bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction between endothelium and CD4+CD45RA+ lymphocytes. Role of the human CD38 molecule.
Authors: Dianzani U , Funaro A , DiFranco D , Garbarino G , Bragardo M , Redoglia V , Buonfiglio D , De Monte L B , Pileri A , Malavasi F
Published: December 2022

CD38 is a type II transmembrane glycoprotein, which is widely used as a marker for immature and activated lymphocytes, as well as plasma cells. Although its functional role and natural ligand are not known, CD38 has been shown to transduce activation signals to lymphocytes. Our work shows that CD38 is preferentially expressed by CD4+CD45RA+ cells, but not by CD4+CD45R0+ cells. CD4+CD45RA+ cells are reported to respond poorly to stimuli acting through the CD3/TCR in vitro and to display unique migration pathways in vivo. Cross-linking of CD38 by mAb did not overcome the hyporesponsiveness of CD4+ resting/naive cells to several activation stimuli; in contrast, CD38 engagement by mAb specifically inhibited their binding with human vein endothelial cells. These data suggest that CD38 may play a role in lymphocyte migration. The same inhibitory effect was detected on the (human x mouse) hybrid cell line CP410.A10, which expresses human CD38, but not on its CD38- subclone CP14. CD38 mAb did not inhibit the conventional binding assay between endothelium and several human CD38+ T and B cell lines. However, the inhibition was apparent when the binding assay was performed at 4 degrees C on a rocking shelf, conditions that minimized integrin function. These data suggest that CD38 mediates weak cell binding to endothelium, which is effective even in dynamic conditions. These features are reminiscent of those exerted by selectins, which are adhesion molecules that account for leukocyte rolling on vascular endothelial cells and play an important role in lymphocyte homing.
Engagement of CD14 on monocytes inhibits the synthesis of human Igs, including IgE.
Authors: Jabara H H , Vercelli D
Published: December 2022

Engagement of CD14 on normal human monocytes results in monocyte activation and, furthermore, delivers a negative signal that terminates T cell proliferation. We show herein that engagement of selected CD14 epitopes by specific mAbs inhibited PWM-induced IgM and IgG synthesis, as well as IL-4-dependent IgE synthesis by human PBMCs. Inhibition by anti-CD14 mAb was still evident when the Ab was added after 6 days of culture, suggesting that inhibition targets a late B cell activation event. In experiments that focused on the role of CD14 in IgE regulation, suppression of IgE synthesis after CD14 engagement did not result from the release of inhibitory monokines (IL-10, TGF-beta, and PG). In fact, neutralizing Abs and specific inhibitors did not restore the IgE response and anti-CD14-conditioned monocyte supernatants did not inhibit IgE synthesis. On the other hand, inhibition of IgE synthesis by anti-CD14 mAb was not likely to be caused by the lack of soluble factors with amplificatory effects on the IgE response, because addition of either rIL-6 or rTNF-alpha did not overcome CD14-dependent inhibition of IgE production. IgE inhibition after CD14 engagement was exerted at the B cell level, inasmuch as it was observed not only in T cell-dependent IgE induction i.e., mononuclear cells stimulated with IL-4, but also in T cell-independent systems, i.e., T cell-depleted populations stimulated with IL-4 plus anti-CD40 mAb or IL-4 plus hydrocortisone. These data indicate that CD14 plays a regulatory role in B cell responses.
Function of bone marrow stromal cell lines derived from nude mice.
Authors: Dong Z , Wortis H H
Published: December 2022

We previously reported that in double deficient nude.xid mice B cells failed to develop and their bone marrow did not produce mature B cells in vitro. However, when progenitors from nude.xid bone marrow were placed on a preestablished normal stromal cell line (AC6) they differentiated into surface IgM+ cells. This raised the possibility of a deficiency of nude and nude.xid stromal cells such that they were incapable of supporting the maturation of X-linked immune deficiency (xid) B cells. Here we ask whether bone marrow stromal cells from nude and nude.xid mice have the ability to support xid B cell lymphopoiesis. A primary stromal cell layer derived from nude mice supported xid B cell differentiation in vitro. We derived panels of stromal cell lines by transfection of primary stromal cell layers with a retrovirus encoding SV40 large T Ag. Several bone marrow stromal cell lines derived from nude and nude.xid mice supported xid B cell differentiation from CD43+/CD45 (B220-) to CD45 (B220+) and from CD45 (B220+)/surface IgM- to surface IgM+. Supporting cell lines expressed both IL-7 and insulin-like growth factor I. The frequencies of bone marrow stromal cells capable of supporting xid B cell differentiation were similar in normal, xid, nude, and nude.xid mice. These results demonstrate that nude and nude.xid mice have bone marrow stromal cells with normal abilities to support B cell maturation.
Biologic activity of low dosage IL-2 treatment in vivo. Molecular assessment of cytokine network interaction.
Authors: Hladik F , Tratkiewicz J A , Tilg H , Vogel W , Schwulera U , Krönke M , Aulitzky W E , Huber C
Published: December 2022

The sequence of activation of the components of the cytokine network subsequent to in vivo application of different dosages of IL-2 is still poorly understood. Although side effects of IL-2 therapy are dose dependent, the dose-response relationship for induction of potentially beneficial or harmful cytokine genes still remains to be studied. We examined the patterns of cytokine gene expression after treatment of chronic hepatitis B patients with various doses of IL-2 in a phase 1 trial. Total RNAs were isolated from PBMC harvested at various time points after s.c. injection of natural IL-2 ranging from 30,000 to 1,000,000 U. Dose-dependent effects on mRNA expression of IL-2, GM-CSF, IFN-gamma, TNF-alpha, and IL-6 were assessed using Northern blotting and slot blotting techniques. A single application of 30,000 U nIL-2 induced selective and long-lasting expression of IL-2, IFN-gamma, and GM-CSF genes, which was not accompanied by accumulation of TNF-alpha and IL-6 mRNAs. Larger dosages of IL-2 induced activation of monokine genes and were associated with systemic side effects. mRNA levels of the different cytokines related to biologic activity and correlated with expression of specific proteins and cellular parameters: IL-2 mRNA with soluble IL-2R serum levels and induction of lymphopenia, GM-CSF mRNA with induction of neutrophilia, and IL-6 mRNA with c-reactive protein serum concentrations. Taken together these data indicate unexpected immunoregulatory activities of very low and nontoxic dosages of IL-2 in vivo.
In vitro potentiation of lymphocyte activation by dehydroepiandrosterone, androstenediol, and androstenetriol.
Authors: Padgett D A , Loria R M
Published: December 2022

Dehydroepiandrosterone (5-androstene-3 beta-ol-17-one, DHEA) has been shown to protect mice from lethal viral and bacterial infections. However, recently, androstenediol (5-androstene-3 beta-17 beta-diol, AED) and androstenetriol (5-androstene-3 beta-7 beta-triol, AET), metabolites of DHEA, have each shown to be more potent endocrine regulators of the immune response. In contrast to glucocorticosteroids, in vivo, these steroid hormones up-regulated the cellular immune response of the host to limit virus-mediated pathology. These experiments first examined the in vitro influences of DHEA, and AED, or AET on mitogen-stimulated activation of murine lymphocytes. From physiologic to pharmacologic doses, DHEA suppressed proliferation of mixed splenocyte cultures activated with Con A or LPS by 20 to 70% whereas AED had little influence on the response. In sharp contrast, AET potentiated the response with both mitogens to 50 to 70% above control. Analogous to these observations was the regulation of IL-2 and IL-3 secretion from Con A-activated lymphocytes by each of these hormones which again was depressed analyzed. The suppressive effects of hydrocortisone on Con A-induced lymphocyte proliferation and cytokine production were strongly counteracted by coculture with AET. DHEA did not counteract hydrocortisone activity whereas AED showed moderate antiglucocorticoid function.
Tumor-induced regulation of suppressor macrophage nitric oxide and TNF-alpha production. Role of tumor-derived IL-10, TGF-beta, and prostaglandin E2.
Authors: Alleva D G , Burger C J , Elgert K D
Published: December 2022

In vitro-activated macrophages (Mphi) co-express cytotoxicity for tumor cells and suppression of lymphocyte proliferation. These Mphi functions increase during tumor growth and are mediated by soluble molecules. Because Mphi-derived nitric oxide (NO) and TNF-alpha mediate both cytotoxicity and suppression, we determined whether fibrosarcoma (Meth-KDE) growth increased Mphi-mediated suppression of T cell proliferation by increasing Mphi NO and TNF-alpha production. Tumor-bearing host peritoneal Mphi produced more NO and TNF-alpha than normal host Mphi when activated with IFN-gamma or LPS, respectively. This tumor-induced increase in Mphi NO and TNF-alpha production mediated suppression of alloantigen-driven T cell proliferation, because treatment with either NG-monomethyl-L-arginine or anti-TNF-alpha Ab blocked tumor-bearing host Mphi-mediated suppression. TNF-alpha did not directly suppress T cells, but it induced Mphi NO production that down-regulated proliferation. When non-tumor-infiltrating peritoneal Mphi were cultured with Meth-KDE cell supernatants, Mphi production of NO and TNF-alpha was strongly down-regulated. The tumor-derived molecules responsible for this inhibition were IL-10, TGF-beta 1, and prostaglandin E2. The experimental evidence leading to this conclusion included: 1) The Meth-KDE cells produced significant levels of these cytokines. 2) Recombinant forms of these cytokines suppressed NO and TNF-alpha production. 3) Ab-mediated absorption of these cytokines from tumor cell supernatants restored NO and TNF-alpha production. 4) Anti-IL-10 and anti-TGF-beta 1 Ab addition to IFN-gamma-stimulated Mphi restored NO production. Culture supernatants of two human carcinoma cell lines and another murine fibrosarcoma suppressed Mphi NO and TNF-alpha production, which was partly mediated by TGF-beta 1 and prostaglandin E2. Collectively, these results suggest that tumor growth promotes distal Mphi suppressor activity by increasing Mphi production of cytotoxic molecules and concomitantly down-regulating the local production of these antitumor molecules.
IL-12 inhibits Th2 cytokine responses induced by eggs of Schistosoma mansoni.
Authors: Oswald I P , Caspar P , Jankovic D , Wynn T A , Pearce E J , Sher A
Published: December 2022

In the mouse, infection with the helminth parasite Schistosoma mansoni results in the selective induction of CD4+ T lymphocytes belonging to the Th2 subset. Schistosome ova are responsible for the development of Th2 responses seen in patently infected animals and the injection of eggs s.c. into the footpad leads to the development of elevated Th2 cytokine production by T cells in the draining popliteal lymph node. Using the egg injection model, we have shown that IL-12 suppresses schistosome egg-induced Th2 responses as evidenced by decreased IL-4, IL-5, and IL-10 secretion in vitro while increasing the production of the Th1 cytokine IFN-gamma. Similar responses were obtained using either total lymph node cells or purified CD4+ T cells, indicating that IL-12 acts at the T cell level. When given as a single injection IL-12 was most effective at inhibiting Th2 responses when administered 2 days after egg inoculation, a time when T cells are still in a Th0 phase. The suppression of Th2 responses induced by IL-12 was blocked when the animals were simultaneously injected with neutralizing anti-IFN-gamma mAb, either systemically or systemically plus locally. Anti-IFN-gamma also inhibited the enhancement of IFN-gamma responses induced by IL-12 but only if the mAb was administered systemically plus locally. NK cells are likely to be a major source of the immunoregulatory IFN-gamma, because the effects of IL-12 on Th2 cytokine production were suppressed in mice treated with anti-asialo-GM1 Abs. Together these results suggest that IL-12 may have potential use in preventing or treating parasite-induced pathology resulting from Th2 cytokine production.
Human polymorphonuclear leukocytes lack detectable nitric oxide synthase activity.
Authors: Yan L , Vandivier R W , Suffredini A F , Danner R L
Published: December 2022

Nitric oxide regulates polymorphonuclear leukocyte (PMN) function, but whether or not human PMNs express nitric oxide synthase (NOS) activity is controversial. We studied NOS activity in human PMNs by using human aortic endothelial cells (HAECs) for comparison. The conversion of L-arginine to L-citrulline, a relatively specific measure of NOS activity, was easily measured and inducible in fractionated HAECs, and > 90% of all L-arginine conversion was blocked by the NOS inhibitor, N omega-amino-L-arginine (L-NAA). In fractionated PMNs, L-arginine conversion was low and was unaffected by L-NAA. In addition, NOS activity was not induced in PMNs by LPS, IL-1 beta, or IFN-gamma. In a whole-cell assay, total L-arginine conversion was much lower in human PMNs compared with HAECs (3.38 +/- 0.21 vs 157.5 +/- 10.28 pmol/h/10(6) cells, respectively; p < 0.01). This conversion in whole PMNs was not increased in vitro by LPS, IFN-gamma, IL-1 beta, or TNF-alpha nor decreased by W13, a calmodulin inhibitor. Furthermore, PMNs isolated from four volunteers before and after challenge with i.v. LPS (4 ng/kg) showed no increase in L-arginine to L-citrulline conversion. Nitrite and nitrate release from human PMNs was 35-fold lower than for HAECs and was not inhibited by L-NAA. These data suggest that human PMNs do not express NOS activity.
A cultured malignant B-1 line serves as a model for Richter's syndrome.
Authors: Peng B , Sherr D H , Mahboudi F , Hardin J , Wu Y H , Sharer L , Raveché E S
Published: December 2022

Human chronic lymphocytic leukemia (CLL) is a malignancy of B-1 cells characterized by the accumulation of mature appearing, long lived, slow growing B-1 cells in peripheral blood. CLL occasionally evolves into an aggressive large cell lymphoma termed Richter's syndrome. NZB mice can be used to model the early stage of CLL because aged NZB mice can spontaneously develop slow growing malignant B-1 cell clones. The malignant NZB B-1 clones fail to grow in culture and are typically carried in vivo as passaged lines. During serial passage, an aggressive lymphoma developed as a result of a continued transformation of the original B-1 clone, similar to the development of Richter's syndrome. An in vitro cell line was established from the aggressive lymphoma, which was stromal dependent and could rapidly metastasize when passaged into recipient animals. Analysis of adhesion molecules did not reveal any consistent characteristics that could account for the metastatic potential of the Richter's-like cells. In addition, the aggressive in vitro line had the identical heavy chain sequence as the slow growing NZB malignant B-1 clones. The in vitro and in vivo aggressive B-1 cells had very high levels of IL-10 message, and underwent more apoptosis in response to anti-IgM than did nonaggressive B-1 clones. Taking these characteristics together, we have composed a comprehensive animal model system for human CLL that includes both the aged NZB mice for the early stage and the recipients of the in vitro B-1 line for the late stage or Richter's syndrome. This model system can be used to study, not only the ontogeny and genetic linkage of CLL, but also the regulatory factors involved in transformation and growth both in vivo and in vitro.
IFN-gamma receptor-deficient mice generate antiviral Th1-characteristic cytokine profiles but altered antibody responses.
Authors: Schijns V E , Haagmans B L , Rijke E O , Huang S , Aguet M , Horzinek M C
Published: December 2022

The lymphokine IFN-gamma is a pleiotropic immunomodulator and possesses intrinsic antiviral activity. We studied its significance in the development of antiviral immune responses by using IFN-gamma receptor-deficient (IFN-gamma R-/-) mice. After inoculation with live attenuated pseudorabies virus (PRV), the mutant mice showed no infectivity titers in various tissues, and transient viral Ag expression only in the spleen, similar as in wild-type mice. However, the absence of the IFN-gamma R resulted in increased proliferative splenocyte responses. The PRV-immune animals showed a normal IFN-gamma and IL-2 production, without detectable IL-4, and with decreased IL-10 secretion in response to viral Ag or Con A. Immunohistochemically, an increased ratio of IFN-gamma:IL-4-producing spleen cells was found. After immunization with either live attenuated or inactivated PRV, IFN-gamma R-/- mice produced significantly less antiviral Ab, and more succumbed to challenge infection than the intact control animals. The reduction in Ab titers in the mutant mice correlated with lower protection by their sera in transfer experiments. Our data demonstrate that ablation of the IFN-gamma receptor surprisingly does not inhibit the generation of antiviral Th1-type and increase Th2-type cytokine responses. However, it profoundly impairs the generation of protective antiviral Ab.
NF-kappa B and Sp1 regulate transcription of the human monocyte chemoattractant protein-1 gene.
Authors: Ueda A , Okuda K , Ohno S , Shirai A , Igarashi T , Matsunaga K , Fukushima J , Kawamoto S , Ishigatsubo Y , Okubo T
Published: December 2022

Expression of the human monocyte chemoattractant protein-1 (hMCP-1) is ubiquitous in various cell types and is increased by a wide variety of stimuli. We initially found that the effects of various stimuli, including IL-1 beta, TNF-alpha, and 2-O-tetradecanoylphorbol 13-acetate, on the expression of hMCP-1 mRNA were quite different among A172 glioblastoma cells, HT1080 fibrosarcoma cells, and SKLMS1 leiomyosarcoma cells. These findings suggested that hMCP-1 expression is regulated both in a stimulus-specific and a tissue-specific manner. To elucidate the mechanism underlying this stimulus-specific and tissue-specific regulation, we isolated a hMCP-1 5'-flanking genomic DNA fragment and sequenced it extensively up to bp 3011 upstream from the transcriptional start site. Among many putative cis-elements, we identified two cis-elements critical for the transcription of the hMCP-1 gene. The first element is a remote kappa B binding site located far upstream between bp -2612 and -2603 that was important for IL-1 beta-, TNF-alpha-, and 2-O-tetradecanoylphorbol 13-acetate-induced enhancer activity. Mutation at the kappa B consensus site resulted in a complete loss of these stimulus-induced enhancer activities. The second element is a GC box located between bp -64 and -59 that was important for the maintenance of basal transcriptional activity. Overexpression of rSp1 resulted in increased hMCP-1 transcriptional activity, possibly suggesting the role of Sp1 in controlling basal hMCP-1 transcription via this GC box. These results together indicate that hMCP-1 expression is controlled by at least two distinct regulatory elements: a kappa B site and a GC box that seem to be associated with stimulus-specific and tissue-specific regulation, respectively.
Mechanisms of IL-4-mediated suppression of IP-10 gene expression in murine macrophages.
Authors: Deng W , Ohmori Y , Hamilton T A
Published: December 2022

The mechanisms involved in negative regulation of IFN-gamma-induced IP-10 mRNA expression by IL-4 have been examined in an immortalized murine macrophage cell line (ANA-1). As in primary peritoneal macrophages, IL-4 selectively inhibits the production of IP-10 mRNA by IFN-gamma-treated ANA-1 cells. Expression of another IFN-gamma-inducible gene (D3) was not affected by co-treatment with IL-4. Suppression of IFN-gamma-induced IP-10 mRNA expression by IL-4 is mediated at the level of transcription. IFN-gamma-induced transcription of CAT expression driven by a 243-bp IP-10 promoter fragment was also sensitive to the suppressive effects of IL-4. In contrast, LPS-induced CAT expression was unaffected under identical experimental conditions. The positive transcriptional response to IFN-gamma required an interferon stimulus response element (ISRE) sequence motif located approximately 230 bp upstream from the transcription start site of the IP-10 gene. That this site is the target of the suppressive action of IL-4 is indicated by the ability of IL-4 to inhibit IFN-gamma-mediated transcription from this ISRE sequence in the context of a heterologous promoter. Finally, both IFN-gamma and IL-4 can enhance or induce expression of distinct nuclear factors that exhibit ISRE-specific binding activity. IL-4 does not suppress the IFN-gamma-induced ISRE binding activity. Together, these findings demonstrate that IL-4 inhibits IP-10 mRNA production in murine macrophages by suppressing the formation of new transcripts. Both positive and negative transcriptional activity appears dependent on activation of factors that recognize the ISRE.
Different signaling pathways for CD18-mediated adhesion and Fc-mediated phagocytosis. Response of neutrophils to LPS.
Authors: Detmers P A , Zhou D , Powell D E
Published: December 2022

The regulation of CD11b/CD18 adhesive and phagocytic functions on human polymorphonuclear leukocytes (PMN) in response to LPS was examined. Adhesion of PMN to surfaces coated with LPS had little or no effect on the cells, but pretreating the LPS-coated surfaces with either diluted serum or LPS-binding protein strongly enhanced their ability to bind C3bi-coated E (EC3bi), a ligand for CD11b/CD18. LPS-binding protein is known to enable responses of cells to LPS by facilitating binding of LPS to CD14. Consistent with this, we found that preformed complexes of LPS with soluble rCD14 stimulated binding of ligand by CD11b/CD18 in a concentration-dependent manner. Known agonists that stimulate CD11b/CD18 binding activity on PMN all cause simultaneous enhancement of Fc-mediated phagocytosis. However, LPS presented in complex with either serum proteins or CD14 failed to stimulate the ingestion of ElgG by PMN. The number of FcRs and their ability to bind ligand were not affected by treatment with LPS, nor were they compromised in their ability to respond to other agonists. These results suggest that LPS generates intracellular signals that alter the ability of CD11b/CD18 to bind ligand, but this alteration is not sufficient to promote phagocytosis of IgG-coated particle. This conclusion was confirmed by showing that PMN treated with LPS and serum produced a lipid with the properties of integrin-modulating factor 1: acetone extracts of these cells stimulated CD11b/CD18 adhesive capacity on PMN. However, the lipid did not enhance Fc-mediated phagocytosis. These studies suggest that CD14 affects CD11b/CD18 function by inducing the synthesis of a lipid such as IMF-1, and that this lipid affects only the binding activity, not the phagocytosis-promoting capacity of CD11b/CD18.
Eosinophil transendothelial migration induced by cytokines. III. Effect of the chemokine RANTES.
Authors: Ebisawa M , Yamada T , Bickel C , Klunk D , Schleimer R P
Published: December 2022

Selective eosinophil recruitment occurs after experimental Ag challenge and in tissue sites of allergic diseases. The mechanisms of selective eosinophil migration are still unknown. In our study, we examined the ability of chemokines to induce transendothelial migration (TEM) of eosinophils in vitro. Among the chemokines tested, only RANTES induced eosinophil TEM. RANTES failed to induce TEM of neutrophils. Interestingly, IL-8 induced neutrophil TEM and had no effect on eosinophil TEM. RANTES-induced TEM was concentration-dependent and was inhibited by Abs directed against the beta 2 integrin CD18. When IL-1-activated endothelial cells were utilized, RANTES-induced TEM also involved the eosinophil beta 1 integrin VLA-4. RANTES did not increase eosinophil adhesion to either resting or IL-1-activated endothelial cells, nor did the chemokine increase CD11b or decrease L-selectin expression. A gradient of RANTES appears to be required for eosinophil TEM. Pre-exposure of eosinophils to IL-5 dramatically potentiated the TEM response to RANTES. These findings suggest that the chemokine RANTES is a potent and selective inducer of eosinophil TEM. Because RANTES appears to be produced in vivo during allergic reactions or in allergic diseases, we speculate that these findings may have some direct relevance to the mechanism of selective eosinophil recruitment in vivo in humans.
Tenidap modulates cytoplasmic pH and inhibits anion transport in vitro. II. Inhibition of IL-1 beta production from ATP-treated monocytes and macrophages.
Authors: Laliberte R , Perregaux D , Svensson L , Pazoles C J , Gabel C A
Published: December 2022

IL-1 beta is an important inflammatory mediator produced by monocytes and macrophages after LPS stimulation. In the absence of a secondary stimulus, however, little IL-1 beta is released into the medium. Previously, ATP was shown to promote the release and proteolytic maturation of IL-1 beta from LPS-stimulated murine peritoneal macrophages. Tenidap, a new anti-inflammatory and antiarthritic agent, inhibited the release and maturation of IL-1 beta induced in vitro by ATP treatment of murine peritoneal macrophages. Tenidap's inhibitory activity was mimicked by other agents that blocked anion transport, such as UK5099 and DIDS. In contrast, cyclooxygenase-inhibiting nonsteroidal anti-inflammatory drugs, such as piroxicam and naproxen, did not impair ATP-induced post-translational processing. Human monocytes responded to LPS to produce IL-1 beta, but externalized little of their newly synthesized cytokine. ATP at concentrations > or = 2 mM promoted IL-1 beta release from these cells. The degree to which the released cytokine was proteolytically processed to its biologically active 17-kDa species, however, depended on the pH of the medium; a greater processing efficiency was observed at slightly acidic (pH 6.9) values. Tenidap and other anion transport inhibitors effectively prevented the ATP response of cultured human monocytes. Likewise, LPS-stimulated human alveolar macrophages responded to ATP by releasing 17-kDa IL-1 beta, and tenidap inhibited this response. The ATP-induced release and maturation of IL-1 beta from human monocytes and macrophages, therefore, was suppressed by anion transport inhibitors, suggesting that anion conductance is a necessary component of the ATP-promoted externalization mechanism. In view of IL-1's importance as an inflammatory mediator, tenidap may demonstrate novel anti-inflammatory activities by virtue of its inhibition of the post-translational release and maturation of this cytokine.
Transforming growth factor-beta prevents stem cell factor-mediated rescue of mast cells from apoptosis after IL-3 deprivation.
Authors: Mekori Y A , Metcalfe D D
Published: December 2022

IL-3-dependent mast cells undergo apoptosis upon removal of IL-3, an event that is prevented by the addition of stem cell factor (SCF) acting through its receptor c-kit, suggesting that SCF provides a mechanism to allow mast cells to survive and to differentiate in tissues in the relative absence of IL-3. This observation is consistent with the thesis that the microenvironment, in part, controls mast cell number and viability by modulating SCF production and release. The purpose of the present study was to determine whether a second factor, TGF-beta 1, was capable of modifying the SCF-mediated survival pathway. TGF-beta 1 (1 and 10 ng/ml), known to be an important regulator of cell growth and function, did inhibit the SCF-mediated rescue from apoptosis in IL-3-deprived mast cells. TGF-beta 1 exerted its inhibitory effect on SCF-mediated rescue from apoptosis, even when added 4 h after the addition of SCF. In contrast, TGF-beta 1 had no substantial effect on the viability of mast cells that were grown in the presence of IL-3. TGF-beta 1 also had no noticeable effect on viability and proliferation of a growth factor-independent mast cell line. The inhibitory effect of TGF-beta 1 was neutralized by specific anti-TGF-beta mAb. TGF-beta 1 did not affect the expression of c-kit, as determined by using flow cytometric analysis of mast cells labeled with FITC-conjugated anti-c-kit. These results demonstrate how SCF and TGF-beta may act in concert to regulate mast cell numbers under physiologic or pathologic conditions.
Effective graft-versus-leukemia effects independent of graft-versus-host disease after T cell-depleted allogeneic bone marrow transplantation in a murine model of B cell leukemia/lymphoma. Role of cell therapy and recombinant IL-2.
Authors: Weiss L , Lubin I , Factorowich I , Lapidot Z , Reich S , Reisner Y , Slavin S
Published: December 2022

After allogeneic bone marrow transplantation (BMT) for leukemia, beneficial graft-vs-leukemia (GVL) effects are usually accompanied by potentially serious graft-vs-host disease (GVHD). Because T cell depletion is the only effective way to prevent GVHD it seems important to understand whether effective GVL can develop after BMT with T cell depletion in GVHD-free recipients. Well-established C57BL/6-->BALB/c chimeras that were free of GVHD, reconstituted with T cell-depleted allogeneic bone marrow cells, and inoculated 3 mo after BMT with a high inoculation of murine B cell leukemia (BCL1) showed no evidence of disease, whereas all control mice developed leukemia and died within 58 days. Results from adoptive transfer experiments in secondary naive BALB/c recipients indicated that all BCL1 cells were eliminated in the chimeras within 14 days. Hence, complete resistance to BCL1 developed in the chimeras despite complete tolerance to host alloantigens. The GVL effects observed in tolerant chimeras were further amplified by administration of immunocompetent allogeneic C57BL/6 spleen cells, low dose rIL-2, or both for 5 days. Our data suggest that GVL effects can develop even after T cell depletion in the absence of clinically overt GVHD and that GVL can be further amplified by rIL-2, either with or without use of additional immunocompetent donor T cells. Our data may provide the basis for new approaches to induce effective GVL after allogeneic BMT with cell therapy and rIL-2 at the stage of minimal residual disease, while avoiding early GVHD induced by the BMT procedure.
IL-1 beta-converting enzyme is present in monocytic cells as an inactive 45-kDa precursor.
Authors: Ayala J M , Yamin T T , Egger L A , Chin J , Kostura M J , Miller D K
Published: December 2022

The major form of IL-1 beta-converting enzyme (ICE) identified in THP.1 monocytic cells and human monocytes is the 45-kDa precursor protein (p45), which is found in the cytoplasm. Cytoplasmic extracts of these cells show no pIL-1 beta cleavage activity, indicating that the p45 has no detectable catalytic activity. pIL-1 beta cleavage activity can only be observed after incubation in vitro when p45 breaks down to the active p20 form of the enzyme. LPS stimulation of human monocytes or THP.1 monocytic cells results in no change in the amount of p45 or its activity and no detectable appearance of p20 ICE. Immunoprecipitation of [35S]Met-labeled LPS-stimulated monocyte extracts revealed only p45 with no other co-precipitating protein. The inability to identify active ICE in stimulated monocytic cells was probably a reflection of the very low levels of active ICE present.
Acquisition of iron bound to low molecular weight chelates by human monocyte-derived macrophages.
Authors: Olakanmi O , Stokes J B , Britigan B E
Published: December 2022

The iron content of tissue macrophages increases under conditions of elevated extracellular iron. Studies of macrophage iron acquisition have generally focused on iron uptake from transferrin via receptor-mediated endocytosis. However, in vivo macrophages are also exposed to extracellular low m.w. iron chelates, particularly under conditions of iron overload. Therefore, we examined the mechanism of iron acquisition from low m.w. chelates by human monocyte-derived macrophages. Iron acquisition was influenced by the nature of the iron chelate: Fe-ascorbate > Fe-citrate > Fe-nitrilotriacetate (NTA) = Fe-ADP > Fe-glycyl-L-histidyl-L-lysine > Fe-diethylenetriamine pentaacetic acid > Fe-EDTA = Fe-deferoxamine. With the exception of Fe-EDTA and Fe-deferoxamine, iron acquisition was greater than that with diferric transferrin. As assessed by using iron acquisition from NTA as a model, the process is temperature dependent, but pH independent (pH 5 to 8), and is influenced by the medium in which the cells are suspended. Acquisition is not affected by NaF, 2-deoxy-D-glucose, NaCN, or monocyte-derived macrophage exposure to trypsin, pronase, phenylarsine oxide, dihydrocytochalasin B, filipin, nystatin, or digitonin. The rate of iron acquisition from NTA is induced by iron pre-exposure as well as aluminum. In contrast, NTA chelates of other transition metals (Cd, Cu, Ga, Mn, and Zn) inhibited iron uptake by 20 to 80%. Unlike results obtained with Ga-NTA, Ga(NO3)3 increased iron uptake from NTA. Data obtained with neutrophils, and undifferentiated U937 and HL-60 cells were similar, which suggests that myeloid cells share this pathway for iron acquisition. Iron acquisition via this mechanism may allow macrophages and other leukocytes to clear local states of iron overload in vivo.
Severe combined immunodeficient mice engrafted with human splenocytes have functional human T cells and reject human allografts.
Authors: Alegre M L , Peterson L J , Jeyarajah D R , Weiser M , Bluestone J A , Thistlethwaite J R
Published: December 2022

Previous studies have shown that human hemopoietic cells can be adoptively transferred into immunodeficient C.B-17 scid/scid (SCID) mice that lack autologous T and B lymphocytes, to generate chimeric animals. The future development of novel immunomodulatory drugs in transplantation will depend increasingly on experimental animal models to investigate the properties of the agents on human cells before starting clinical trials. However, in previous models of SCID mice engrafted with human PBLs, human T cells have been found either to be in an unresponsive state, unable to respond to mitogenic stimulations in vitro, or to mediate skin graft rejection only when HLA-primed in vivo before their adoptive transfer into SCID mice. In addition, T cells and other leukocyte subsets engraft quite poorly in the lymphoid tissues of the animals. In an attempt to develop a useful model for transplantation research, we have inoculated SCID mice with fresh human splenocytes from cadaveric organ donors (hu-Spl-SCID mice). In this model, various leukocyte subsets engraft effectively in different lymphoid compartments. In addition, human T cells retain their proliferative responses to mitogens and to alloantigens when tested 3 wk after engraftment into SCID mice. Finally, mice engrafted with unprimed human spleen cells acutely reject human foreskin allografts. Treatment of hu-Spl-SCID mice with OKT3, an immunosuppressive mAb directed against the human CD3 complex associated with the TCR, prevents the rejection of most human skin allografts, indicating a major role for human T cells in this phenomenon. Thus, this hu-Spl-SCID model may be useful for the study of immunosuppressive therapies in a preclinical in vivo setting.
Thymic stroma exposed to arylhydrocarbon receptor-binding xenobiotics fails to support proliferation of early thymocytes but induces differentiation.
Authors: Kremer J , Gleichmann E , Esser C
Published: December 2022

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 3,3',4,4'-tetrachlorobiphenyl (TCB), two ubiquitous environmental pollutants, accelerate thymocyte maturation, and eventually lead to thymus atrophy. These processes are mediated by binding of TCDD or TCB to the cytosolic arylhydrocarbon receptor (AhR) abundant in the thymus, which acts as a ligand-activated transcription factor. At several stages of their maturation thymocytes need the interaction with thymus stroma. We tested whether thymocytes themselves or the thymus stroma are targets of AhR-binding compounds for interference with thymocyte maturation. We depleted fetal thymus lobes from proliferating cells, i.e., thymocytes, by treatment with deoxyguanosine and recultivated them with immature thymocytes (CD4-CD8-), exposing either the stroma or the thymocytes to TCDD or TCB before recultivation. Although CD4-CD8- immature thymocytes could differentiate in TCB-treated stroma, expansion of the cells was severely impaired. Selective exposure of thymocytes to AhR-binding compounds likewise did not impair the capacity of differentiation of CD4-CD8- thymocytes. These cells, however, could expand when transferred into new lobes that had not been exposed to TCDD or TCB. TCB treatment of fetal thymi leads to an accumulation of phenotypically mature CD4-CD8+ cells. We show here that these cells do not belong to the transient CD4-CD8+ thymocytes, as previously suggested, because in recultivation experiments they do not give rise to any thymocyte subset further down the maturation pathway.
Establishment and characterization of nurse cell-like clones from human skin. Nurse cell-like clones can stimulate autologous mixed lymphocyte reaction.
Authors: Iwagami S , Furue S , Toyosaki T , Horikawa T , Doi H , Satomi S , Itoh T , Sakata T , Suzuki R
Published: December 2022

We have established nurse cell-like clones from long-term cultures of the human skin. These human skin nurse cell (HSNC)-like clones were type I collagen+, type IV collagen-, vimentin+, cytokeratin-, CD44+, CD54+, and weakly positive for VCAM-1, and easily identified by the pseudoemperipolesis that allowed T lymphocytes to migrate beneath the HSNCs. HSNCs and various T cell lines formed a typical complex in the hanging drop culture system. The majority of human and murine T cells, and some of the tumor cell lines other than T cells, including B lymphoma and myeloblastoma cells, migrated beneath the HSNC clones. HSNC clones produced various cytokines, including IL-6, IL-7, IL-8, IL-9, granulocyte CSF (G-CSF), granulocyte-macrophage CSF (GM-CSF), macrophage CSF (CSF-1), TGF-beta 1, and c-kit ligand, but could not produce IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, TNF-alpha, or TNF-beta. These characteristics were similar to those of nurse cells established from the murine thymus. Furthermore, IFN-gamma-pretreated HSNC clones that expressed MHC class II Ags induced autologous mixed lymphocyte reaction (AMLR) in autologous PBMCs to proliferate and exhibit the cytotoxicity against altered autologous cells and various tumor cells. These results suggest that HSNCs play an important role in the immunoregulation at skin tissues.
Staphylococcal protein A induces biased production of Ig by VH3-expressing B lymphocytes.
Authors: Kristiansen S V , Pascual V , Lipsky P E
Published: December 2022

Staphylococcal protein A (SPA) is known to bind preferentially to the VH3 family of Ig heavy chain variable gene products. The current studies were conducted to examine the functional capacity of SPA to induce Ig production by VH3-expressing human B cells preferentially. Human peripheral blood B cells stimulated with anti-CD3-activated T cells or the Wood 46 strain of Staphylococcus aureus, which lacks SPA, expressed IgM containing all VH families, as detected by reverse transcription-PCR. In contrast, stimulation with SPA containing S. aureus or SPA-Sepharose resulted in biased expression of VH3 containing IgM. Similarly, cord blood B cells that require costimulation for Ig production induced by anti-CD3-activated T cells produced only VH3 containing IgM on costimulation with SPA containing S. aureus. The sequences of 21 VH3 IgM gene products derived from different B cell sources were determined and found to include at least nine members of the VH3 family, which were both in germ-line configuration and somatically mutated. Use of DH and JH was diverse. Analysis of these VH3 gene products revealed conserved residues in FR1 and 3'CDR2/FR3, which are candidates to play a role in SPA-mediated activation of VH3-expressing B cells. Like T cell superantigens, SPA probably interacts with residues in the partially solvent exposed regions of the heavy chain molecule outside the classical Ag binding site and provides a signal that can lead to Ig production by human B cells expressing VH3.
Studies with MHC-deficient knock-out mice reveal impact of both MHC I- and MHC II-dependent T cell responses on Listeria monocytogenes infection.
Authors: Ladel C H , Flesch I E , Arnoldi J , Kaufmann S H
Published: December 2022

Mutant mice with a defined genetic defect in the beta 2-microglobulin (beta 2m) or the H2-I-A beta chain, which are virtually devoid of functional CD8 or CD4 alpha beta T cells, respectively, were employed for analyzing immune mechanisms involved in acquired resistance against Listeria monocytogenes. Although the lethal dose of L. monocytogenes was markedly lower for either mouse mutant as compared with their heterozygous control littermates, both beta m -/- and A beta -/- mutants were able to resolve low dose infection. However, in both mouse mutants, the course of disease was exacerbated and clearance was markedly delayed. Vaccine induced immunity against a secondary high dose infection lethal for naive animals was also impaired in beta 2m -/- and A beta -/- mice. However, both mutant mice were still capable of controlling secondary infection. Based on numbers of L. monocytogenes organisms in spleens, beta 2m -/- mutants suffered more dramatically from primary and secondary infection than A beta -/- mice. Ag-induced IFN-gamma secretion was impaired during the early phase of infection in beta 2m -/- mice and at later stages in A beta -/- mice. Modulation of gamma delta T cells by mAb treatment led to significant increase in bacterial load of spleens in both beta 2m -/- and A beta -/- mice. Finally, the development of granulomatous lesions was markedly affected in both mutants. In beta 2m -/- mutants, infiltrative lesions appeared and in A beta -/- mice few inflammatory islets with necrotic centers developed. These data demonstrate the importance of both MHC I- and MHC II-dependent immune mechanisms in acquired resistance to L. monocytogenes and point to the necessity of a coordinated interaction between CD8 and CD4 alpha beta T cells (and probably gamma delta T cells) in anti-L. monocytogenes resistance.
Hepatic and extrahepatic biosynthesis of complement factor C6 in the rat.
Authors: Brauer R B , Baldwin W M , Wang D , Horwitz L R , Hess A D , Klein A S , Sanfilippo F
Published: December 2022

The relative contributions of liver and bone marrow (BM) constituents to systemic C6 production were compared in a rat model. Liver grafts were transplanted from C6-sufficient PVG (RT1c) rats (PVG (C+)) to profoundly C6-deficient PVG rats (PVG (C-)). C6 levels were restored to 32% within 24 h and reached more than 80% of that of the PVG (C+) donor within 7 days post-grafting, which indicates that the liver is a primary source of systemic C6 production. When livers were transplanted from PVG (C-) to PVG (C+) rats (n = 3), levels of C6 dropped to 42% of pretransplant levels within 24 h and remained between 30 and 40% for more than 100 days after grafting. To determine the source of extrahepatic C6 production, BM was transplanted from PVG (C+) to PVG (C-) rats after total body irradiation. Levels of C6 increased from undetectable levels to 5% of C6 levels of the donor PVG (C+) rat within 60 days. Replenishing PVG (C-) recipients with BM after treatment of recipients with busulfan, which preferentially allows reconstitution with donor myelomonocyte stem cells, resulted in restoration of C activity. Treatment with cyclophosphamide before BM transplantation, which preferentially allows reconstitution of lymphoid stem cells, did not restore hemolytic C activity in PVG (C-) rats. These results were confirmed directly by the successful restoration of C activity with BM depleted of lymphocytes by counterflow centrifugal elutriation from PVG (C+) rats. These in vivo experiments demonstrate that the liver is a primary, but not the sole, source of C6 in the rat and that extrahepatic sources, such as myelomonocytes, and not lymphoid cells in the BM produce a significant amount of systemic C6 in the rat.
CD40 ligand (CD40L) expression and B cell function in agammaglobulinemia with normal or elevated levels of IgM (HIM). Comparison of X-linked, autosomal recessive, and non-X-linked forms of the disease, and obligate carriers.
Authors: Callard R E , Smith S H , Herbert J , Morgan G , Padayachee M , Lederman S , Chess L , Kroczek R A , Fanslow W C , Armitage R J
Published: December 2022

Hyper-IgM syndrome is a rare immunodeficiency characterized by low or absent IgG, IgA, and IgE with normal or elevated levels of IgM. It can occur as an acquired or familial disorder with either X-linked or autosomal modes of inheritance. The X-linked form (HIGM1) is a result of mutations in the CD40 ligand (CD40L) gene, but the defect in non-X-linked forms of the disease (HIM) has not been determined. We show here that CD40L expression on activated T cells from non-X-linked patients can be detected by CD40Fc, 5c8 Mab, and anti-TRAP, whereas activated T cells from HIGM1 patients either had no detectable CD40L (Type I), or stained with anti-TRAP but not CD40Fc or 5c8 (Type II). Activated T cells from obligate carriers varied from low to normal expression of CD40L. B cells from HIGM1 and non-X-linked HIM patients proliferated in response to CD40L. Costimulation of B cells from HIGM1, from sporadic HIM, or from non-X-linked HIM patients with CD40L plus IL-2 resulted in some IgM production, but no significant IgG or IgA. Costimulation with CD40L plus IL-10 resulted in significant IgG and/or IgA secretion by B cells from some HIGM1 patients, but consistently failed to stimulate IgG or IgA secretion by B cells from non-X-linked patients. In addition, costimulation with CD40L and IL-4 failed to induce IgE secretion by B cells from one non-X-linked HIM patient, and induced a weak response in another. These results suggest that patients with non-X-linked forms of HIM may have an intrinsic B cell defect preventing heavy chain switching, which is not related to expression of CD40L.
Induction of T helper cell hyporesponsiveness in an experimental model of autoimmunity by using nonmitogenic anti-CD3 monoclonal antibody.
Authors: Hughes C , Wolos J A , Giannini E H , Hirsch R
Published: December 2022

Autoimmune diseases can be characterized by increases in Th cell activities, suggesting that inhibition of Th cell function might ameliorate autoimmunity. We have recently reported that administration of nonmitogenic anti-CD3 mAb (nmCD3) to nonautoimmune mice can induce long-term Th cell hyporesponsiveness, reflected by reduced IL-2 secretion upon re-exposure to Ag. This study was designed to determine the effects of nmCD3 on autoimmunity by using the murine collagen-induced arthritis model. Treatment of DBA/1 mice with nmCD3 delayed the onset and reduced the severity of arthritis in mice immunized with type II collagen (CII). This effect was not caused by depletion of T cells or modulation of TCR. The observed inhibition of arthritis was not caused by decreased Ab production, as anti-CII titers were not affected. Rather, lymph node cells from CII-immunized mice treated with nmCD3 were hyporesponsive to in vitro stimulation with CII. This hyporesponsiveness was reflected by a marked decrease in secretion of IL-2 and IFN-gamma, but not of IL-4, which suggests that nmCD3 had its principal effect on Th1 cells. The hyporesponsiveness was not Ag-specific, because IL-2 and IFN-gamma production in response to a pan-T cell mitogen was also reduced. These results demonstrate that induction of Th1 cell hyporesponsiveness with nmCD3 can significantly alter the course of CIA and suggest that IL-2 and/or IFN-gamma play a crucial role in disease pathogenesis.