was inhibited by a soluble CD40.G1 construct. Moreover, neonatal T cells supported the secretion of significantly more IgG, IgA, and IgE than CD40L-deficient hyper-IgM T cell clones. In addition, neonatal CD4+ T cells induced CD86 B7-2) expression by neonatal B cells that was blocked by anti-CD40L but not by control mAb. The results indicate that neonatal T cells activated through the TCR/CD3 complex express CD40L and use it to promote CD86 expression, Ig secretion and heavy chain isotype switching by neonatal B cells.
Molecular cloning and expression of cDNA encoding human macrophage C-type lectin. Its unique carbohydrate binding specificity for Tn antigen.
Authors: Suzuki N , Yamamoto K , Toyoshima S , Osawa T , Irimura T
Published: December 2022

A human macrophage calcium-dependent (C-type) lectin cDNA clone was obtained from a library derived from IL-2-treated peripheral blood monocytes. The cDNA cloning was based on the structural homology to hepatic asialoglycoprotein receptors. The nucleotide sequence of this cDNA clone was homologous to those of the galactose- and N-acetylgalactosamine-specific C-type macrophage lectins of rodents. In the putative carbohydrate recognition domain, deduced amino acid sequence revealed 60 and 63% homology to galactose- and N-acetylgalactosamine-specific C-type macrophage lectins of mice and rats, respectively. The cDNA clone was ligated into a mammalian expression vector and transfected into COS-1 cells. In the lysates of these cells, an MR 38,000 component, which bound to galactose-Sepharose, was identified after electrophoretic separation by its interaction with polyclonal antisera against synthetic polypeptides representing a portion of the carbohydrate recognition domain. The carbohydrate-binding specificity of the recombinant macrophage lectin was investigated by comparing elution profiles of various glycopeptides having defined carbohydrate structures from immobilized macrophage lectins. When N-terminal octapeptides from human glycophorin A that bore NeuAc alpha 2-3 Gal beta 1-3 (NeuAc alpha 2-6) GalNAc and its sequentially deglycosylated derivatives were compared, glycopeptides carrying three constitutive GalNAc-Ser/Thr (Tn Ag) strongly bound to the recombinant human macrophage lectin. This is the first study to demonstrate that human macrophage cell surface lectin recognizes Tn Ag, a well-known human carcinoma-associated epitope.
The role of NK cells during in vivo antigen-specific antibody responses.
Authors: Wilder J A , Koh C Y , Yuan D
Published: December 2022

Investigations into the role of NK cells in regulating Ab responses have yielded variable results, some suggesting that NK cells can down-regulate Ag-specific Ig production and others proposing an enhancing effect. These apparently inconsistent findings may stem partially from the specificity of reagents used in purifying cell populations and/or the nature of the in vitro systems used to study these events. We chose to investigate the ability of either resting or poly(I:C)-activated NK cells to alter an in vivo Ab response in mice given a T-independent (TNP-LPS) or T-dependent (TNP-keyhole limpet hemocyanin (KLH)) Ag. By using a more specific Ab, anti-NK-1.1, to deplete NK cells, we were able to clearly show that resting, endogenous NK cells do not affect either type of response, as measured by serum Ag-specific Ig levels quantitated by isotype-specific ELISA. In contrast, activation of NK cells by poly(I:C) increased Ag-specific IgC2a as well as IgG1 levels. Interestingly, only the effect on IgG2a production is reversible by depletion of NK cells.
Induction of microvariant-specific CTL lines reactive to a single amino acid mismatch in bulk cultures using a transfectant expressing a single HLA class I molecule.
Authors: Cereb N , Yang S Y
Published: December 2022

Alloreactivity against micromismatches in MHC class I molecules is difficult to measure. Here, we describe an in vitro model with which it is possible to examine alloreactivity against a single HLA class I allotype. The HLA class I- and class II-negative myelocytic leukemia cell line K562 was transfected with a genomic DNA clone carrying B*4403 to express a single allotype. CTL lines were generated from normal individuals carrying B*4402, B*4403, or unrelated HLA-B alleles by stimulation with B*4403- transfected K562. The bulk CTL lines generated from B*4402+ T cells against B*4403 that carry a single amino acid disparity at position 156 were specific for B*4403+ targets and did not react with targets carrying any other HLA allotype. However, the CTL lines generated from B44-negative individuals exhibited killing of the targets bearing not only B44, but also B44 CREG and a few other B alleles. Reverse transcriptase-PCR analysis of TCRs, expressed in the CTL clones uniquely specific for B*4403, showed that TCR V beta usage of alloreactive T cells directed against B*4403 was diverse but nonrandom and was affected by the HLA background of the responder. Thus, the K562-HLA transfectant system provides a useful in vitro tool to analyze alloreactivity against a single class I allele and to aid in the prediction of alloreactivity in unrelated marrow transplantation.
B cells from p50/NF-kappa B knockout mice have selective defects in proliferation, differentiation, germ-line CH transcription, and Ig class switching.
Authors: Snapper C M , Zelazowski P , Rosas F R , Kehry M R , Tian M , Baltimore D , Sha W C
Published: December 2022

To better understand the role of NF-kappaB in normal B cell physiology, we used a purified population of resting B cells from p50/NF-kappa B knockout (p50-/-) mice to determine their ability to proliferate, secrete Ig, express germ-line CHRNA, and undergo Ig isotype switching in vitro in response to a number of distinct stimuli. p50-/- B cells proliferated normally in response to dextran-anti-IgD Abs (alpha delta-dex) and membrane-bound, but not soluble, CD40 ligand (CD40), and they were virtually unresponsive to LPS when compared with control B cells. p50-/- B cells secreted markedly reduced Ig in response to alpha delta-dex or mCD40L in the presence of IL-4 + IL-5, despite their relatively normal proliferative rates, whereas normal Ig secretion was restored by the combination of alpha delta-dex and CD40L. p50-/- B cells expressed normal steady-state levels of germ-line CH gamma 1 and CH alpha RNA but markedly reduced germ-line CH gamma 3 and CH epsilon RNA upon appropriate stimulation. Although p50-/- B cells underwent substantial switching to IgG1, a marked reduction in the switch to IgG3 and IgE, as IgA, was observed. These data are the first to demonstrate key, independent roles for p50/NF-kappaB in normal B cell maturation to Ig secretion, germ-line CH gene activation, and Ig class switching, as well as mitogenesis, and provide a powerful and well-defined in vitro model system for studying the role of p50/NF-kappaB in a wide range of normal cellular functions.
Susceptibility to TNF in the presence of inhibitors of transcription or translation is dependent on the activity of cytosolic phospholipase A2 in human melanoma tumor cells.
Authors: Voelkel-Johnson C , Thorne T E , Laster S M
Published: December 2022

In this study, we have examined the relationship between the expression of the high molecular weight, cytosolic form of PLA2 (cPLA2) and ability of inhibitors of transcription or translation (ITT) to induce susceptibility to TNF. S Susceptibility to lysis was assayed by 51CR release, and the expression of cPLA2 was assayed by activity assay and by Western blot. The panel of cells that we examined included two murine cell lines, six human melanoma-derived cell lines, two samples of freshly explanted melanoma tumor tissue, and a culture of normal epidermal melanocytes. Our experiments revealed a near perfect correlation between the activity of cPLA2 per cell and susceptibility to TNF in the presence of either cycloheximide (CHI) or actinomycin D (r = 0.97). These results suggest that the activity of cPLA2 is both necessary and rate-limiting in this form of programmed cell death, conclusions that were confirmed in transfection experiments and in experiments with antisense oligonucleotides. Over-expression of cPLA2 in two melanoma-derived cell lines, WM793 and SK-MEL-131, led to enhanced susceptibility to TNF and CHI. Conversely, suppression of cPLA2 with antisense oligonucleotides dramatically decreased susceptibility to TNF and CHI in C3HA fibroblasts. These experiments also revealed a coupled, transformation-released change in the expression of cPLA2 and susceptibility to lysis. Normal melanocytes contained the lowest levels of cPLA2 and were completely resistant to sensitization with ITT. In contrast, all of the melanoma-derived cell lines and samples of melanoma tumor tissue we examined has higher levels of cPLA2 and could be killed, to some extent, by treatment with TNF and ITT.
Neuroinvasive properties of herpes simplex virus type 1 glycoprotein variants are controlled by the immune response.
Authors: Mitchell B M , Stevens J G
Published: December 2022

Neuroinvasiveness is a property central to the pathogenesis of herpes simplex virus (HSV) and most isolates demonstrate this property. Exceptions are HSV strains KOS and ANG, for which we have previously shown that the non-neuroinvasive phenotype is referable to single amino acid changes in glycoprotein D for ANG or glycoprotein B for KOS. Because glycoproteins B and D are immunologically significant, the possibility that the phenotype has an immunologic basis was examined. Nonimmunosuppressed mice could not be killed with any dose of these non-neuroinvasive viruses after footpad inoculation, but in cyclophosphamide-suppressed animals, the ratios of plaque-forming units to LD50 decreased by at least four orders of magnitude to levels comparable with that of ANG-path, a neuroinvasive derivative of ANG. KOS and ANG induced a more rapid circulating neutralizing Ab response than did ANG-path, and mice were protected when these agents were co-infected with the neuroinvasive strain. The noninvasive viruses engendered an enhanced mononuclear cell infiltrate in infected spinal ganglia which consisted of increased numbers of CD4+ and CD8+ T cells and an increased production and secretion of IgG. HSV-specific Ab-secreting cells were also observed. In addition, passive transfer of anti-HSV mouse serum protected immunosuppressed mice from lethal HSV challenge. Selective in vivo depletion of T lymphocytes increased the detectable levels of both KOS and ANG viruses in the spinal ganglia at 6 days postinfection, but it did not alter the ratios of plaque-forming units to LD50 or affect the HSV-induced increase in ganglionic IgG. Taken together, these data indicate that in these systems there is an immunologic basis for the control of HSV-1 neuroinvasiveness and that humoral, rather than cell-mediated immunity, is playing the major role.
IL-12 protects immunocompetent and immunodeficient neonatal mice against infection with Cryptosporidium parvum.
Authors: Urban J F , Fayer R , Chen S J , Gause W C , Gately M K , Finkelman F D
Published: December 2022

The protozoan parasite Cryptosporidium parvum (Cp) causes diarrhea that can be acutely severe in immunocompetent persons and can become chronic and life-threatening in immunocompromised individuals. Because studies in mice have implicated IFN-gamma in protection against this parasite, and IL-12 can induce IFN-gamma production, we examined the ability of IL-12 to prevent or cure Cp infection in neonatal BALB/c and SCID mice. Treatment of both immunocompetent and immunodeficient mice with IL-12 before experimental inoculation with Cp oocysts prevented or greatly reduced the severity of infection. Intestinal epithelial cell invasion and/or early intracellular development of Cp was inhibited by exogenous IL-12. The protective effect of IL-12 was completely blocked by anti-IFN-gamma mAb. Established infections were associated with elevated IFN-gamma gene expression and were not ameliorated by IL-12 treatment, even though such treatment further enhanced IFN-gamma gene expression. The severity of Cp infection was, however, exacerbated by treatment with anti-IL-12 Ab. These observations provide the first evidence that treatment with exogenous IL-12 can prevent Cp infection through an IFN-gamma-dependent, specific immune system-independent mechanism, and that endogenous IL-12 production has a role in limiting Cp infection.
IL-3 augments TCR-mediated responses of type 2 CD4 T cells.
Authors: Dan Y , Katakura Y , Ametani A , Kaminogawa S , Asano Y
Published: December 2022

A subset of type 2, but not type 1, CD4 T cell clones expresses IL-3R and can be stimulated by IL-3. Expression of IL-3R on these type 2 T cell clones is induced by TCR stimulation, and subsequent stimulation by IL-3 augmented the proliferation of and IL-4 production by these cells. This augmented response is inhibited by anti-IL-4 mAb, suggesting the involvement of IL-4 in this response. In place of TCR stimulation, treatment of these type 2 CD4 T cell clones with PMA rendered them responsive to further stimulation of proliferation by IL-3, indicating the cooperation between the IL-3R-elicited signals and PKC-mediated signals in stimulating proliferation. Although the augmentation of the TCR-mediated proliferative response by IL-3 was mainly due to the increased production of IL-4, we also demonstrated the presence of IL-4-independent mechanism mediating the response to IL-3. In situ, we found that splenic T cells could be induced to respond to Il-3 by TCR stimulation. Thus, IL-3 can stimulate a specific population of T cells and influence the immune response.
Mast cell tryptase is a mitogen for epithelial cells. Stimulation of IL-8 production and intercellular adhesion molecule-1 expression.
Authors: Cairns J A , Walls A F
Published: December 2022

Tryptase, a protease unique to the mast cell secretory granule, is released in substantial quantities into the respiratory tract of patients with inflammatory disease of the airways. We have investigated the potential of tryptase to act as a mitogen for bronchial epithelial cells and to stimulate release of IL-8 and expression of ICAM-1. Tryptase was isolated from extracts of human lung tissue using ammonium sulphate precipitation, octyl agarose, and heparin agarose chromatography. Purified tryptase stimulated DNA synthesis in the human epithelial cell line H292, as measured by [3H] thymidine incorporation. Maximal growth was observed after 24 h using 25 mU/ml of tryptase (where 1 micron is defined as that which can hydrolyze 1 mumol of the peptide substrate N-alpha-benzoyl-DL-arginine p-nitroanilide hydrochloride per minute at 25 degrees C), a concentration that is likely to be achieved in vivo. Inhibitors of tryptase activity, including leupeptin and benzamidine hydrochloride, significantly decreased tryptase-induced stimulation of DNA synthesis, indicating the requirement for an active catalytic site. Tryptase stimulated a catalytic site-dependent release of IL-8 from epithelial cells after 24 h, and this was associated with up-regulation of ICAM-1 expression, as revealed by FACS analysis. Tryptase may play a critical role in epithelial repair and in the recruitment of granulocytes following mast cell activation.
Urokinase-type plasminogen activator receptor reversibly dissociates from complement receptor type 3 (alpha M beta 2' CD11b/CD18) during neutrophil polarization.
Authors: Kindzelskii A L , Laska Z O , Todd R F , Petty H R
Published: December 2022

Previous studies have shown that the leukocyte integrin CR3 (CD11b/CD18) is physically associated with the urokinase-type plasminogen activator receptor (uPAR;CD87), a glycosyl-phosphatidylinositol (GPI)-linked protein, in resting neutrophil membranes. We now show that uPAR-to-CR3 interactions are reversible, correlating with cell shape. Neutrophils were first labeled with fluorescein conjugates of anti-CR3 F(ab')2 fragments followed by capping using a second-step F(ab')2 directed against murine F(ab')2s. Cells were then probed using rhodamine-conjugated anti-uPAR F(ab')2s. Although uPAR co-caps with CR3 on resting cells, uPAR was found to dissociate or "uncap" coincident with spontaneous cell polarization for migration. CR3 caps transformed into uropods while uPAR accumulated at lamellipodia of polarized cells. Capping was unnecessary for the observed distribution of CR3 and uPAR since the anti-CR3 and anti-uPAR F(ab')2s traffic to the uropod and lamellipodium, respectively, during polarization of uncapped cells. These receptors reassociate when cells return to a spherical morphology. In contrast to uPAR, Fc gamma RIIIB did not dissociate from CR3 caps during cell polarization. Resonance energy transfer (RET) microscopy was used to image the spatial distribution of RET and to follow the kinetics of association and dissociation. Initial levels of RET dramatically fell during cell polarization, but did not change on cells fixed with paraformaldehyde. Receptor reassociation was a biphasic process with initial reassociation about the perimeter of a cap, followed by a plateau and a slower rise in RET within a cap. We suggest that cells regulate receptor-receptor associations depending upon their physiologic activities.
Cholera toxin increases IL-6 synthesis and decreases TNF-alpha production by rat peritoneal mast cells.
Authors: Leal-Berumen I , Snider D P , Barajas-Lopez C , Marshall J S
Published: December 2022

Mast cells have been traditionally associated with an acute allergic response. However, their role in regulating chronic inflammatory processes must also be considered in view of evidence that mast cells synthesize and release a number of cytokines. In this study, we have examined the effect of cholera toxin (CT) on peritoneal mast cell IL-6 and TNF-alpha production. Highly purified, freshly isolated, rat peritoneal mast cells from Brown Norway rats were cultured in the presence of CT or its B subunit (CTB) alone or in combination with anti-IgE or bacterial LPS. Histamine release was measured after 10 min; IL-16 and TNF-alpha production was assessed in supernatants after 18 h. We found that CT or CTB alone did not affect histamine release; however, mast cell IL-6 production was significantly enhanced by CT but not by CTB. In contrast, constitutive production of TNF-alpha was inhibited by CT. The effects of CT were similar to our previous observations of the actions of prostaglandin E2 on mast cells. We also examined the effects of CT in combination with other mast cell activating agents. CT had no significant effect on anti-IgE-induced histamine release. An additive effect on IL-6 production was observed in the context of LPS. Forskolin, an agent known to increase intracellular cAMP levels, also induced a significant increase in IL-6 production, whereas TNF-alpha production was decreased. These data have important implications for our understanding of the regulation of mast cell cytokine production and the effects of CT on local cytokine production.
Dehydroepiandrosterone modulation of lipopolysaccharide-stimulated monocyte cytotoxicity.
Authors: McLachlan J A , Serkin C D , Bakouche O
Published: December 2022

Dehydroepiandrosterone (DHEA), the predominant androgen secreted by the adrenal cortex, can be converted to both potent androgens and estrogens. In addition to its role as a precursor for other steroid hormones, DHEA has been proposed to play an important role in immunity. This study has investigated DHEA modulation of LPS-induced monocyte cytotoxicity. Cytotoxicity markers assessed include tumor cell killing, IL-1 secretion, reactive oxygen intermediate release, nitric oxide synthetase activity as measured by the release of reactive nitrogen intermediates, complement receptor-1 cell surface protein, and TNF-alpha protein presence. Monocytes stimulated with LPS concentrations of 1.0 micrograms/ml displayed the above cytotoxic markers, whereas monocytes stimulated with DHEA alone or with LPS at a lower concentration of 0.2 ng/ml did not. However, when used simultaneously, DHEA and LPS 0.2 ng/ml displayed a synergistic effect on monocyte cytotoxicity against cancerous cell lines, IL-1 secretion, reactive nitrogen intermediate release, complement receptor-1 cell-surface protein, and TNF-alpha protein to levels comparable with levels obtained using LPS 1.0 microgram/ml. Finally, Scatchard plot analysis demonstrated the presence of a DHEA receptor in monocytes, suggesting that DHEA effects on LPS-stimulated monocytes are mediated through a receptor-dependent process.
In vivo response of mature T cells to Mlsa antigens. Long-term progeny of dividing cells include cells with a naive phenotype.
Authors: Hayden K A , Tough D F , Webb S R
Published: December 2022

To characterize T cells surviving elimination following strong in vivo responses, bromodeoxyuridine (BrdU) was used to mark the V beta 6+ CD4+ T cells proliferating after exposure to MLsa superantigens. Though a portion of the surviving V beta 6+ cells failed to label with BrdU, many were BrdU+, indicating that some proliferating cells escape elimination. Both the portion of V beta 6+ cells responding to MLs Ag in vivo and the number of responding cells that escaped elimination were influenced by the MHC haplotype; weaker responses correlated with enhanced survival of BrdU+ V beta 6+ cells. As expected, some of these cells expressed cell surface markers generally associated with an activated/memory phenotype. Significantly, however, a large proportion of BrdU+ V beta 6+ cells were phenotypically indistinguishable from naive cells.
Macrophages synthesize and secrete a 25-kilodalton protein that binds insulin-like growth factor-I.
Authors: Li Y M , Arkins S , McCusker R H , Donovan S M , Liu Q , Jayaraman S , Dantzer R , Kelley K W
Published: December 2022

Primary (thymus) and secondary (spleen) murine lymphoid tissues express a 25-kDa protein that binds IGF-I. To determine the cellular source of this insulin-like growth factor binding protein (IGFBP), 11 murine or human cell lines representing T, B, and myeloid cells at various stages of differentiation were characterized by IGF-I affinity cross-linkage and Western ligand blotting. Mature myeloid cells, but not T or B cells, secrete a 25-kDa protein that is capable of binding IGF-I. CSF-1-derived bone marrow macrophages also synthesize this 25-kDa IGFBP. Thymic macrophages, which were estimated to secrete 2 ng of binding protein/10(6) cells-h, were used in conjunction with [125I] IGF-I affinity cross-linking to develop a protein binding immunomobility-shift assay to identify which IGFBP is produced by these cells. An anti-IGFBP-4Ab, but not an anti-IGFBP-2 Ab or normal rabbit serum, shifted the [125I] IGF-IGFBP complex to a higher m.w. position, indicating that the single 25-kDa IGFBP is IGFBP-4. Northern blotting confirmed that transcripts for IGFBP-4 as well as IGF-I are expressed in thymic macrophages. A putative 278-bp IGFBP-4 cDNA fragment (residues 341-618) of rat) that contains two unique cysteine residues found only in IGFBP-4 was cloned and sequenced from thymic macrophages. These clones differed from the rat sequence at only six residues (97% homology), and the deduced amino acid sequence from the murine cDNA was identical with that of the rat sequence. Subsequent studies revealed that IGF-I stimulates DNA synthesis in thymic macrophages. However, two different IGF-I analogues differing in the amino-terminus that bind equally well to the IGF-I receptor but poorly to IGFBPs are as effective as IGF-I at 100-fold lower concentrations. These data demonstrate that murine macrophages are a source of a single 25-kDa secreted protein that binds IGF-, that the molecular identity of this protein is IGFBP-4, and that this binding protein may antagonize the extracellular effects of IGF-I.
Analysis of CD28 cytoplasmic tail tyrosine residues as regulators and substrates for the protein tyrosine kinases, EMT and LCK.
Authors: King P D , Sadra A , Teng J M , Xiao-Rong L , Han A , Selvakumar A , August A , Dupont B
Published: December 2022

The CD28 cell surface receptor provides an important costimulatory signal for T cells necessary for their response to Ag. Early events in CD28 signaling include recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase) and activation of the protein tyrosine kinases (PTKs), LCK and EMT. Recruitment and activation of PI3-kinase is known to be dependent upon phosphorylation of tyrosine 173 of the CD28 cytoplasmic tail contained within a YMNM motif. By contrast, little is known of which residues of the CD28 tail, including tyrosines, are required for the activation of PTKs. To address this we studied the ability of truncation mutants and tyrosine to phenylalanine substitution mutants of the CD28 cytoplasmic tail to activate LCK and EMT in Jurkat T leukemia cells. Our results indicate that 1) activation of EMT is partially dependent upon tyrosine 173 of the CD28 tail, although it does not require PI3-kinase activation; 2) activation of LCK is independent of CD28 cytoplasmic tail tyrosine residues; and 3) elements sufficient for the activation of both kinases are contained within the first half of the tail. In addition we studied the CD28 tail as a substrate for both PTKs in in vitro kinase assays. We demonstrate that EMT can phosphorylate all four tyrosines of the CD28 tail, in contrast to LCK, which phosphorylates only tyrosine 173. Together with evidence that in vivo, tyrosines other than tyrosine 173 become phosphorylated following CD28 stimulation, this finding suggests that, like LCK, one function of EMT during CD28 signaling is phosphorylation of the receptor.
Carrier-independent hapten recognition and promiscuous MHC restriction by CD4 T cells induced by trinitrophenylated peptides.
Authors: Kohler J , Hartmann U , Grimm R , Pflugfelder U , Weltzien H U
Published: December 2022

The elucidation of mechanisms underlying the recognition of haptens by class II MHC-restricted T cells is instrumental for the understanding of chemical- and drug-induced allergies. We have previously demonstrated that trinitrophenyl (TNP) peptides represent dominant antigenic epitopes for CD8+ and CD4+ mouse T cells triggered by chemically TNP-modified APC. Here, we report the characterization of TNP-specific, CD4+ mouse T cell lines and hybridomas that were induced in vivo and in vitro by defined hapten-conjugated peptides. These peptides, which we had previously shown to induce contact sensitivity to picryl chloride in vivo regardless of sequence homologies to mouse proteins, were found to activate carrier-independent TNP-specific T cells in vitro. We interpret these findings to support our view that carrier-independent T cells, reactive to particularly repetitive hapten epitopes, may play a crucial role in allergies to chemicals and drugs. In addition to carrier independence, one of our hybridomas (IT-H6/A11) exhibited a striking promiscuity of MHC restriction. Although absolutely dependent in its TNP reactivity on the presence of MHC class II molecules, the IT H6/A11 hybridoma completely ignored class II polymorphism and even reacted to TNP peptides presented on human DR molecules. Regarding hapten allergies in humans with a heterozygous situation for three types of class II molecules (DR, DP, and DQ), such promiscuous MHC restriction should lead to the presentation of even higher epitope densities to the respective T cell clones. Hybridoma IT-H6/A11, reacting to TNP independent of carrier peptide and of MHC haplotype, also allowed for an unusually systematic study of the minimal requirements for TNP recognition. Despite an almost complete ignorance of amino acid side chains on the carrier peptide, our data indicate a clearly position-specific interaction of hapten and TCR.
T cell-derived IL-4 and dendritic cell-derived IL-12 regulate the lymphokine-producing phenotype of alloantigen-primed naive human CD4 T cells.
Authors: Ohshima Y , Delespesse G
Published: December 2022

Previous studies on human Th subset development were restricted to the analysis of naive T cells activated with anti-CD3 mAb in the absence of physiologic APC. In this study, we have analyzed the role of cytokines and physiologic APC on T cell maturation in an Ag-specific system, in which naive neonatal CD4 T cells were primed with allogeneic dendritic cells (DC). We found that the cytokine profile of primed cells was dependent upon 1) the ratio between T cells and allogeneic DC and 2) the endogenous production of IL-4 and IL-12. Neutralization of IL-4 during primary MLR increased IFN-gamma production at priming and shifted the phenotype of primed cells from Th0 to Th1. These effects were IL-12 dependent, in that they were suppressed by anti-IL-12 Abs. The production of IL-12 in primary MLR was further evidenced by the presence of IL-12 p40 in the culture supernatant fluids. IL-12 production was suppressed by exogenous IL-4 and increased by anti-IL-4 blocking mAbs, indicating that endogenous IL-4 down-regulated IL-12 production by DC. Finally, IL-12 was produced as a result of T cell/DC interaction involving the CD40/CD40 ligand and CD28/B7 costimulation pathways, as revealed by the inhibitory effect of anti-CD40 ligand mAb and CTLA-4Ig. These observations suggest that in neutral conditions, Ag presentation by DC results in the coordinate production of naive T cell-derived IL-4 and DC-derived IL-12 that in concert shape the cytokine profile of Th cells.
Visualization of antigen-specific T cell activation and cytokine expression in vivo.
Authors: Rogers W O , Weaver C T , Kraus L A , Li J , Li L , Bucy R P
Published: December 2022

A transgenic TCR adoptive transfer system was used to visualize Ag-specific T cell activation and cytokine expression in vivo. After s.c. injection of peptide in adjuvant the entire Ag-specific population up-regulated IL-2 receptor alpha-chain expression, underwent blast transformation, and developed a memory-surface phenotype. A minority of the Ag-specific T cells produced predominantly IL-2 mRNA and localized at the T cell/B cell junction in draining lymph nodes. In the secondary response, a mixed cytokine pattern of both Th1 and Th2 cytokines was demonstrated. When peptide was administered i.v. without adjuvant, 50% of the Ag-specific cells expressed IL-2, but the peak of expression occurred before IL-2 receptor alpha-chain up-regulation, and only a minority of the Ag-specific T cells underwent blast transformation.
CD28 costimulation promotes the production of Th2 cytokines.
Authors: Rulifson I C , Sperling A I , Fields P E , Fitch F W , Bluestone J A
Published: December 2022

CD28 ligation augments TCR-mediated proliferation, IL-2 production, and T cell survival. However, the role of CD28 costimulation in T cell differentiation remains controversial. To address this issue, CD28+ and CD28-deficient TCR alphabeta transgenic (Tg) mice were used to examine cytokine production by T cells following antigenic stimulation. Increasing CD28 ligation resulted in increased production of IL-4 and IL-5, consistent with differentiation toward a Th2 phenotype, in both CD4+ TCR Tg T cells and CD8+ TCR Tg T cells. The same result was obtained with CD4+ TCR Tg mice bred to RAG2-deficient mice, indicating that the Th2 differentiation observed with increased CD28 ligation was not due to the presence of memory T cells. Although CD28 costimulation is an essential factor regulating IL-2 synthesis, differentiation toward a Th2-like phenotype by CD28 ligation was not an indirect effect of enhanced IL-2 production. In contrast, blockade of IL-4 during the primary cultures of the T cells resulted in a profound inability to produce Th2-type cytokines upon restimulation. The critical role of IL-4 was confirmed by the finding that CD28-deficient TCR alphabeta Tg+ T cells cultured with rIL-4 differentiated into Th2-like T cells. Therefore, CD28 ligation promotes the production of Th2-type cytokines by naive murine T cells via an IL-4-dependent mechanism.
B220 expression by T lymphoid progenitor cells in mouse fetal liver.
Authors: Sagara S , Sugaya K , Tokoro Y , Tanaka S , Takano H , Kodama H , Nakauchi H , Takahama Y
Published: December 2022

The present study has characterized T lymphoid progenitor cells that reside in mouse fetal liver. Day 14 fetal liver contains progenitor cells that can differentiate into mature T cells upon being transferred into the thymus by hanging drop cultures. Fractionation of fetal liver cells indicated that T progenitor cells were confined in TER119- CD45+ FcR(low) cells. To our surprise, B220+ rather than B220- fraction in TER119- CD45+ FcR(low) fetal liver cells exhibited efficient progenitor activity generating T cells. Progenitor activity by the B220+ fetal liver cells was restricted to T cells, B cells, and macrophages at frequency approximately 1/10, approximately 1/10, and approximately 1/20, respectively, of isolated B220+ cells. B220+ fetal liver cells did not contain detectable D-J rearrangement of TCR-beta gene and were c-kit+ IL-7R+ Thy-1- CD3- CD4(low) CD8- CD25- CD44+. B220+ fetal liver cells expressed mRNAs encoding TCR-beta, pT alpha, Ig alpha, and VpreB. Interestingly, TCR beta-chains were expressed by B220+ fetal liver cells in the VDJ-rearranged TCR-beta-transgenic mice, indicating that TCR-beta transcription and B220 expression are activated simultaneously by the transgenic B220+ fetal liver cells. These results indicate that B220 is expressed by fetal liver lymphoid progenitor cells that can become T cells, and suggest that lymphoid progenitor cells in fetal liver concurrently undergo T- and B-specific molecular events within a single cell.
Integrin-associated protein (CD47) is a comitogenic molecule on CD3-activated human T cells.
Authors: Ticchioni M , Deckert M , Mary F , Bernard G , Brown E J , Bernard A
Published: December 2022

IAP is a glycoprotein functionally and physically associated with some integrins, i.e., the leukocyte response integrin and the beta3 integrin chain on placenta, platelets, and polymorphonuclear cells. IAP may act as a transducer element in activation mediated via these integrins. Since IAP is present at high density on peripheral T lymphocytes we have investigated its involvement in T cell activation. We tested three mAbs against IAP, namely B6H12, BRIC126, and 2D3, which recognize two distinct epitopes. IAP cross-linking with B6H12 or BRIC126, but not 2D3, transduces costimulatory signals within highly purified CD3-activated T lymphocytes, i.e., enhancement of proliferation, CD25 expression, and IL-2 secretion, while no effect was observed upon CD2 stimulation. However, we could not observe any functional association between IAP and integrins on peripheral T cells. In an attempt to explore further the activation signal delivered by IAP, we show here that IAP cross-linking with the comitogenic B6H12 mAb induces the phosphorylation on tyrosine of several proteins, one of which is identified as p56(lck) protein tyrosine kinase. Moreover, we observed that IAP is associated with p56(lck) on PMA-activated, but not on resting, T cells. These data suggest that on T cells, IAP may be involved directly via a specific ligand in cell-matrix or cell-cell interactions. Such interactions could trigger protein tyrosine phosphorylation pathways, which play an important role in both maturation and activation of T cells.
Antigen-presenting cells in the thymus that can negatively select MHC class II-restricted T cells recognizing a circulating self antigen.
Authors: Volkmann A , Zal T , Stockinger B
Published: December 2022

We investigated Ag presentation of an extracellular self Ag (C5), which only reaches the thymus via the blood circulation, for negative selection of MHC class II-restricted, C5-specific T cells. Thymic APC were introduced into fetal thymic reaggregation culture with thymocytes from C5-specific TCR transgenic mice to follow the development of C5-specific T cells in the presence or the absence of self Ag presented by various APC. To mimic the physiologic distribution of C5 peptide/MHC class II complexes on thymic APC as closely as possible, they were isolated from thymi of C5+ mice, so that the amount of C5 peptide bound to MHC class II on their surface would reflect the amount of self Ag they have access to and process normally in vivo. This circumvented the problems related to artificially high doses of Ag or peptide in vivo or in vitro, that might obscure physiologic differences such as the capacity to internalize and process Ag. The results show that not only thymic dendritic cells, but also cortical and medullary epithelial cells were able to induce negative selection of C5-specific thymocytes with similar efficiency. In contrast, thymic macrophages were unable to influence the development of C5-specific T cells. Their failure to present exogenous self Ag for negative selection suggests that macrophages concentrate on their primary function in the thymus, the disposal of dying thymocytes.
Constitutive expression of terminal deoxynucleotidyl transferase in transgenic mice is sufficient for N region diversity to occur at any Ig locus throughout B cell differentiation.
Authors: Bentolila L A , Wu G E , Nourrit F , Fanton d'Andon M , Rougeon F , Doyen N
Published: December 2022

N region diversity in Ag receptors is a developmentally regulated process in B and T cells that correlates with the differential expression of terminal deoxynucleotidyl transferase (TdT). Absent in fetal and newborn mice, TdT expression is restricted to early T and pro-B cells in adults. To extend the TdT expression pattern throughout B cell ontogenesis, we generated transgenic mice carrying a TdT cDNA under the regulatory elements of the N-myc gene and the IgH enhancer. High expression was observed in secondary lymphoid organs consistent with TdT activity beyond the pre-B cell stage. This suggests that TdT transgene expression is not down-regulated as is the endogenous gene. Unlike normal mice, extensive N region diversity was found in rearranged lambda light chain genes of adult transgenic animals. Therefore, expression of TdT appears sufficient for N region diversity to occur at any Ig locus. More importantly, expression of the transgene takes place during fetal development. As a consequence, the potential fetal B cell repertoire is modified as both rearranged heavy and light chain genes now show N region additions. Constitutive expression of TdT throughout B cell differentiation does not therefore appear deleterious and suggests that TdT is recruited only to participate in the V(D)J recombination process.
Identification of IL-4 promoter elements conferring Th2-restricted expression during T helper cell subset development.
Authors: Wenner C A , Szabo S J , Murphy K M
Published: December 2022

Selective cytokine gene expression by T cell subsets underlies polarization of cellular and humoral immune responses. Our interest has been to define the molecular basis for restricted cytokine expression by Th1 and Th2 cells. IL-4 is selectively expressed by Th2 cells, providing a model for Th2-specific gene expression. To allow for promoter analysis during the process of Th1/Th2 differentiation within a normal cellular context, we have taken a transgenic approach. We generated a series of murine transgenic lines harboring both the DO11.10 alphabeta-TCR transgene and the luciferase gene driven by regions of the IL-4 promoter. The results identify proximal promoter regions that provide significantly Th2-restricted IL-4 gene expression. The IL-4 -741- to +60-bp region allows, on the average, 40-fold higher inducible reporter activity in Th2 cells than in Th1 cells. When trimerized, the region spanning -88 to -61 bp, containing a composite NF-AT/AP-1 site, also confers significant Th2-specific reporter activity. These results suggest that trans-acting factors binding this NF-AT/AP-1 composite site cooperate to allow substantial Th2-selective reporter expression. Finally, because reporter expression is low relative to endogenous IL-4 mRNA in activated Th2 cells, we suggest that additional control elements outside of the IL-4 promoter may be required to enhance overall IL-4 gene activity.
Infection of human vascular endothelial cells with Staphylococcus aureus induces hyperadhesiveness for human monocytes and granulocytes.
Authors: Beekhuizen H , van de Gevel J S , Olsson B , van Benten I J , van Furth R
Published: December 2022

The consequences of internalization of Staphylococcus aureus by HUVEC with respect to their adhesiveness for human monocytes and granulocytes were investigated. Viable and UV-killed, but not heat-killed, S. aureus were internalized by HUVEC, which required participation of the endothelial cytoskeleton. S. aureus-infected HUVEC displayed increased surface expression of CD106 (VCAM-1), CD54 (ICAM-1), and MHC I molecules. Expression of CD62P (P-selectin), CD62E (E-selectin), CD31 (PECAM-1), and CD102 (ICAM-2) was not affected. Concomitantly, these HUVEC expressed a time- and inoculum size-dependent hyperadhesiveness for monocytes and granulocytes. Monocyte adhesion reached maximal levels (approximately 60% adhesion) 23 h after the initial 1 h period of infection of HUVEC with about 50 bacteria per single HUVEC. To induce maximal (approximately 20%) adhesion of granulocytes, five times higher concentrations of HUVEC-infecting bacteria were required. Using the appropriate mAb, granulocyte adhesion to S. aureus-infected HUVEC was shown to be entirely mediated by the beta2 (CD11/CD18) integrins. Monocyte adhesion to these HUVEC was largely (approximately 70%) dependent on both CD11a/CD18 (LFA-1) and CD49d/CD29 (VLA-4). This demonstrates that infection of HUVEC with S. aureus potentiates CD11/CD18-mediated granulocyte adhesion and shifts the mechanism of monocyte adhesion from being completely CD11/CD18 dependent to one that also utilizes the VLA-4/VCAM-1 dependent pathway. Together, these findings indicate that in response to internalization of S. aureus, vascular endothelial cells may initiate recruitment of monocytes and granulocytes, which may be an important initial event in the pathogenesis of endovascular diseases.