bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and characterization of IL-2 hyper-responsive NZB/WF1 CD5- B cells.
Authors: Hirose S , Nakanishi K , Yoshimoto T , Kashiwamura S , Hiroishi K , Matsumoto H , Hamaoka T , Shinka S , Higashino K
Published: December 2022

The responsiveness of CD5- and CD5+ B cells of BALB/c and NZB/WF1 mice to various cytokines was examined with respect to their growth and differentiation. BALB/c splenic CD5- B cells required longer incubation with IL-2 or pretreatment with IL-4 to respond to IL-2 by DNA synthesis, whereas NZB/WF1 CD5- B cells were highly competent to IL-2. Flow cytometric analysis demonstrated that NZB/WF1 and BALB/c CD5- B cells had higher and intermediate proportions of B cells positive for IL-2R beta, respectively. On the other hand, BALB/c and NZB/WF1 splenic CD5+ B cells consisted of lower proportion of B cells positive for IL-2R beta than did their corresponding CD5- B cells and grew meagerly in response to IL-2. Peritoneal exudative CD5+ B cells of NZB/WF1 mice lacked IL-2R beta mRNA expression and failed to respond to IL-2. Although both BALB/c and NZB/WF1 CD5- B cells pretreated with anti-IgM, IL-4, and IL-5 responded to IL-2 by DNA synthesis, only BALB/c CD5- B cells developed into IgM-producing cells. Furthermore, BALB/c, but not NZB/WF1 CD5-, B cells pretreated with anti-IgM and IL-5 responded to IL-2 by IgM production without DNA synthesis. Thus, cross-talk between IL-4 and IL-2 operated in the growth responses of both BALB/c and NZB/WF1 splenic CD5- B cells, whereas cross-talk between IL-5 and IL-2 operated only in the differentiation of BALB/c CD5- B cells, providing us with another intriguing functional abnormality of NZB/WF1 B cells.
Targeted lymph node immunization with simian immunodeficiency virus p27 antigen to elicit genital, rectal, and urinary immune responses in nonhuman primates.
Authors: Lehner T , Bergmeier L A , Tao L , Panagiotidi C , Klavinskis L S , Hussain L , Ward R G , Meyers N , Adams S E , Gearing A J
Published: December 2022

A s.c. route of immunization was developed in non-human primates, which targets the genitourinary-rectal associated lymphoid tissue. A vaccine consisting of rSIV gag p27, expressed as hybrid Ty virus-like particles (p27: Ty-VLP) was administered in the proximity of the internal iliac lymph nodes. Secretory IgA and IgG Abs to the p27 Ag were elicited in the vaginal, male urethral, rectal and seminal fluids, urine and serum. Two or more immunodominant B cell epitopes were identified within peptides 51-90 and 121-170 of the sequence of p27, using serum or biliary IgA and IgG Abs. CD4+ T cell proliferative responses to p27 were elicited predominantly in the targeted internal iliac, as well as the inferior mesenteric lymph nodes and the spleen, but not in the unrelated lymph nodes. These cells were then studied for helper function in p27 specific B cell Ab synthesis. Specific IgA and IgG Abs were detected in the same lymphoid tissues as those that displayed proliferative responses. However, cross-over reconstitution experiments between splenic and iliac lymph node B and CD4+ T cells suggest that the iliac B cells are essential for specific IgA Ab synthesis, whereas splenic B cells preferentially synthesize IgG Ab. The targeted lymph node (TLN) route of immunization gave comparable B cell, proliferative T cell, and Th cell responses to the vaginal, male genitourinary, and rectal mucosal routes, which were augmented by oral immunization. However, the TLN route induced urinary and seminal fluid sIgA and IgG Abs in addition to genital and rectal Abs. Generating secretory IgA and IgG Abs at the mucosal surfaces, and T and B cell immunity in the regional draining lymph nodes, spleen and circulation by TLN immunization may prevent transmission of virus through the mucosa, dissemination of the virus, and the formation of a latent reservoir of infection.
Treatment with dextran-conjugated anti-IgD delays the development of autoimmunity in MRL-lpr/lpr mice.
Authors: Shirai A , Aoki I , Otani M , Mond J J , Klinman D M
Published: December 2022

The onset of clinical disease in autoimmune MRL-lpr/lpr mice is preceded by a switch from predominantly IgM production to IgG production. Previous studies have shown that IgG autoantibodies play a central role in the development of life-threatening glomerulonephritis in this strain. Delaying or preventing the switch from IgM to IgG production might therefore be of therapeutic benefit. We previously documented similarities in the B cell repertoire expressed by young MRL-lpr/lpr mice and normal mice treated with the polyclonal activator LPS. Recent in vivo studies indicate that cross-linking membrane IgM or IgD can suppress LPS-dependent IgG production in normal animals. These observations led us to examine whether membrane cross-linking could also lower serum IgG levels in MRL-lpr/lpr mice. Lupus-prone animals were treated with multivalent anti-IgD conjugated to high m.w. dextran. This anti-IgD dextran conjugate was previously shown to reduce IgG production in LPS-stimulated normal animals. Treatment of young lupus-prone MRL-lpr/lpr mice resulted in a significant reduction in the total number of B cells secreting IgG and lower serum titers of IgG anti-DNA and IgG anti-histone autoantibodies. Anti-IgD dextran treatment also delayed the development of glomerulonephritis and improved survival. Thus, anti-IgD dextran interfered with autoantibody-dependent disease progression, perhaps by inhibiting the switch from IgM to IgG autoantibody production.
Epstein-Barr virus induces actin polymerization in human B cells.
Authors: Melamed I , Stein L , Roifman C M
Published: December 2022

The EBV selectively infects human and primate lymphocytes. This selective tropism occurs as a result of live virus infection through permissive membrane receptors. Once EBV has entered the cell, it induces proliferation and immortalization of these cells. The mechanism of EBV infection, however, remains largely unknown. We demonstrate here that a transforming strain, but not a nontransforming strain, of EBV stimulates the conversion of globular actin (G-actin) to filamentous actin (F-actin), a process that has been associated with activation and transformation of other cell types. Preincubation of B cells with botulinum C2 toxin or cytochalasin, which block the conversion of G-actin to F-actin, resulted in the inhibition of EBV-induced proliferation. These findings indicate that actin rearrangement is essential for infection of B cells by EBV.
CD1+ human thymocytes proliferate in response to superantigen staphylococcal enterotoxin B1.
Authors: Todd S C , Baccala R , Hedrick J A , Theofilopoulos A N , Tsoukas C D
Published: December 2022

Exposure of human thymocytes to superantigens results in the deletion of thymocytes expressing specific TCR-V beta genes. The factors that contribute to this deletion may relate to the inherent nature of the T cell at a given stage of development. In this paper, we demonstrate that CD1+ human cortical thymocytes are capable of proliferating in response to a bacterial superantigen (staphylococcal enterotoxin B (SEB)) in the presence of autologous CD2-/low thymic APCs. Phenotypic analysis of the responding populations revealed that the majority of the CD1+ cells were CD4+CD8low or CD8+CD4low cells. The response is triggered by low concentrations of SEB, requires the participation of the TCR and IL-2R molecules, and is inhibited by cyclosporin A. Thymocytes that express specific V beta genes are expanded, which results in an engagement profile that parallels that found in PBLs. Additionally, four V beta-chains that have not been reported previously are shown to engage SEB. Once stimulated, the thymocytes failed to respond to additional SEB; however, they could be induced to proliferative with IL-2, which suggests that these expanded populations had become anergic. These data represent the first demonstration of a human cortical thymocyte subpopulation that responds to superantigen by proliferation and subsequent anergy.
Effect of aging on murine macrophages. Diminished response to IFN-gamma for enhanced oxidative metabolism.
Authors: Ding A , Hwang S , Schwab R
Published: December 2022

The ability of macrophages to secrete reactive oxygen intermediates, as well as reactive nitrogen intermediates, correlates closely with their capacity to perform two critical effector functions: intracellular killing of microorganisms and lysis of tumor cells. In this study, age-associated changes in the ability of caseinate-elicited peritoneal macrophages to release hydrogen peroxide were determined. Macrophages from aged BALB/c mice produced 50% less hydrogen peroxide than those from young mice in response to PMA or opsonized zymosan. In contrast, the production of macrophage-activating cytokines including IFN-gamma was not diminished in splenocyte supernatants from the aged group. Furthermore, no difference was detected in surface expression of IFN-gamma receptor in old and young mice. Macrophage responses to IFN-gamma, however, declined with aging. In vitro, IFN-gamma-induced release of hydrogen peroxide and nitric oxide was 50% lower in old mice than in young mice. IFN-gamma-induced tyrosine phosphorylation of MAPK, an early activation event, was undetectable in macrophages from the aged mice. These data demonstrate that diminished responses of macrophages to activating signals are one aspect of the impaired immune response in aged mice.
Natural autoantibody against apolipoprotein A-I. Detection and characterization of the monoclonal antibody established from normal unimmunized BALB/c mice.
Authors: Imai H , Suzuki S , Uchida K , Kikuchi K , Sugiyama H , Kohno H , Umeda M , Inoue K
Published: December 2022

During the course of studying the immunogenicity of soybean lipids, we observed frequent production of the mAbs that bind to apolipoprotein A-I (apoA-I) when spleen cells from unimmunized normal BALB/c mice were employed for fusion. Of the 986 colonies from six fusions, 38 (3.9% of the total) were directed against apoA-I and 13 mAbs (IgM) were established for further analysis. The following lines of evidence indicate that this family of mAb may form a novel family of natural autoantibodies against apoA-I: 1) The mAbs were shown to bind effectively to high density lipoprotein from various species, including BALB/c mouse, and immunoblotting analyses revealed that the mAbs bound specifically to the 28-kDa protein of high density lipoprotein. 2) The 28-kDa protein was purified to homogeneity and identified as apoA-I by amino acid sequence analyses and by its cross-reactivity with a xenogenic anti-apoA-I mAb (clone A/11). 3) Differing from the xenogenic anti-apoA-I mAb, the present mAb did not bind to native apoA-I, whereas an effective binding was observed only when the apoA-I had formed a complex with neutral lipids containing polyunsaturated fatty acids such as trilinoleoylglycerol and 5-cholesten-3 beta-ol 3-linoleate. 4) Sera from unimmunized BALB/c mice had readily detectable Abs against apoA-I and the majority of the serum autoantibodies were of the IgA and IgM isotype. 5) The anti-apoA-I mAbs displayed a functional heterogeneity in their reactivity with polyanionic substances and some of the mAbs established showed an extensive cross-reaction with polyanionic substances such as ssDNA and cardiolipin.
B cell Ag receptor mediates different types of signals in the protein kinase activity between immature B cell and mature B cell.
Authors: Igarashi H , Kuwahara K , Nomura J , Matsuda A , Kikuchi K , Inui S , Sakaguchi N
Published: December 2022

Ig receptor (IgR) on the surface of B cells mediates the Ag-specific stimulatory signal for B cell proliferation and differentiation. In immature B cells, the stimulatory signal causes an inhibitory effect which is believed to be a key phenomenon in B cell tolerance or B cell anergy. Here, we studied the molecular mechanism of the inhibitory response of the IgR-mediated signal transduction that results in the programmed cell death of immature B cells. To analyze the downstream molecules of the IgR-mediated signal transduction, we prepared a mAb against a 160-kDa membrane protein (p160) that can coprecipitate the kinase molecule(s) acting on serine, threonine, and tyrosine residues. Anti-IgR stimulation induces the increase of the kinase activity coprecipitated with the p160 protein in mature B cell BAL17 and normal adult spleen B cells. This result suggest that the p160-associated kinase activity is one of the downstream events of the IgR-mediated signal transduction cascade. Interestingly, immature B cell lymphoma WEHI-231 and the neonatal spleen B cells showed the adverse reaction of the p160-associated kinase which results in the transient loss of the kinase activity. Moreover, the transient decrease of the p160-associated kinase was caused by the tyrosine phosphatase activity induced by the stimulation of IgR in WEHI-231. The results suggest that this molecular difference in the downstream events of the IgR-mediated signal transduction between immature B cells and mature B cells already begins at the transmembrane level in the IgR-mediated signal transduction pathway.
Co-receptor (CD4/CD8) engagement enhances CD3-induced apoptosis in thymocytes. Implications for negative selection.
Authors: McConkey D J , Fosdick L , D'Adamio L , Jondal M , Orrenius S
Published: December 2022

Negative selection of self-reactive immature T cells is mediated by TCR engagement and is thought to occur via apoptosis (programmed cell death). The requirement for the co-receptors CD4 and CD8 in negative selection has been demonstrated, but the biochemical mechanisms underlying their involvement in this process remain undefined. Here we present evidence that co-receptor engagement dramatically enhances CD3-induced endonuclease activation and cell death characteristic of apoptosis in immature thymocytes. The responses are associated with increased tyrosine phosphorylation of a number of cellular substrates, including the gamma isoform of phospholipase C, and with increased association of tyrosine phosphoproteins, including the protein tyrosine kinase p56lck, with the TCR complex. Co-receptor engagement also potentiated CD3-mediated Ca2+ increases via a mechanism dependent upon tyrosine kinase activation. Sustained Ca2+ availability was found to be necessary for endonuclease activation and apoptosis to occur. We suggest that CD4 and CD8 may participate in negative selection by enhancing TCR/CD3-induced tyrosine kinase activation and sustained Ca2+ increases that lead to endonuclease activation and apoptosis in self-reactive CD4+ CD8+ thymocytes.
CD59 costimulation of T cell activation. CD58 dependence and requirement for glycosylation.
Authors: Menu E , Tsai B C , Bothwell A L , Sims P J , Bierer B E
Published: December 2022

In addition to the TCR-CD3 complex, T lymphocytes can be activated via another surface glycoprotein, the CD2 molecule. CD58 is the principal ligand for human CD2; CD59 and CD48 are two additional, low affinity ligands that have been defined for CD2. In this study, we have explored the role of CD59 in T cell activation. We have expressed human rCD58 and rCD59 molecules in Chinese hamster oocytes (CHO), and tested paraformaldehyde-treated transfectants for the ability to promote proliferation of and IL-2 secretion from PBMC and human purified T cells. We have shown that CD59 enhanced CD58-dependent T cell proliferation and IL-2 secretion in the presence of suboptimal concentration of PHA or a submitogenic combination of stimulatory anti-CD2 mAbs, presence of suboptimal concentration of PHA or a submitogenic combination of stimulatory anti-CD2 mAbs, T11-2 + T11-3. CD59-dependent costimulation was dependent on several factors including the level of co-expression of CD58, the ratio of CHO cell transfectants to T cells added in culture, the concentration of mitogen, and also donor-dependent differences. As expected, CD59 costimulation of CD58-dependent T cell proliferation was inhibited by Abs directed against CD59, CD58, and CD2. In our hands, the CD59 molecule itself, in the absence of CD58, was unable to support proliferation alone even in the presence of exogenous recombinant IL-1, IL-2, or IL-6. Finally, the ability of CD59 to enhance CD58-dependent T cell responses was shown to be dependent on N-glycosylation of CD59 at amino acid Asn18.
The role of the Fas lytic pathway in a perforin-less CTL hybridoma.
Authors: Walsh C M , Glass A A , Chiu V , Clark W R
Published: December 2022

The murine CTL hybridoma PMMI has been shown by the most sensitive techniques to be devoid of perforin. We thus used PMMI activated with PMA and ionomycin, to investigate possible alternate lytic pathways in CTLs in the absence of perforin. We found that PMMI is equipped with membrane TNF-alpha as a potential lytic mechanism, but TNF-alpha is unlikely to be involved in acute (4 h) lytic reactions. On the other hand, PMMI readily lyses target cells expressing the gene for the Fas Ag, but does not lyse target cells expressing fas antisense DNA. The generation of fas-dependent lysis required protein synthesis in PMMI, but target cell protein synthesis was not required for lysis. Lysis of Fas-positive target cells by PMMI was accompanied by DNA fragmentation, and both lysis and DNA fragmentation were blocked by inhibition of protein synthesis in the effector cell. We find the relative extent and kinetics of fas-dependent lysis and DNA fragmentation indistinguishable from that seen in "classical" CTL lytic assays. Both fas- and perforin-dependent lysis were blocked by inhibitors of poly(ADP) ribosylation. We found very little difference in the sequence of events in target cells lysed by the fas pathway compared with the classical (probably perforin) lytic pathway. Given the widespread distribution of fas, particularly in hematopoietic target cells, caution may be required in interpreting the relationship between parameters such as DNA fragmentation and 51Cr-release solely on the basis of the granule exocytosis model.
Secretion of TNF from a rat mast cell line is a brefeldin A-sensitive and a calcium/protein kinase C-regulated process.
Authors: Baumgartner R A , Yamada K , Deramo V A , Beaven M A
Published: December 2022

Stimulated mast cells and cognate cultured cell lines produce and secrete a variety of cytokines including TNF. Because the mechanism by which cytokines are delivered to the external milieu is unknown, the release of TNF was studied in a rat mast cell line (RBL-2H3 cells). In these cells, TNF was not constitutively expressed nor incorporated into secretory granules but was generated de novo upon cell stimulation. It was then released by a process analogous to constitutive secretion in that brefeldin A, an agent known to disrupt Golgi membranes in these cells, inhibited this release without inhibiting release of secretory granules. Unlike constitutive secretion, however, the secretion of TNF was highly regulated by Ca2+ and protein kinase C. Studies with various stimulants and inhibitors indicated that simultaneous mobilization of Ca2+ and activation of protein kinase C were sufficient signals for secretion although optimal production of TNF may be dependent on additional synergistic signals. Because suppression of Ca2+ mobilization or inhibition of protein kinase C alone abrogated TNF secretion, the process may be amenable to therapeutic intervention.
Systemic release and protective role of IL-10 in staphylococcal enterotoxin B-induced shock in mice.
Authors: Florquin S , Amraoui Z , Abramowicz D , Goldman M
Published: December 2022

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that induces the production of several pro-inflammatory cytokines, leading to a self-limited shock. In the present study, we show that SEB also triggers the systemic release of IL-10, an anti-inflammatory and immunosuppressive cytokine. Serum IL-10 was undetectable (< 1000 pg/ml) in control BALB/c mice and rose to 8500 +/- 2850 pg/ml (mean +/- SEM) 4 h after injection of 100 micrograms SEB. Cell depletion experiments and analysis of IL-10 mRNA expression indicated that CD4+ cells played a major role in SEB-induced IL-10 production. Pretreatment of mice with neutralizing anti-IL-10 mAb before SEB challenge did not modify the release of TNF but led to increased and sustained IL-2 and IFN-gamma serum levels. Furthermore, although no lethality occurred in mice injected with SEB and control mAb, injection of anti-IL-10 mAb before SEB resulted in a 30% lethality (p < 0.05). This lethality was completely prevented by anti-IFN-gamma mAb injection, indicating that IFN-gamma plays a crucial role in the increased toxicity of SEB in anti-IL-10 mAb-injected mice. We conclude that SEB induces the production of IL-10 by CD4+ cells in vivo and that endogenous IL-10 plays an important immunoregulatory role in this model by down-regulating IL-2 and IFN-gamma production.
Eosinophil granule proteins increase microvascular macromolecular transport in the hamster cheek pouch.
Authors: Minnicozzi M , Durán W N , Gleich G J , Egan R W
Published: December 2022

By using microscopic, fluorometric, and immunologic methods, we have assessed the effects of eosinophil granule proteins on the microcirculation of the hamster cheek pouch. The plasma clearance of FITC-dextran 150 (FITC-dx 150) was used to quantify macromolecular transport. Topical application of major basic protein (MaBP) at 0.1 and 0.5 nM increased the clearance of FITC-dx 150 from a base line of 591 to 1283 and 1966 nl/60 min/g, respectively. Numerous muscle fasciculations were also observed with the 0.5 nM dose of MaBP. Eosinophil cationic protein (ECP) was as potent as MaBP and caused an increase in the clearance of FITC-dx 150, 0.5 nM eliciting 2156 nl/60 min/g. In contrast, topical application of 0.5 nM eosinophil peroxidase (EPO) increased clearance of FITC-dx 150 to a significantly lower level, 1113 nl/60 min/g. Supplementing 0.5 nM EPO with 1 nM H2O2 enhanced the clearance of FITC-dx 150 to 2404 nl/60 min/g, suggesting separate cationic charge and enzymatic activity-related effects. Compared with these eosinophil granule proteins, eosinophil-derived neurotoxin (EDN) required a 2000-fold higher concentration (1 microM) to elicit a significant increase in the clearance of FITC-dx 150 (1505 nl/60 min/g). Neither EPO, EPO+H2O2, ECP, nor EDN at 1 mM caused muscle fasciculations. Quantitative analysis of the suffusates from the preparations exposed to these eosinophil proteins did not contain histamine. Our results demonstrate that MaBP, ECP, EPO, and EDN increase microvascular transport in the hamster cheek pouch and that this increase is independent of endogenous histamine release. The concentrations of eosinophil granule proteins causing increased vascular permeability are achieved in many pathologic conditions suggesting that the granule proteins play an important role in disease.
Activation of human monocytes by the pineal hormone melatonin.
Authors: Morrey K M , McLachlan J A , Serkin C D , Bakouche O
Published: December 2022

To determine the effects of the pineal hormone melatonin on human monocytes, human monocytes were activated by different concentrations of melatonin. Above the activation threshold of 5 x 10(-11) M, melatonin was able to induce the cytotoxicity of human monocytes, the secretion of IL-1, and the production of reactive oxygen intermediates. Melatonin and LPS seemed to have a synergistic effect on human monocyte activation. Indeed, below their respective monocyte activation threshold (5 x 10(-11) M and 0.625 ng/ml), melatonin (10(-12) M) in association with LPS (0.2 ng/ml) was able to induce cytotoxicity, IL-1 secretion, and reactive oxygen intermediates production. Melatonin alone at 10(-12) M or LPS alone at 0.2 ng/ml did not activate monocytes. Furthermore, melatonin was able to prime the monocytes for a subsequent activation by LPS. When monocytes were activated by LPS (0.25 ng/ml) at the time that they were plated and then activated by melatonin (10(-12) M) 8 h later, no IL-1 secretion and no cytotoxicity were detected. However, when the cells were first activated by melatonin (10(-12) M), and then 8 h later by LPS (0.25 ng/ml), IL-1 secretion and monocyte cytotoxicity were observed. Above its monocyte activation threshold, melatonin induces both cell-associated IL-1 alpha and IL-1 beta activities. Below this activation threshold, i.e., at 10(-12) M, melatonin does not induce the cell-associated IL-1 alpha and IL-1 beta activities, but does induce the mRNA for both IL-1 (alpha and beta). It seems that melatonin activates monocytes through protein kinase C. These data suggest that melatonin activates monocytes and induces their cytotoxic properties, along with the IL-1 secretion.
The chemokines IL-8, monocyte chemoattractant protein-1, and I-309 are monomers at physiologically relevant concentrations.
Authors: Paolini J F , Willard D , Consler T , Luther M , Krangel M S
Published: December 2022

The chemokines are a family of immune mediators involved in a wide range of inflammatory processes, most importantly as chemoattractants of monocytes, neutrophils, lymphocytes, and fibroblasts to sites of inflammation. Nuclear magnetic resonance and x-ray crystallographic studies have shown that IL-8 and macrophage-inflammatory protein-1 beta (MIP-1 beta) form noncovalent dimers and that platelet factor-4 (PF-4) forms noncovalent dimers and tetramers, leading to the assumption that, as a family, the chemokines would form multimeric structures. In this study, we analyze the association states of the chemokines IL-8, monocyte chemoattractant protein-1 (MCP-1), and I-309, by using a combination of size exclusion HPLC, sedimentation equilibrium ultracentrifugation, and chemical cross-linking. We find that the association states of MCP-1 and IL-8 are characterized by an equilibrium between monomers and dimers: although dimers predominate at concentrations above 100 microM, these chemokines are almost exclusively monomeric at the nanomolar concentrations at which they display maximal chemotactic activity. I-309, by contrast, remains a monomer at all concentrations tested. I-309 contains two additional cysteine residues (C26 and C68) that are not found in any other members of the chemokine family. We used cyanogen bromide and trypsin digestion strategies to demonstrate that these two residues are linked in a unique intramolecular disulfide bond. Furthermore, by using site-directed mutagenesis, we show that the integrity of this bond is crucial for protein secretion.
Potential indirect anti-inflammatory effects of IL-4. Stimulation of human monocytes, macrophages, and endothelial cells by IL-4 increases aminopeptidase-N activity (CD13; EC 3.4.11.2).
Authors: van Hal P T , Hopstaken-Broos J P , Prins A , Favaloro E J , Huijbens R J , Hilvering C , Figdor C G , Hoogsteden H C
Published: December 2022

IL-4 up-regulates various monocytic properties that are associated with pro-inflammatory functions. Paradoxically, IL-4 may also act as an anti-inflammatory agent by down-regulating the production of several inflammatory mediators. As the activity of some mediators has recently been shown to be regulated by peptidases, we examined whether IL-4 was able to modulate the expression of a cell membrane-associated peptidase, aminopeptidase-N (CD13). IL-4 caused a dose-dependent increase in the expression of CD13 Ag on highly purified human blood monocytes. Maximal expression was observed around 48 h of culture. This IL-4-induced increase was completely blocked by anti-IL-4 antiserum. Furthermore, the increase in surface expression was preceded by increased mRNA levels of CD13, which was maximal around 24 h of culture. We also observed that CD13-mediated leucine-aminopeptidase activity of monocytes was induced by IL-4. Other CD13-expressing cells were also sensitive to IL-4, as CD13 Ag expression and CD13 mRNA levels were up-regulated in human alveolar macrophages and endothelial cells upon IL-4 treatment. The increased expression of cell membrane aminopeptidase-N represents a potentially increased cellular ability to inactivate inflammatory mediators. Therefore, these findings represent further evidence of IL-4-mediated anti-inflammatory actions. We postulate that up-regulation of aminopeptidase-N expression may be an indirect mechanism of IL-4 to modulate the action of bioactive peptides. This mechanism may underlie, at least partially, the anti-inflammatory effects of IL-4 in vivo.
Primary biliary cirrhosis. High proportions of B cells in blood and liver tissue produce anti-mitochondrial antibodies of several Ig classes.
Authors: Björkland A , Lööf L , Mendel-Hartvig I , Tötterman T H
Published: December 2022

Anti-mitochondrial Abs (AMA) of the M2 type are recognized as being specific for primary biliary cirrhosis (PBC). We developed a highly specific assay to detect single AMA-producing cells using pyruvate dehydrogenase (PDH), the major Ag for AMA. Total Ab and AMA production of in vivo activated B cells was measured in peripheral blood from 13 PBC patients, 22 patients with other liver diseases, and 15 healthy controls. Anti-PDH-producing cells, PDH spot-forming cells (PDH-SFC), were detected in 12 of 13 PBC patients and represented a very high proportion of circulating Ig-producing lymphocytes (9 +/- 8.6%; mean +/- SD). The autoantibodies were mainly of IgG and IgM Ig classes. The number of PDH-SFC was positively correlated with the numbers of total Ig-SFC within each Ig class. An increased number of total IgM SFC in blood was found in PBC patients compared with controls, and IgM SFC correlated with the elevated serum levels of IgM typical for this disease. Among the lymphocytes extracted from PBC liver tissue (n = 5), we detected PDH-SFC of IgG (range 0 to 33% of total SFC), IgM (0 to 42%), and IgA types (1 to 30%). IgA PDH-SFC were found in five of five livers, but only in two of thirteen blood samples investigated. Taken together, our data reveal that very high numbers of B cells spontaneously produce disease-specific autoantibodies in blood and liver tissue in PBC. Furthermore, the Ig class distribution of produced autoantibody differs between these two compartments, and this may have pathogenetic significance.
Monocyte adhesion in patients with bone marrow fibrosis is required for the production of fibrogenic cytokines. Potential role for interleukin-1 and TGF-beta.
Authors: Rameshwar P , Denny T N , Stein D , Gascón P
Published: December 2022

Idiopathic myelofibrosis (IMF) is a hemologic disorder characterized by bone marrow (BM) fibrosis. The BM contains excessive deposits of extracellular matrix proteins and exhibits neovascularization. The fibrosis is hypothesized to be a reactive phenomenon secondary to a clonal myeloid disorder. Growth factors such as platelet-derived growth factor (PDGF), TGF-beta, and epidermal growth factor have been postulated as potential agents involved in BM fibrosis. We studied the induction of two fibrogenic cytokines, IL-1 and TGF-beta, in IMF monocytes. High levels of both cytokines were produced in unstimulated IMF monocytes, compared with background levels produced in normal controls. Most of the TGF-beta produced by IMF monocytes was in its active form. The spontaneous induction of IL-1 alpha, IL-1 beta, and TGF-beta in IMF monocytes parallels an increase in their steady state mRNA. Although high levels of cytoplasmic IL-1 alpha, IL-1 beta, and TGF-beta protein were detected in monocytes that were not subjected to any form of adherence, the secretion of these cytokines required adhesion. High levels of fibronectin, hyaluronic acid, and collagen, all potential ligands for the CD44 adhesion molecule, have been reported in the circulation of IMF patients. However, the Ab-binding capacity of CD44 in IMF monocytes was reduced by 50% when compared with normal controls. Our results indicate that monocytes and adhesion molecules may play a role in the induction of fibrogenic cytokines. These parameters may be important to the pathophysiology of BM fibrosis.
Subset of CD4+ T cell clones expressing IL-3 receptor alpha-chains uses IL-3 as a cofactor in autocrine growth.
Authors: Mueller D L , Chen Z M , Schwartz R H , Gorman D M , Kennedy M K
Published: December 2022

In this paper we demonstrate that IL-3 can act as a cofactor for the growth of some CD4+ T cells. This lymphokine synergized with IL-4 to induce both a unique set of protein tyrosine phosphorylations and the vigorous proliferation of the keyhole limpet hemocyanin-specific and I-Ab-restricted CD4+ Th0 cell clone, E6. In addition, neutralizing anti-IL-3 Abs specifically inhibited the growth of E6 T cells to Ag or anti-CD3 mAb stimulation. Finally, this T cell clone was shown to express both the IL-3R alpha-chain and an IL-3R beta-chain (AlC2A). An examination of other CD4+ T cell clones determined that one Th1 clone (A.E7), two Th0 clones (16B.2 and L9A.1), and one Th2 clone (D10.G4.1) were not influenced by the addition of rIL-3. However, proliferation of the Th2 clones CDC25 and CDC35 to CD3-stimulation was significantly enhanced by IL-3. The sensitivity of these latter two clones to IL-3 was also found to be associated with expression of IL-3R alpha-chains. Because E6 T cells are highly dependent on IL-4 for autocrine growth similar to Th2 cells, these results suggest that IL-3 may synergize with IL-4 to enhance the proliferation of a subset of IL-4-dependent CD4+ T cells, and the study indicates that IL-3R alpha-chain expression may be a specific marker of this CD4+ T cell subset.
Quantitation of L-selectin and CD18 expression on rabbit neutrophils during CD18-independent and CD18-dependent emigration in the lung.
Authors: Burns A R , Doerschuk C M
Published: December 2022

In the systemic circulation, the leukocyte adhesion molecule, L-selectin facilitates the initial adhesion of the neutrophil to the inflamed endothelium, whereas CD11/CD18 is essential to transendothelial migration. Previous work from our laboratory showed that neutrophil emigration in the lung occurs through either a CD18-independent or CD18-dependent mechanism, depending on the inflammatory stimulus. This study quantitated and compared the surface expression of L-selectin and CD18 on neutrophils in the lungs of rabbits during emigration toward Streptococcus pneumoniae (a CD18-independent stimulus) and Escherichia coli endotoxin (a CD18-dependent stimulus). Ultrathin frozen lung tissue sections were immunogold labeled for 1-selectin and CD18, and gold particles were quantitated on intravascular, interstitial, and airspace neutrophils by transmission electron microscopy. The results show that CD18-independent neutrophil emigration was associated with L-selectin down-modulation (78%) and CD18 up-modulation (260%) on intravascular neutrophils, before emigration. A similar alteration in the expression of L-selectin and CD18 was observed during CD18-dependent neutrophil emigration, but only on neutrophils that emigrated into the interstitium and airspace. In emigration induced by either stimulus, alterations in L-selectin and CD18 expression were restricted to the inflammatory focus and emigrated airspace neutrophils consistently expressed greater levels of CD18 than intravascular and interstitial neutrophils. We conclude that before emigration, L-selectin and CD18 expression on intravascular neutrophils is altered only during CD18-independent emigration and only on those neutrophils within the inflammatory focus. The increased CD18 expression on airspace neutrophils may facilitate bacterial phagocytosis.
Antibodies specific for human thyrotropin receptor induce MHC antigen expression in thyroid cells.
Authors: Ropars A , Marion S , Takorabet L , Braun J , Charreire J
Published: December 2022

Autoantibodies (AAbs) to hormone receptors are found in autoimmune diseases such as Graves' disease (GD) or myasthenia gravis. A structural link between hormone receptor and MHC genes has been documented, suggesting a possible co-regulation of MHC and hormone receptor genes. Thus, in vitro experiments were designed to search for a pathologic role for AAbs. In a model study, we investigated whether adding murine anti-human thyrotropin receptor mAbs would affect MHC gene expression in either cloned human thyroid epithelial cell or primary murine thyroid epithelial cell cultures. We found that two anti-human thyrotropin receptor monoclonal AAbs, 11E7 and 34A, induced, with an intensity comparable to that of IFN-gamma, transcription and expression of class I and class II/Ii chain proteins in human and murine thyroid epithelial cells. Two other anti-human thyrotropin receptor mAbs, 12E3 and 243-3, were ineffective. These data suggest a new role for autoantibodies in the pathology of autoimmune endocrinopathies.
Prevention of anti-CD3 monoclonal antibody-induced thymic apoptosis by protein tyrosine kinase inhibitors.
Authors: Migita K , Eguchi K , Kawabe Y , Mizokami A , Tsukada T , Nagataki S
Published: December 2022

The thymus gland is crucial for the formation of thymocytes of diverse TCR specificity. Recent studies have demonstrated that deletion (negative selection) of autoreactive thymocytes occurs through the process of apoptosis in which TCR activates cell death by DNA fragmentation. In addition, in vitro stimulation of thymocytes with anti-CD3 mAb, calcium ionophore, or glucocorticoids results in DNA fragmentation followed by cell death. The availability of various substances capable of inhibiting activation-induced programmed cell death of thymocytes may be used as a tool to help identify several important events occurring during the process of apoptosis. We investigated the effect of protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, on thymocyte apoptosis induced by stimulation of anti-CD3 mAb or glucocorticoid. Anti-CD3 mAb stimulation resulted in removal of CD4+CD8+ thymocytes by DNA fragmentation. However, in PTK inhibitor-pretreated thymocytes, there was a minimal deletion of double positive thymocytes. In contrast, PTK inhibitors did not prevent glucocorticoid-induced thymic apoptosis. Our results suggest that anti-CD3 mAb-induced thymic apoptosis depends on PTK activation via TCR, and that glucocorticoid-induced thymic apoptosis is PTK-independent.
Single IL-2-secreting precursor CD4 T cell can develop into either Th1 or Th2 cytokine secretion phenotype.
Authors: Sad S , Mosmann T R
Published: December 2022

Immunocompetent T lymphocytes in peripheral tissues mainly secrete IL-2 when first stimulated, whereas effector T lymphocytes generated during an immune response secrete different cytokine patterns, such as the Th1, Th2, or other phenotypes displayed by in vitro T cell clones. In this paper, we have examined whether the cell populations that have distinctive cytokine-producing capabilities represent different cell lineages or whether they are derived from the same uncommitted precursor T cell. During allostimulation of CD4+ spleen T cells, TGF-beta inhibited the development of T cells that could produce the Th2 cytokines IL-4 and IL-5. Anti-IFN-gamma inhibited the development of Th1-like IFN-gamma-secreting cells, and the combination of TGF-beta and anti-IFN-gamma resulted in the proliferation of a cell population that produced IL-2, but not IFN-gamma, IL-4, or IL-5, on restimulation. These IL-2-producing T cells expressed low levels of CD45RB and MEL14 and high levels of CD44. Clones of IL-2-producing T cells were isolated, and either bulk culture or clonal IL-2-producing populations acquired the ability to secrete Th1 or Th2 cytokine patterns when restimulated in the presence of TGF-beta or IL-4, respectively. These results demonstrate that both Th1- and Th2-like cells can be derived from a bipotential IL-2-producing precursor CD4+ T cell.
Functional analysis of the serpin domain of C1 inhibitor.
Authors: Coutinho M , Aulak K S , Davis A E
Published: December 2022

To analyze the role of the heavily glycosylated amino-terminal domain of C1 inhibitor in protease inhibitory activity, two truncated C1 inhibitor molecules were constructed. The abilities of the recombinant truncated inhibitors to complex with target proteases were compared with that of the wild-type recombinant protein. One recombinant truncated molecule consisted of amino acid residues 76 to 478 (C-serp(76)) and the other of residues 98 to 478 (C-serp(98)). The recombinant proteins were each expressed in similar quantities. The thermal denaturation profiles of the two truncated proteins were similar to that of the wild-type protein. Identical binding of C1s, C1r, kallikrein, and beta factor XIIa was observed with the three molecules. Furthermore, the truncated molecules also effectively inhibited C1 activity in hemolytic assays. These studies therefore clearly demonstrate that the amino-terminal domain of C1 inhibitor does not influence complex formation with target proteases.
Selective inhibitory effects of the anticoagulant activated protein C on the responses of human mononuclear phagocytes to LPS, IFN-gamma, or phorbol ester.
Authors: Grey S T , Tsuchida A , Hau H , Orthner C L , Salem H H , Hancock W W
Published: December 2022

Recent studies have shown that infusion of the anticoagulant protein, activated protein C (APC), can ameliorate many of the systemic effects of endotoxemia in experimental animals, although the mechanisms in this action are unknown. We investigated the effects of APC on the responses of blood monocytes, alveolar macrophages, and cells of the monocyte line, THP-1, to stimulation in vitro by LPS, IFN-gamma, or PMA. Mononuclear phagocyte (MO) activation was associated with rapid production of TNF-alpha, down-regulation of the glycophosphatidylinositol-linked protein CD14 (the key MO receptor for complexes of LPS and LPS-binding protein responsible for intracellular signaling), and down-regulation of the related LPS-binding proteins CD11b and CD18. Addition of APC, but not the zymogen, PC, or active site-blocked APC, inhibited selected MO responses involving the CD14-dependent LPS-induced pathway of MO activation, or activation induced by IFN-gamma or PMA. Thus, APC inhibited the production of TNF-alpha and prevented down-regulation of membrane CD11b, CD14, and CD18, but had no effect on up-regulation of MHC class II, ICAM-1, or IL-2R, down-regulation of MO expression of another glycophosphatidylinositol-linked protein, CD59, or production of reactive oxygen intermediates. These data show that APC inhibits host cytokine production but maintains MO responses associated with adhesion, phagocytosis, and killing of Gram-negative bacteria, such that use of APC may be a logical and potent adjunctive therapy in select inflammatory diseases involving MO activation and damaging host cytokine overproduction.
Relationship of TSG-14 protein to the pentraxin family of major acute phase proteins.
Authors: Lee G W , Goodman A R , Lee T H , Vilcek J
Published: December 2022

TNF-stimulated gene-14 (TSG-14) encodes a secreted glycoprotein with significant sequence homology to C-reactive protein (CRP) and serum amyloid P component (SAP), members of the pentraxin family of acute phase proteins. TSG-14 mRNA was elevated in human FS-4 fibroblasts by treatment with TNF, IL-1, or bacterial LPS, and weakly by dexamethasone. Abs to recombinant TSG-14 immunoprecipitated a 42-kDa protein from the culture supernatants of TNF- or IL-1-stimulated FS-4 cells. TSG-14 protein was also inducible in the Hep3B human hepatoma cell line by TNF, IL-1, IL-6, or dexamethasone. CRP protein, identified by immunoprecipitation of a 25-kDa band with Abs to CRP, was induced in Hep3B cells by IL-1, IL-6, or dexamethasone. Immunoprecipitations with polyclonal Abs to TSG-14 and CRP suggested that the two proteins are immunologically cross-reactive. Appearance of TSG-14 protein was demonstrated in the serum of mice after injection with LPS. No TSG-14 mRNA was detected in the liver of LPS-injected mice, suggesting that hepatocytes are not the major site of TSG-14 synthesis. Thus, in the intact organism the main cellular sources of TSG-14 and classical acute phase proteins appear to be different.
Stem cell factor is a chemotactic factor for human mast cells.
Authors: Nilsson G , Butterfield J H , Nilsson K , Siegbahn A
Published: December 2022

The mast cell is one of the major effector cells in inflammatory reactions and can be found in most tissues throughout the body. During inflammation, an increase in the number of mast cells can be seen, e.g., in the intraepithelial cell layer after a provoked allergic reaction. Such accumulation probably requires directed migration of mature mast cells or their precursors. To study the migration of human mast cells we used as a model the human mast cell line, HMC-1, and stem cell factor-dependent (also referred to as mast cell growth factor or Kit ligand) cord blood-derived mast cells. The results show that stem cell factor is a potent chemotactic factor for human mast cells in vitro. The chemotactic response to SCF was found to be dose dependent, reaching a maximum at 50 ng/ml. The activity of SCF could be blocked by anti-SCF Abs. We also tested the effect of different intercrines, i.e., IL-8, MIP-1 alpha, MIP-1 beta, RANTES, and MCAF (also referred to as monocyte chemotactic protein 1), on human mast cell migration. Only RANTES was chemotactic for in vitro-developed mast cells. None of the tested intercrines induced migration of HMC-1 cells. For migration, the mast cells were dependent on binding to an extracellular matrix protein. Thus, coating of the filters with fibronectin was required, whereas collagen or laminin did not promote migration. Adhesion of HMC-1 cells to fibronectin could also be shown in an adhesion assay. In addition, expression of receptors for fibronectin could be detected on the surface of the mast cells. These results show that SCF is not only a growth and differentiation factor for human mast cells in vitro but also a potent chemoattractant for such cells.
Expression of a novel autoantibody defined by the VH3-15 gene in inflammatory bowel disease and Campylobacter jejuni enterocolitis.
Authors: Berberian L S , Valles-Ayoub Y , Gordon L K , Targan S R , Braun J
Published: December 2022

This study newly introduces anti-VH mAbs to assess the role of clonal B cell activity in inflammatory bowel disease. Immunohistochemistry of colonic biopsies in ulcerative colitis (UC) and Crohn's disease (CD), but not unaffected individuals, demonstrated uniform staining of intravascular erythrocytes with BK2, a monoclonal specific for the VH3-15 Ig heavy chain gene product. Staining was caused by erythrocytes opsinized in vivo by anti-erythrocyte Abs present in patient sera and by using the VH3-15 gene product. The erythrocyte Ag was identified by immunoprecipitation as 22- and 28-kDa membrane proteins. A direct flow cytometric assay was developed to measure this serum autoantibody and was tested in 101 individuals with UC, CD, other acute or chronic colitis, and healthy controls. Compared with normal subjects, BK2+ anti-erythrocyte Abs were elevated in most sera from patients with CD and UC (including postcolectomy). BK2+ anti-erythrocyte Abs also were elevated in 10 of 38 noninflammatory bowel disease patients, all of whom had Campylobacter jejuni enterocolitis. These findings suggest that a common immunopathogenetic factor, manifested by VH3-15 B cell activation may be shared in UC, CD, and Campylobacter jejuni enterocolitis.