bluuebunny/crossref_metadata_2025 huggingface dataset:
Lymphoma models for B cell activation and tolerance. I. Conditions for the anti-mu-dependent stimulation of growth in NBL, a nude B cell lymphoma.
Authors: Ling M , Livnat D , Pillai P S , Scott D W
Published: January 2023

NBL is a spontaneous B cell lymphoma that originated in NIH Swiss nude mouse, and has been maintained as an in vitro line for 4 yr in our laboratory. It is surface IgM positive and expresses several B cell markers including Fc receptors, as well as Ly-1. Although clones of NBL will grow in serum-containing medium, this cell line enters a quiescent state in serum-free culture. However, in the presence of affinity-purified or monoclonal anti-mu reagents, NBL increases its rate of proliferation as measured by thymidine incorporation or in absolute cell numbers. This stimulation is specific for mu-chain, because it does not occur with anti-beta 2 microglobulin, irrelevant nonbinding antibodies, or with monoclonal anti-B cell lineage markers. Bivalent anti-mu is required, and no consistent Fc-mediated inhibition of growth has been detected. Stimulation of NBL occurs optimally at critical cell densities (greater than 3 X 10(3)/well) in the absence of serum. Therefore, we reasoned that NBL either produced or was receptive to known B cell growth factors. Although no classic IL 1 was detected in NBL supernatants, some BCGF-I-like activity was found. Finally, in the presence of LPS, both spontaneous and anti-mu-stimulated NBL growth was inhibited, a result suggesting maturation of this lymphoma. These results suggest that NBL represents an excellent model to study the growth and differentiation of B cell subsets.
A monoclonal antibody to a differentiation antigen present on mature human macrophages and absent from monocytes.
Authors: Zwadlo G , Bröcker E B , von Bassewitz D B , Feige U , Sorg C
Published: January 2023

A monoclonal antibody is described that has been generated in the mouse against cultured human blood monocytes/macrophages. The antibody, designated 25F9, belongs to the IgG1 subclass, detects antigens of m.w. 86,000, and does not react with freshly isolated blood monocytes but reacts with monocytes after 3 days of culture. The expression of the 25F9 antigen on macrophages increases with culture time. Furthermore, the antibody is negative on platelets, granulocytes, lymphocytes, and a large number of human cell lines except the two melanoma lines MeWo and Mel 57. In cryostat sections of normal human tissue (skin, lung, liver, thymus) and of inflammatory or neoplastic tissue (cutaneous lymphoma, eczema, BCG-granuloma, and melanoma), the antibody reacts with scattered macrophages in the dermis but not with epidermal Langerhans cells, with alveolar macrophages, with liver Kupffer cells, and with scattered macrophages in the cortex and medulla of thymus. In eczema, BCG-granuloma, and cutaneous lymphoma, only a few infiltrating macrophages were stained. On the other hand, a large number of macrophages and melanophages reacted positively in melanoma. In some cases melanoma cells also stained weakly positive. Thus, the antibody detects a differentiation antigen preferentially expressed on mature, tissue-fixed macrophages and absent from blood monocytes.
In vivo activity of affinity-purified helper factor from antigen-specific helper clone.
Authors: Azar Y , Falek P R , Kagan J , Ben-Sasson S Z
Published: January 2023

Supernatant from culture of a virally transformed OVA-specific helper T clone (C-41) was examined for the presence of soluble helper factor. Inoculation of helper clone supernatant into DNP-KLH-primed mice enhanced the IgG anti-DNP response when given with DNP-OVA. The C-41 supernatant did not trigger the DNP-primed B cells in mice when injected with hapten (DNP) coupled to an unrelated carrier (BSA). The carrier-dependent helper activity of C-41 supernatant in vivo demonstrates the presence of an antigen-specific T helper factor in the media of the cultured helper clone. Extensive immunization of F1(C57BL X BALB/c) mice with the helper clone resulted in the production of anti C-41 antibodies. Monoclonal antibodies prepared from the immunized mice were screened for specificity of binding to other transformed T lines and clones, some specific to OVA. Monoclonal antibodies that stained the C-41 cells exclusively were considered clone-specific. Supernatants of the helper clone were passed over columns of anti-clone-specific antibodies. The eluates from three antibodies were active as antigen-specific helper factor, i.e., they elevated the IgG anti-DNP response in vivo in a linked recognition fashion in the presence of DNP-OVA. The affinity-purified factor was inactive when injected with DNP-BSA or DNP-BSA + OVA. Thus, we describe the antigen-specific immune function of a clone-produced helper factor in normal mice.
Human eosinophil peroxidase: purification and characterization.
Authors: Carlson M G , Peterson C G , Venge P
Published: January 2023

Human eosinophil peroxidase (EPO) was isolated from granules from granulocytes of a patient with hypereosinophilia. The granules were extracted by means of 0.2 M NaAc, pH 4.0. The purification steps included gel filtration chromatography on Sephadex G-75 superfine and ion-exchange chromatography on CM-Sephadex G-50. The purified protein showed one band on agarose-electrophoresis, a high peroxidase activity, and a 415-nm/280 nm ratio of 1.15. After reduction, EPO showed two bands on SDS-PAGE of m.w. 52,000 and 15,000, respectively. On gel filtration, the unreduced protein had a m.w. of approximately 77,000. Amino acid analyses showed a high content of arginine and aspartic acid. Monospecific antibodies to EPO were prepared in rabbits, and a specific radioimmunoassay was developed. There was an almost linear correlation between the content of EPO measured by the radioimmunoassay and the number of eosinophils in a mixed cell extract from reference material, indicating the eosinophil origin of EPO. The content of EPO was estimated to be 15.0 micrograms/10(6) eosinophils.
Oxidant membrane injury by the neutrophil myeloperoxidase system. II. Injury by stimulated neutrophils and protection by lipid-soluble antioxidants.
Authors: Sepe S M , Clark R A
Published: January 2023

The stimulated human neutrophil can damage a variety of target cells, and in some models, a mechanism involving secretion of myeloperoxidase and H2O2 has been demonstrated. We explored the characteristics of this cell-cell interaction by using neutrophils and our recently described liposome model target cell system. Exposure of 51Cr-labeled liposomes to phorbol myristate acetate-stimulated human neutrophils resulted in release of 25 to 30% of the radioactivity. 51Cr release was abrogated by omission of the neutrophils, the phorbol ester or halide (iodide), replacement of the phorbol by an inactive congener, or addition of azide, cyanide, or catalase. Neutrophils from patients with hereditary absence of myeloperoxidase (MPO) or a failure of H2O2 formation (chronic granulomatous disease) did not cause liposome lysis unless purified MPO or a source of H2O2, respectively, was added. These data indicate that 51Cr release from liposomes is a consequence of the secretion of MPO and H2O2, which combine with extracellular halides to form a membrane lytic system. The influence of liposome composition on injury was then examined, with a focus on physiologically relevant lipid soluble antioxidants. Liposomes containing either alpha-tocopherol (0.33 to 1.67% of molar fraction of lipid) or beta-carotene (1.67% of molar fraction of lipid) were markedly resistant to lysis by the cellfree MPO-H2O2-chloride system. When the major structural lipid phosphatidyl choline was replaced by dipalmitoyl phosphatidyl choline, a synthetic phospholipid with no oxidizable double bonds, the resultant liposomes were totally resistant to lysis by the MPO-H2O2-chloride system. The addition of iodide to this system (i.e., both chloride and iodide present) changed the pattern of protection dramatically in that alpha-tocopherol and beta-carotene were no longer protective and the resistance of dipalmitoyl phosphatidyl choline liposomes was partial rather than complete. In contrast to iodide, the addition of bromide or thiocyanate did not have a major effect on the protection by antioxidants. Finally, we demonstrated protection by alpha-tocopherol or dipalmitoyl phosphatidyl choline against liposome lysis by phorbol-activated neutrophils. These studies illustrate the use of model phospholipid membranes in the characterization of oxygen-dependent cell-mediated cytotoxicity. Activated neutrophils lyse liposome targets through a MPO-dependent mechanism. Target properties, especially the content of lipid-soluble antioxidants, have a marked influence on susceptibility to lysis.(ABSTRACT TRUNCATED AT 400 WORDS)
Selective localization of tumor-immune spleen cells at the tumor challenge site after adoptive transfer of line 10 tumor immunity in strain 2 guinea pigs.
Authors: de Jong W H , van de Plas M M , Steerenberg P A , Kruizinga W , Ruitenberg E J
Published: January 2023

In this study, the distribution of immune spleen cells was investigated after adoptive transfer of immunity in inbred strain 2 guinea pigs. Spleen cells obtained from line 10 immune donor animals became specifically restimulated in vitro with 3 M KCl-extracted line 10 soluble proteins, but not with 3 M KCl-extracted line 1 or liver proteins. After 4 days culture in vitro, these specifically restimulated immune spleen cells retained their antitumor activity in vivo after adoptive transfer. The specifically restimulated immune spleen cells were radiolabeled with [3H]thymidine, 1 X 10(8) viable cells were adoptively transferred in tumor-bearing guinea pigs, and their distribution was investigated. As controls for the specific localization of the immune cells at the line 10 tumor, the presence of labeled cells was studied in the contralateral transplanted line 1 hepatoma as well as in cellular inflammatory reactions elicited by injection with incomplete Freund's adjuvant (IFA) and complete Freund's adjuvant (CFA). A significantly higher localization of the labeled immune spleen cells in the line 10 tumor and the first and second draining lymph nodes of the line 10 challenge site were found when compared to the influx of these cells in the line 1 tumor and the nontumor antigen-related inflammatory reactions. Because our immune donor animals were immunized with a mixture of line 10 cells and BCG, these animals are immune to both. Line 10 immune spleen cells were restimulated in vitro with PPD and were radiolabeled. These PPD-restimulated immune spleen cells showed no preferential localization at the line 10 tumor challenge site but, as expected, a tendency for localization at the CFA (H37Ra) injection site. Furthermore, PPD-reactive spleen cells from BCG-immunized guinea pigs showed a significantly higher accumulation at the CFA injection site compared to the IFA injection site and the line 10 and line 1 tumor challenge site. From the results, it is concluded that line 10 tumor-immune and BCG-immune spleen cells are two distinct cell populations, and that the existence of cross-reacting antigens between BCG and the line 10 hepatocarcinoma are of no importance for the rejection of the line 10 tumor by immune spleen cells.
Antigen-induced proliferation and immunoglobulin A secretion by a human-human hybridoma.
Authors: Splawski J B , Lipsky P E
Published: January 2023

The capacity of membrane immunoglobulin A (IgA)-bearing B cells to respond to specific antigen in the absence of T cell influences has not been defined. A human-human hybridoma, constructed from an Epstein-Barr virus transformed tonsil B cell that secreted IgA anti-phosphorycholine (PC) and a human plasmacytoma cell, was utilized to examine this issue. The cloned hybridoma expressed membrane IgA and secreted IgA specific for PC. Stimulation of the hybridoma cells with PC conjugated to Sepharose beads (PC-Sepharose) but not glycine-conjugated Sepharose resulted in an increase in DNA synthesis. Affinity purified goat anti-human IgA bound to Sepharose also augmented DNA synthesis. Soluble PC did not increase DNA synthesis and inhibited the increase in DNA synthesis resulting from PC-Sepharose. IgA secretion was augmented in response to PC-Sepharose, as demonstrated by an increase in the number of Ig-secreting cells detected by a reverse hemolytic plaque assay and by quantitation of the IgA secreted per cell by enzyme-linked immunosorbent assay. Mitogen-stimulated T cell supernatants increased IgA secretion of the hybridoma cells but did not cause synergistic stimulation of the cells in the presence of PC-Sepharose. These data indicate that Sepharose-bound antigen was sufficient to induce proliferation and augment IgA secretion by this membrane IgA anti-PC-bearing hybridoma. The results suggest that cross-linking of membrane IgA by specific antigen may be a sufficient stimulus for proliferation and differentiation of B cells at this stage of maturation.
Inhibitory effect of herpes simplex virus infection to target cells on recognition of minor histocompatibility antigens by cytotoxic T lymphocytes.
Authors: Kuzushima K , Isobe K , Morishima T , Takatsuki A , Nakashima I
Published: January 2023

Target cell lysis by CTL specific for minor histocompatibility Ag (minor HA), which were generated in (C3H/He x BALB/c)F1 mice immunized with A/J mouse spleen cells, was dramatically reduced by infection of HSV to Neuro-2a (A/J mouse origin) cells as target. The reduction was apparent at 5 h after infection of HSV to target cells, when many viral proteins were produced in the cells. Conversely, MHC-restricted HSV-specific CTL-mediated cell lysis increased time dependently. Using an RNA virus, vesicular stomatitis virus, significant reduction of minor specific CTL-mediated target cell lysis was also found. During the time when this reduction of target cell lysis by HSV occurred, the surface expression of class I H-2Dd molecules was maintained, and anti-H-2a allo-MHC-specific CTL lysed HSV-infected Neuro-2a cells as strongly as uninfected Neuro-2a cells. When HSV-infected or uninfected Neuro-2a cells were treated with Brefeldin A that selectively blocks transportation of newly synthesized proteins out of endoplasmic reticulum, both HSV- and minor HA-specific CTL-mediated cell lyses were blocked. These observations demonstrated that minor HA are continuously synthesized and associated with class I molecules at pre-Golgi and transported via trans Golgi system with quick turnover, and that newly synthesized HSV Ag, which are also associated with class I molecules and transported via the same system, should take the place of intrinsic minor HA and be presented on the surface of the cells to be recognized by MHC-restricted CTL.
Expression of c-fos proto-oncogene in tumor-associated macrophages.
Authors: Bottazzi B , Nobili N , Mantovani A
Published: January 2023

Tumor-associated macrophages (TAM) have peculiar membrane phenotype and functional properties. In an effort to unravel possible molecular determinants associated with the reprogramming of mononuclear phagocytes that infiltrate tumors, we have investigated the expression of immediate-early genes in TAM from murine sarcomas. c-fos is a prototypic immediate oncogene, with transregulatory function on gene transcription, expressed by myelomonocytic cells and induced by certain activation signals. TAM from three murine sarcomas expressed basal levels of c-fos transcripts considerably higher than those of peritoneal exudate macrophages (PEM). Activation of myelomonocytic cells by bacterial LPS is associated with an early transient increase in c-fos transcription. Unlike PEM, TAM did not show any increase in c-fos expression after exposure to LPS. A similar unresponsiveness to LPS stimulation was observed in macrophages isolated from peritoneal ascitic tumors. c-fos expression in macrophages can be induced via protein kinase C activation or via an increase in cAMP levels. Unlike PEM, TAM did not respond to the protein kinase C activator PMA and to cholera toxin. After culture for 18 to 20 h, c-fos expression in TAM declined, and concomitantly, TAM completely recovered responsiveness to LPS in terms of augmented oncogene mRNA levels. These results demonstrate that TAM from murine sarcomas have an altered expression of the c-fos proto-oncogene, with high basal levels and unresponsiveness to augmenting signals, reversible upon in vitro culture. The altered c-fos expression in TAM may reflect exposure to cytokines present in the tumor microenvironment and may underlie at least some of the peculiar properties of TAM.
Immunodominance: intermolecular competition between MHC class II molecules by covalently linked T cell epitopes.
Authors: Wang Y , Smith J A , Gefter M L , Perkins D L
Published: January 2023

The T cell response to complex protein Ag typically focuses on a few, and frequently a single, immunodominant epitope. Several groups have proposed that the mechanism of immunodominance is determined by the steps of Ag processing and presentation including protein unfolding, the sites of proteolytic cleavage, and the affinity of binding to MHC molecules. Also, the failure of the TCR repertoire to recognize MHC-bound peptides, termed a hole in the repertoire, can prevent recognition of a potentially dominant processed peptide. In the present study, we demonstrate that immunodominance can be determined by intermolecular competition for binding to MHC class II molecules between covalently linked T cell epitopes. In addition, we have analyzed the factors controlling T cell recognition of the covalently linked epitopes. In our system, T cell recognition of the dominant epitope is not altered by Ag processing, and is not simply a function of MHC-binding affinity. We propose that adjacent sequences can subtly alter the conformation of an epitope, creating significant changes in T cell recognition. These observations are discussed in terms of the mechanisms of immunodominance and in terms of the development of synthetic peptide vaccines.
Persistent productive HIV-1 infection of a CD4- human fetal thymocyte line.
Authors: Hatch W C , Tanaka K E , Calvelli T , Rashbaum W K , Kress Y , Lyman W D
Published: January 2023

Human fetal thymuses were obtained from abortuses of HIV-1 seronegative females. Thymocytes were isolated and cultured for 2 days with PHA. Thereafter, the culture was divided and half of the cells were exposed to the HIV-1 RF isolate for 4 h. After this incubation period, the HIV-1 exposed and nonexposed control cells were cultured in RPMI 1640 supplemented with IL-2 for 30 days and subsequently maintained in RPMI without the addition of growth factors. Long term culture of both HIV-1 exposed and control thymocytes has yielded two cell lines that have been maintained for more than 3 yr without the addition of growth factors. Flow cytometry using mAb that recognize T cell differentiation markers was used to analyze cell phenotypes. The HIV-1 exposed thymocyte cell line (E88/RF) was shown to be HIV-1 infected by p24 ELISA, reverse transcriptase activity, immunocytochemistry, in situ hybridization, polymerase chain reaction, electron microscopy, and to produce infectious particles by a syncytial forming assay. The non-HIV-1-exposed thymocyte cell line (T412) has remained negative by all criteria for HIV-1 infection. Flow cytometry showed the T412 cells to be positive for the T cell markers CD45, CD38, and CD4 but negative for all other markers tested. The E88/RF cells are positive for CD45 and CD38 but negative for CD4 and other markers. These data report the isolation of two human fetal thymocyte cell lines; one uninfected and susceptible to HIV-1 infection, and the other persistently and productively infected with HIV-1 with little cytopathology. These findings suggest that HIV-1 can persistently infect early T cells and may alter T cell differentiation.
Yersinia enterocolitica activates a T helper type 1-like T cell subset in reactive arthritis.
Authors: Lahesmaa R , Yssel H , Batsford S , Luukkainen R , Möttönen T , Steinman L , Peltz G
Published: January 2023

The profile of lymphokines secreted by 14 T cell clones and 24 T cell lines reactive with Yersinia Ag isolated from the synovial fluid cells of two HLA-B27+ patients with Yersinia-triggered reactive arthritis was characterized. In response to Ag-specific or -nonspecific stimulation, all of the Yersinia-reactive T cell clones and lines had a pattern of lymphokine secretion resembling that of murine (Th1) cells. A total of 50% of T cell lines and clones randomly isolated from a reactive arthritis patient, without prior in vitro stimulation with Yersinia Ag, also exhibited a Th1-like profile of cytokine secretion upon nonspecific activation. This indicates that the selective expansion of this subset of T cells had already occurred in vivo. The possibility that the predominance of Th1-like T cells was an artefact generated by the T cell cloning procedure was excluded; 50% of the randomly isolated T cell clones and lines produced IL-4, IL-5, or both cytokines upon nonspecific activation. These results indicate that Yersinia Ag selectively activate a Th1-like subset of T cells in patients with Yersinia-triggered reactive arthritis. Accumulation of such cells in the synovial tissue of patients with reactive arthritis may play a key role in the pathogenesis of this disease.
Lung injury after deposition of IgA immune complexes. Requirements for CD18 and L-arginine.
Authors: Mulligan M S , Warren J S , Smith C W , Anderson D C , Yeh C G , Rudolph A R , Ward P A
Published: January 2023

Acute lung injury produced by deposition of IgA immune complexes is complement-dependent, neutrophil-independent, oxygen radical-mediated, and may be a result of the formation of the hydroxyl radical (HO) generated directly or indirectly from activated lung macrophages. The current studies were designed to evaluate further the pathophysiologic events that occur after intrapulmonary deposition of IgA immune complexes. Pretreatment of rats with the human recombinant soluble complement receptor-1 resulted in marked attenuation of IgA immune complex-induced lung injury. Intravenous administration of antibody to CD18, but not antibody to CD11b, was highly protective against lung injury. Treatment of animals with either anti-endothelial leukocyte-adhesion molecule-1 or anti-TNF-alpha, both of which were highly protective against IgG immune complex-induced lung injury, had no protective effects in the model of IgA immune complex-induced lung injury. Immunohistochemical analysis revealed up-regulation of the endothelial leukocyte adhesion molecule-1 in the pulmonary vasculature after deposition of IgA immune complexes. This up-regulation was TNF-alpha-dependent. The arginine analog, NG-monomethyl-L-arginine, was highly protective against IgA immune complex-induced lung injury. This protective effect was reversed by the co-presence of L-arginine (but not D-arginine). Protective interventions against IgA immune complex-induced lung injury were inversely correlated with the numbers of macrophages that could be retrieved by lung lavage. These data suggest fundamental differences in the pathogenesis of lung injury after intrapulmonary deposition of IgA immune complexes, as compared with injury caused by deposition of IgG immune complexes. In the latter, neutrophils, intrapulmonary generation of TNF-alpha, and up-regulation of pulmonary vascular endothelial leukocyte-adhesion molecule-1 are required for the full development of lung injury, whereas no such requirements appear in the case of IgA immune complex-induced lung injury. Full expression of IgA immune complex-induced lung injury also appears to require L-arginine, suggesting a possible role for nitric oxide or its derivatives in events ultimately leading to injury.
Difference in signal transduction pathway for IL-2 and IL-4 production in T helper 1 and T helper 2 cell clones in response to anti-CD3.
Authors: Tamura T , Yanagida T , Nariuchi H
Published: January 2023

To elucidate Th2 cell clone activation mechanism through TCR-CD3 complex, we examined the reactivity of Th2 cell clones to soluble anti-CD3 in the absence of accessory cells or costimulator. The soluble anti-CD3 stimulated IL-4 production of Th2 cell clones as efficiently as specific Ag. IL-4 production of Th2 cell clones was consistent with the elevation of intracellular free Ca2+ concentration ([Ca2+]i). The elevation was slow and sustained but occurred consistently after the anti-CD3 stimulation in all Th2 cell clones tested. The [Ca2+]i elevation appeared to depend on Ca2+ influx because it could not be observed in Ca(2+)-free medium. Several chemicals such as cholera toxin, neomycin, and herbimycin A, which have been shown to block phosphatidylinositol-4,5-bisphosphate (PIP2) breakdown pathway or protein tyrosine kinase activation, exerted no effect on the IL-4 production. In accordance with these findings, neither PIP2 breakdown nor protein tyrosine phosphorylation was observed in Th2 cell clones stimulated with anti-CD3. The inclusion of anti-CD4 in culture and the depletion of protein kinase C (PKC) did not affect IL-4 production of Th2 cell clones either. These findings support a hypothesis that Th2 cell clones use a signaling pathway for IL-4 production that is independent of protein tyrosine kinase, PIP2 breakdown or PKC, and that the [Ca2+]i elevation is the only pathway common to an IL-2 production of Th1 cell subset.
Role of the B cell antigen receptor in antigen processing and presentation. Involvement of the transmembrane region in intracellular trafficking of receptor/ligand complexes.
Authors: Liu K J , Parikh V S , Tucker P W , Kim B S
Published: January 2023

To study the role of the B cell Ag receptor (membrane-bound Ig [mIg]) in Ag processing and presentation, we have generated several Ig transfectants that express transfected TEPC-15 idiotype (T15-Id) mIgM with phosphorylcholine (PC)-binding specificity. The wild-type Ig transfectant is able to present a specific Ag (e.g., PC-conjugated hen egg-white lysozyme [PC-HEL]) more efficiently than a nonspecific Ag (HEL) to a T cell hybridoma recognizing an epitope on the HEL molecule. A substitution in the entire spacer, transmembrane, and cytoplasmic region of mIg with an equivalent region of I-A alpha chain completely abolishes this mIg-enhanced Ag presentation. Experiments with the wild-type and substituted variant Ig transfectants suggest that this substitution may interfere with normal intracellular trafficking of mIg after cross-linking with specific Ag or antibodies specific for the mIg (anti-T15-Id mAb). Prolonged treatment of the wild-type Ig transfectants with specific Ag or anti-T15-Id mAb reduces the surface expression of T15-Id mIgM and leads to an accumulation of T15-Id mIgM inside the cells. The reduced surface expression and the elevated cytoplasmic accumulation of T15-Id mIgM are not observed in the variant Ig transfectant. Despite the ability of the variant to endocytose ligands similarly to the wild-type Ig transfectant, this variant displays a higher rate of recycling of the mIg/ligand complexes back to the cell surface and a lower rate of degradation of the ligands. These abnormalities may be responsible for the deficiency in mIg-mediated enhancement of Ag presentation in the variant Ig transfectant. Therefore, our results suggest that the transmembrane region of mIg is involved in intracellular trafficking of receptor/ligand complexes and that proper delivery and handling of internalized Ag are required for the enhanced presentation of specific Ag by B cells.
Association of tyrosine and serine kinases with the B cell surface antigen CD20. Induction via CD20 of tyrosine phosphorylation and activation of phospholipase C-gamma 1 and PLC phospholipase C-gamma 2.
Authors: Deans J P , Schieven G L , Shu G L , Valentine M A , Gilliland L A , Aruffo A , Clark E A , Ledbetter J A
Published: January 2023

CD20 is a B cell-specific 35/37 kDa integral membrane protein which modulates proliferation and differentiation of normal resting B cells when stimulated by CD20 antibodies. An increase in c-myc mRNA levels occurs within hours after treatment of resting B cells with CD20 mAb; however earlier events in the CD20 signal transduction pathway have not been described. Here we demonstrate that anti-CD20 mediated induction of c-myc mRNA is inhibited by the tyrosine kinase inhibitor herbimycin A, that CD20 is associated with both tyrosine and serine kinase activity, and that tyrosine phosphorylation of multiple substrates is induced within minutes upon ligation of CD20 with mAb. Association of the tyrosine and serine kinases with CD20 was stable in lysis buffer containing 1% NP40 and 0.25% deoxycholate. Under the same conditions, antibodies against several other B cell surface molecules failed to co-precipitate tyrosine kinase activity, however, a serine kinase was precipitated by the anti-CD19 mAb, B43. Both phospholipase C-gamma 1 and -gamma 2 were phosphorylated on tyrosine after cross-linking of CD20-bound mAb, and this correlated with increases in intracellular calcium that were partially resistant to depletion of extracellular calcium with EGTA. The pattern of tyrosine phosphorylated proteins observed in whole cell lysates after anti-CD20 cross-linking appeared to be a subset of those induced by anti-IgM; however, differences in phosphoproteins induced by anti-IgM and anti-CD20 were detected using a fynSH2-fusion protein.
An immune suppressive factor derived from esophageal squamous carcinoma induces apoptosis in normal and transformed cells of lymphoid lineage.
Authors: O'Mahony A M , O'Sullivan G C , O'Connell J , Cotter T G , Collins J K
Published: January 2023

An immunosuppressive factor produced by an esophageal squamous carcinoma cell line mediates profound irreversible suppression of in vitro proliferative responses of lymphoid cells. Exposure of activated normal PBL to the immune suppressive factor (ISF) resulted in the induction of an irreversible anergic state with apoptosis evident in 20% of those cells. Flow cytometric cell cycle analysis of mitogenically stimulated normal lymphocytes exposed to the ISF revealed that despite exhibiting full activation status (IL-2 production, IL-2R, and transferrin receptor expression) PBL were arrested at the G1/S interphase of the cell cycle. Transformed lymphoid cell lines, NSO and JURKAT, displayed morphologies characteristic of apoptosis within 24 h of exposure to the ISF. Flow cytometric cell cycle analysis of the JURKAT cells incubated with ISF revealed that > 90% of these cells had undergone apoptosis within 24 h. Agarose gel electrophoresis of DNA extracted from the ISF-treated lymphoid cells resolved a DNA fragmentation pattern characteristic of apoptosis in both the NSO cells and to a lesser extent in the activated PBL exposed to the ISF but not in control cells. In JURKAT cells stimulated with anti-CD3 antibodies, Ca2+ mobilization was markedly enhanced in those cells exposed to ISF. Also, ISF independently induced a calcium flux in JURKAT cells. Induction of programmed cell death by ISF may account for the in vivo immune suppression local to the tumor site in squamous carcinoma of the esophagus.
Modulation of murine cytokine responses to mycobacterial antigens by helminth-induced T helper 2 cell responses.
Authors: Pearlman E , Kazura J W , Hazlett F E , Boom W H
Published: January 2023

BALB/c mice inoculated with live Brugia malayi microfilariae (mf), or immunized with a soluble filarial extract (B. malayi Ag (BmA)), develop a pronounced Th2-like response over time. In contrast, single or repeated immunizations with a soluble Mycobacterium tuberculosis Ag preparation (purified protein derivative, PPD) stimulates a Th1, but not Th2 response (IFN-gamma > IL-4, IL-5). To determine if the Th1 response to PPD can be modulated by the ongoing helminth-induced Th2 activity, mice were: 1) immunized simultaneously with BmA and PPD; 2) immunized first with BmA, then with PPD; or 3) inoculated with live mf and immunized with PPD at various times thereafter. Simultaneous immunization with both Ag had no effect on the Th response induced by PPD, i.e., it was strictly Th1. In contrast, establishment of a Th2 response by either inoculation of live mf or immunization with BmA before administration of PPD skewed the PPD-specific Th response such that IL-4 and IL-5 were produced in addition to IFN-gamma. IL-4 and IL-5 levels produced in response to PPD under these conditions were further elevated in vitro in the presence of neutralizing IFN-gamma. Finally, in vivo neutralization of IL-4 diminished induction of Th2 responses to PPD. These results demonstrate that ongoing Th2 responses to helminth Ag modulate the Th response to mycobacterial Ag by an IL-4 dependent mechanism.
Transforming growth factor-beta expression and natural killer cell responses during virus infection of normal, nude, and SCID mice.
Authors: Su H C , Ishikawa R , Biron C A
Published: January 2023

The experiments presented here assess the contribution of T cells to NK cell regulation and transforming growth factor-beta (TGF-beta) production. The kinetics of NK cell activation in the absence of T cells was evaluated by comparing responses in athymic nude and SCID mice to those in normal C3H/HeN, C57BL/6, and BALB/c mice during lymphocytic choriomeningitis virus (LCMV) infection. T cell-deficient mice exhibited high NK cell activity for longer periods of time post-infection. To determine whether the reduction in NK cell activation at later times post-infection of normal mice resulted from induction of negative regulatory elements, mice were treated with the chemical IFN and NK cell inducing agent polyinosinic-polycytidylic acid (poly I:C). Although poly I:C induced plasma IFN in both uninfected and LCMV-infected normal mice, NK cell activity was elevated in the uninfected mice only. In vivo expression of TGF-beta protein in normal and T cell-deficient mice was evaluated by immunohistochemical studies on splenic sections. TGF-beta proteins were not detected in sections from uninfected mice but were induced in all strains during infection. High levels of TGF-beta proteins were localized to discrete cells, and diffuse staining was observed along marginal zones. The ability of the splenic leukocytes to release TGF-beta into conditioned media was evaluated in the Mv 1 Lu biologic assay. Transient acid treatment to release active factor from the latent form and use of specific neutralizing antibodies demonstrated that leukocytes from all strains of infected mice had released latent TGF-beta 1 into conditioned media. However, cells from infected nude or SCID mice produced substantially less active TGF-beta than those isolated from normal mice. Production of biologically active TGF-beta 1 was dependent upon the presence of T cells in normal mice, as in vivo depletion of CD4+ and CD8+ T cells by antibody treatment resulted in augmented NK cell activity and loss of active factor production. The lack of TGF-beta production was accompanied by increased NK cell numbers; at late times post-infection, T cell-deficient C57BL/6-nude mice had almost twice as many NK1.1+CD3- cells per spleen compared with T cell-containing C57BL/6 littermate control mice. Treatment with active TGF-beta 1 reduced the numbers of NK cells per spleen in infected nude mice to the levels found in infected littermate controls.(ABSTRACT TRUNCATED AT 400 WORDS)
Regulation of mouse bone marrow macrophage mannose receptor expression and activation by prostaglandin E and IFN-gamma.
Authors: Schreiber S , Perkins S L , Teitelbaum S L , Chappel J , Stahl P D , Blum J S
Published: January 2023

The macrophage mannose receptor mediates the clearance of microorganisms and glycoproteins containing terminal mannose oligosaccharides. Cell surface expression of this receptor progresses with macrophage differentiation, and thus may be critical to the scavenger function of tissue and circulating macrophages. Bone marrow macrophages, which were used in this study, differentiate in culture and express functional mannose receptors. The cytokine IFN-gamma triggered activation of these macrophages and down-regulated cell surface expression of the mannose receptor after 48 h. Macrophage activation, as assessed by the generation of superoxide radicals, was inversely correlated with mannose receptor expression. The number of surface receptors was diminished by exposure to IFN-gamma, whereas the binding affinity of the mannose receptor remained unchanged. Treatment with IFN-gamma reduced receptor biosynthesis yet did not alter receptor degradation. Mannose receptor biosynthesis is up-regulated by PG of the E series, and these anti-inflammatory agents reversed the effects of IFN-gamma on receptor expression. Down-regulation of the mannose receptor by IFN-gamma was fully reversible by PGE, indicating that receptor levels are dependent on the functional state of the cell rather than being linked to terminal cell differentiation. The regulation of the receptor by cytokines and anti-inflammatory reagents suggests that the mannose receptor plays a critical role in macrophage scavenger functions and potentially in modulating inflammatory reactions.
CD4 mAb therapy modulates alloantibody production and intracardiac graft deposition in association with selective inhibition of Th1 lymphokines.
Authors: Kupiec-Weglinski J W , Wasowska B , Papp I , Schmidbauer G , Sayegh M H , Baldwin W M , Wieder K J , Hancock W W
Published: January 2023

The accelerated (24 h) rejection of (LEWxBN)F1 cardiac allografts (Tx) in LEW rats sensitized with BN skin grafts, is abrogated with CD4 mAb (BWH-4) administration between skin (day -7) and heart (day 0) transplantation (Tx survival ca. 11 days, p < 0.0001). This study analyzed the effects of CD4-targeted therapy upon host IgG and IgM alloantibody (allo-Ab) within the serum by two-color flow cytometry, and within the Tx, by immunohistology. These data were correlated with mRNA and protein production profiles of Th1 (IL-2, IFN-gamma) vs Th2 (IL-4) specific cytokines (polymerase chain reaction and/or immunohistology). Skin grafts elicited a strong systemic IgM allo-Ab response, which peaked at the time of cardiac Tx rejection at 24 h. It was associated with extensive deposits of IgM on Tx endothelium. Treatment with BWH-4 mAb diminished circulating IgM allo-Ab levels, and only low levels of IgM could be detected at the Tx site. Conversely, the low circulating IgG allo-Ab levels during rejection at 24 h in untreated recipients were accompanied by a strong labeling for intra-Tx IgG. BWH-4 mAb therapy did not prevent totally the switch of the IgM to IgG, but the IgG allo-Ab response was earlier, less intense and more transient than in untreated recipients. Accelerated rejection triggered sequential lymphokine mRNA expression in cardiac Tx, with the peak of transcription for IL-2 (6-12 h) preceding that for IL-4 (24 h). Interestingly, although CD4 targeted therapy virtually ablated the induction of IL-2 mRNA, it preserved transcription of the IL-4 gene. BWH-4 mAb therapy decreased otherwise abundant intra-Tx IL-2 and IFN-gamma, but allowed a vigorous elaboration of IL-4, confirming the translation of mRNA to the protein in vivo. Thus, CD4 mAb-mediated abrogation of accelerated cardiac Tx injury correlates with suppression of Th1 responses (depressed IL-2 and IFN-gamma production), but sparing of the Th2 function (enhanced IL-4 elaboration). Indeed, CD4 mAb-induced allo-Ab depression and immunosuppressive effects may reflect selective targeting of proinflammatory Th1-like cells and the multifaceted effects of IL-4 produced by unopposed Th2-like cells.
Complete Freund's adjuvant induces an earlier and more severe arthritis in MRL-lpr mice.
Authors: Ratkay L G , Zhang L , Tonzetich J , Waterfield J D
Published: January 2023

A study was performed on the effect of CFA on the spontaneous arthritis of MRL-lpr mice. The development of swelling and erythema was monitored for 1 mo after injecting 13- to 14-wk-old mice intradermally with CFA, at which time the histopathology of the joints and serologic responses to extracellular matrix proteins were investigated. In a series of six experiments, 67 to 82% of mice showed early clinical evidence of arthritis in contrast to the low percentage observed in control animals. Similarly, the histopathologic analyses on the CFA-injected mice indicated a significantly higher frequency of advanced histopathologic alterations, characterized by cartilage erosion and pannus formation. The serologic evaluation of the adjuvant-injected mice demonstrated a significant enhanced antibody production to type I and type II collagens, DNA, and the Mycobacterium tuberculosis-positive control. This reproducible adjuvant-enhanced model of murine arthritis will be extremely useful in evaluating experimental therapeutic regimes as the arthritis is initiated earlier and exhibits an enhanced frequency and severity compared with the spontaneous arthritis seen in MRL-lpr mice.
Ionizing radiation increases endothelial and epithelial cell production of influenza virus and leukocyte adherence.
Authors: Colden-Stanfield M , Kalinich J F , Gallin E K
Published: January 2023

To characterize the effect of 60Co gamma radiation on cell-cell and pathogen-cell interactions, the adherence of undifferentiated HL-60 cells to HUVEC monolayers was tested in the absence and presence of LPS or influenza virus type A. Basal HL-60 cell adherence to uninfected HUVEC monolayers (3.0 +/- 1.6%, n = 30) was not altered when HUVECs were exposed to 1- to 10-Gy gamma irradiation 4 to 72 h before the adhesion assay. LPS treatment of HUVEC monolayers (0.5 microgram/ml, 4 h) produced a 6.9-fold increase in adherence that was not altered by previous irradiation. However, when HUVEC monolayers were subjected to 1-10 Gy 41 h before influenza virus infection (10(6) pfu/ml) for 7 h, virus-induced adherence was enhanced in a dose-dependent manner. Increased virus hemagglutinin (HA) protein expression mediated the radiation-induced adherence for the following reasons: 1) HA Ag increases paralleled increases in leukocyte adherence. 2) Northern blot analysis demonstrated a time-dependent increase in mRNA HA levels. 3) Anti-HA blocked HL-60 cell adherence to irradiated and virus-infected HUVEC monolayers. These changes were associated with an increased virus titer yield and virus-induced HUVEC killing. In contrast, cytotoxicity produced by vesicular stomatitis virus, which unlike influenza virus replicates cytoplasmically, was not altered by radiation in HUVECs. In related studies, the canine kidney epithelial (MDCK) cell line showed a similar increased influenza virus production after gamma radiation, indicating that the radiation-induced increase in production of influenza virus is not cell-specific and probably involves a nuclear mechanism.
Soluble IgA and IgG aggregates are catabolized by cultured rat mesangial cells and induce production of TNF-alpha and IL-6, and proliferation.
Authors: Gómez-Guerrero C , López-Armada M J , González E , Egido J
Published: January 2023

IgA nephropathy, a primary glomerulonephritis, is principally characterized by mesangial deposits of IgA immune complexes. Recently, it has been demonstrated that cultured glomerular mesangial cells (MC) express Fc alpha and Fc gamma receptors. In this work, we studied whether the interaction of soluble aggregates of IgA and IgG (AIgA and AIgG) with MC triggers a number of responses, including generation and release of inflammatory mediators, cell proliferation, and catabolism of the complexes. Aggregates bound to MC and were catabolized in a time-dependent manner. The percentage of cell-associated or -degraded proteins decreased in the presence of Fc, but not with Fab fragments. Both AIgA and AIgG elicited the synthesis and release of TNF-alpha and IL-6 in a dose-dependent manner, reaching a maximum between 6 and 12 h, respectively. Northern blot analysis showed that both aggregates induced the expression of mRNA encoding TNF-alpha and IL-6. Because MC proliferation is a morphologic feature commonly observed in patients with IgA nephropathy, we examined whether immune aggregates could be involved in this phenomenon. Both AIgA and AIgG induced an increase in MC number, assessed by [3H]thymidine and methylene blue uptake. The presence of anti-TNF-alpha or anti-IL-6 Abs in the medium decreased the proliferative effect triggered by aggregates. These results show that Fc alpha and Fc gamma receptor occupancy of MC induces the synthesis and release of inflammatory cytokines and proliferation, as well as the phagocytosis of stimulatory proteins. These findings could have implications for the understanding of inflammation and repair in IgA nephropathy.
MHC class I molecule-restricted presentation of viral antigen in beta 2-microglobulin-deficient mice.
Authors: Lehmann-Grube F , Dralle H , Utermöhlen O , Löhler J
Published: January 2023

Normally, Ag is presented to CD8+ T lymphocytes as a tripartite complex consisting of peptide epitope, MHC-encoded class I heavy (alpha) chain, and beta 2-microglobulin (beta 2-m) light chain. Although there is agreement about the function of both peptide and alpha-chain, the role of beta 2-m has remained uncertain. In particular, can Ag be presented without its participation? We have sought to obtain an answer by using mice in which the gene for beta 2-m had been disrupted by homologous recombination. As a consequence, no light chains are synthesized and, furthermore, few if any CD8+ T lymphocytes are formed. Elimination of lymphocytic choriomeningitis (LCM) virus from the tissues of acutely infected mice is mediated solely by CD8+ T lymphocytes; hence, in the beta 2-m-lacking mutants the infection cannot be terminated. Here it is shown that infusion of immune spleen cells from syngeneic beta 2-m+/+ mice and from mice compatible in K or D of the MHC resulted in virus clearance. Approximately five times more cells were required to achieve antiviral effects in beta 2-m-deficient than in wild-type mice but attempts to improve elimination by treatment of the former with IFN-gamma or beta 2-m have failed. Depleting the immune splenocytes of CD8+ T lymphocytes but not of CD4+ T lymphocytes abolished the antiviral potential. We conclude that LCM virus-infected murine cells can present viral Ag to CD8+ effector T lymphocytes together with class I MHC molecules K and D, despite the total absence of beta 2-m.
Jaw1, A lymphoid-restricted membrane protein localized to the endoplasmic reticulum.
Authors: Behrens T W , Jagadeesh J , Scherle P , Kearns G , Yewdell J , Staudt L M
Published: January 2023

Jaw1 is a novel lymphoid-restricted gene that is expressed in a developmentally regulated fashion in both the B and T cell lineages. Jaw1 mRNA is abundantly expressed in pre-B and B cell lines with minimal or undetectable expression in plasma cell lines. Pre-T cell lines and normal mouse thymocytes express high levels of Jaw1 mRNA, whereas most mature T cell lines express low levels. Comparison of the mouse and human genes reveals that Jaw1 encodes a 539 amino acid protein with a highly conserved coiled-coil domain in the middle third of the protein and a COOH-terminal transmembrane domain. Jaw1 was localized to the endoplasmic reticulum (ER) of lymphocytes by indirect immunofluorescence and confocal microscopy. When overexpressed in HeLa cells, Jaw1 protein targeted to the ER. In vitro translation of Jaw1 in the presence of canine microsomes demonstrated that Jaw1 is an integral membrane protein of the ER and is oriented on the ER membrane facing the cytosol. Jaw1 is a member of a class of proteins with COOH-terminal hydrophobic membrane anchors and is structurally similar to proteins involved in vesicle targeting and fusion. These findings suggest that the function and/or the structure of the ER in lymphocytes may be modified by lymphoid-restricted resident ER proteins.