bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-1 and IL-4 modulate IL-1 receptor expression in a murine T cell line.
Authors: Lacey D L , Erdmann J M
Published: December 2022

The combination of IL-1 and IL-4 stimulates the proliferation of certain murine T cell populations. Although this effect has been best characterized for a number of murine type 2 Th cell (Th2) clones, the mechanism(s) by which these cytokines effect this response is unclear. We have examined the effects of IL-1 and IL-4 on IL-1R expression by MD10 cells, and IL-1-responsive murine T cell line. These cells bear specific IL-1R, which bind human and murine IL-1 alpha and -beta. The measured apparent IL-1R dissociation constant ranged from 41 to 255 pM using 125I-HrIL-1 alpha. Cross-linking studies demonstrated two different 125I-HrIL-1 alpha binding complexes having Mr of 70,000 and 130,000 to 156,000. When removed from passage conditions and placed in non-growth factor-supplemented media, MD10 IL-1R expression spontaneously increased two- to fourfold over the first 11 to 12 h of culture followed by a decline. This phenomenon is partially inhibitable by cycloheximide suggesting that protein synthesis is involved. In agreement with other reports, HrIL-1 alpha down-regulated the expression of its own receptor with an ED50 of between 1 and 10 pM HrIL-1 alpha for this effect. In most experiments, low amounts of HrIL-1 alpha (1.0, 0.1 pM) significantly augmented IL-1R expression. Scatchard analysis of data obtained with all HrIL-1 alpha treatment conditions showed that the effects were due to a change in receptor number, not affinity. Significantly, purified murine IL-4 (MpIL-4) augmented MD10 IL-1R expression in both a time- and dose-dependent fashion. In the presence of 50 U/ml MpIL-4, MD10 IL-1R expression increased two- to threefold after 24 h without a change in receptor affinity. When MpIL-4 (50 U/ml) and various amounts of HrIL-1 alpha (.01-1000 pM) were co-added, the down-regulatory effect of high levels of HrIL-1 alpha was significantly antagonized. When added to cultures after 24 h of HrIL-1 alpha (100 pM) treatment, MpIL-4 reversed the IL-1R down-regulatory effect induced by high levels of HrIL-1 alpha. Finally, when combined in MD10 proliferation assays, MpIL-4 synergistically enhanced the proliferation of MD10 cells treated with suboptimal levels of HrIL-1 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)
Natural and recombinant human IL-1 receptor antagonists block the effects of IL-1 on bone resorption and prostaglandin production.
Authors: Seckinger P , Klein-Nulend J , Alander C , Thompson R C , Dayer J M , Raisz L G
Published: December 2022

Inhibitory factors towards IL-1 have been identified in the urine and in the supernatants of human monocyte cultures and have been shown to act as receptor antagonists. We have investigated whether a natural inhibitor purified from human urine (uIL-1ra) and a recombinant molecule expressed using the gene for an IL-1 antagonist isolated from monocytes (rIL-1ra) can alter responses to human rIL-1 alpha in organ cultures of fetal rat long bones and neonatal mouse calvariae. The two preparations probably contained similar or identical molecules, because an antibody to rIL-1ra reacted with uIL-1ra by Western blot analysis. uIL-1ra and rIL-1ra specifically blocked stimulation of bone resorption by rIL-1 in both culture systems, as well as the increase in PGE2 production in cultured calvariae. Resorptive effects of parathyroid hormone and TNF-alpha were not blocked. The uIL-1ra preparation had some intrinsic resorbing activity, but on gel chromatography this appeared in fractions that eluted earlier than uIL-1ra. Concentration ratios of rIL-1ra to rIL-1 as low as 10 could block the resorptive response of fetal rat long bones, whereas concentration ratios of 100 to 1000 were required to block IL-1 action on neonatal mouse calvariae. The inhibitory effects appeared to be competitive, because increasing concentrations of IL-1 overcame the block of bone resorption in both systems and the inhibition of PGE2 production in calvariae.
Anti-IL-6 monoclonal antibodies protect against lethal Escherichia coli infection and lethal tumor necrosis factor-alpha challenge in mice.
Authors: Starnes H F , Pearce M K , Tewari A , Yim J H , Zou J C , Abrams J S
Published: December 2022

Potentially fatal physiologic and metabolic derangements can occur in response to bacterial infection in animals and man. Recently it has been shown that alterations in the levels of circulating cytokines such as IL-6 and TNF-alpha occur shortly after bacterial challenge. To understand better the role of IL-6 in inflammation, we investigated the effects of in vivo anti-mouse IL-6 antibody treatment in a mouse model of septic shock. Rat anti-mouse IL-6 neutralizing mAb was produced from splenocytes of an animal immunized with mouse rIL-6. This mAb, MP5-20F3, was a very potent and specific antagonist of mouse IL-6 in vitro bioactivity, demonstrated using the NFS60 myelomonocytic and KD83 plasmacytoma target cell lines, and also immunoprecipitated radiolabeled IL-6. Anti-IL-6 mAb pretreatment of mice subsequently challenged with lethal doses of i.p. Escherichia coli or i.v. TNF-alpha protected mice from death caused by these treatments. Pretreatment of E. coli-challenged mice with anti-IL-6 led to an increase in serum TNF bioactivity, in comparison to isotype control antibody, implicating IL-6 as a negative modulator of TNF in vivo. Anti-TNF-alpha treatment of mice challenged i.p. with live E. coli resulted in a 70% decrease in serum IL-6 levels, determined by immunoenzymetric assay, compared to control antibody, thereby supporting a role for TNF-alpha as a positive regulator of IL-6 levels. We conclude that IL-6 is a mediator in lethal E. coli infection, and suggest that antagonists of IL-6 may be beneficial therapeutically in life-threatening bacterial infection.
Natural mouse and human antibodies bind to a peptide derived from a germline VH chain. Evidence for evolutionary conserved self-binding locus.
Authors: Kaveri S V , Kang C Y , Köhler H
Published: December 2022

Murine antibodies derived from the V1 S107/T15 germline structure combined with Vk 22 L chains express the property of self-binding. Previous studies have shown that the self-binding is mediated by the Fab fragment involving structures of the hapten binding site. The molecular locus of self-binding has also been identified by showing that a peptide derived from the CDR2/FR3 region of the V1 S107 H chain inhibits self-binding. We have addressed the question of whether self-binding antibodies interact with peptides that inhibit self-binding. We found that labeled TEPC15 (T15) binds to immobilized VH (50-73) peptide; the peptide binding is specific because different CDR peptides and other unrelated peptides do not inhibit this binding. Furthermore, the hapten phosphorylcholine is a potent inhibitor for the T15-peptide binding. We have demonstrated the presence of naturally occurring antibodies that bind to the T15H(50-73) peptide in the sera of different strains of mice and also in humans, indicating that the CDR2/FR3 sequence of T15 is a conserved Id determining region. We have isolated peptide-specific antibodies from pooled normal human Ig preparations. Human anti-peptide antibodies have self-binding properties similar to their murine counterparts. This interspecies conserved peptide binding of antibodies that are self-binding indicates the existence of an evolutionarily important and biologically active site.
Proliferative potential of murine peritoneal mast cells after degranulation induced by compound 48/80, substance P, tetradecanoylphorbol acetate, or calcium ionophore A23187.
Authors: Jozaki K , Kuriu A , Waki N , Adachi S , Yamatodani A , Tarui S , Kitamura Y
Published: December 2022

Proliferative potential of degranulated mast cells was investigated. Mast cells were collected from the peritoneal cavity of mice, and degranulation was induced by compound 48/80, substance P, 12-O-tetradecanoylphorbol 13-acetate (TPA), or calcium ionophore A23187. The potentiality of colony formation in methylcellulose was not reduced by treatment of various concentrations of compound 48/80, substance P and TPA. When degranulation was induced by compound 48/80, substance P or TPA, proportion of highly degranulated mast cells containing less than five granules was rather small. In contrast, considerable proportion of highly degranulated mast cells was obtained after the treatment with the low concentration (0.1 microgram/ml) of A23187. These highly degranulated mast cells, which were individually picked up by the micromanipulator, proliferated not only in methylcellulose but also in the skin of mast cell-deficient WBB6F1-W/Wv mice. Inasmuch as we have already shown the proliferation of IgE-sensitized and Ag-stimulated mast cells, degranulated mast cells appear to retain the proliferative potential in general.
Picolinic acid, a catabolite of tryptophan, as the second signal in the activation of IFN-gamma-primed macrophages.
Authors: Varesio L , Clayton M , Blasi E , Ruffman R , Radzioch D
Published: December 2022

We have studied the effects of picolinic acid, a product of tryptophan degradation, on the activation of mouse peritoneal macrophages (M phi). Picolinic acid acts synergistically with IFN-gamma in activating M phi from C57BL/6 mice. Moreover, M phi from C3H/HeJ mice and C3H/HeN that do not become cytotoxic in response to IFN-gamma alone could be fully activated by exposure to picolinate plus IFN-gamma. These results indicate that picolinic acid is a potent costimulator of M phi activation that functions as a second signal. Inasmuch as we have previously demonstrated that the activation of cytotoxic M phi correlates with specific changes in ribosomal RNA (rRNA), we investigated whether picolinic acid could modify M phi RNA metabolism. Picolinic acid inhibited the synthesis of total M phi RNA, the accumulation of newly synthesized 28S rRNA, and augmented the steady state levels of rRNA precursors (pre-rRNA). These changes in RNA metabolism were similar to those previously described in murine M phi activated in vitro or in vivo to express tumoricidal activity. These results demonstrate that picolinic acid is a potent, biologic M phi second signal, suggest that the changes in rRNA are causally connected with the expression of tumoricidal activity, and suggest the existance of an autocrine effect mediated by picolinic acid.
Staphylococcus aureus infection of human endothelial cells potentiates Fc receptor expression.
Authors: Bengualid V , Hatcher V B , Diamond B , Blumberg E A , Lowy F D
Published: December 2022

Vasculitis, a recognized complication of staphylococcal-endovascular infections, may result in part, from the expression of FcR by Staphylococcus aureus-infected endothelial cells. FcR were measured using [51]Cr labeled SRBC preincubated with rabbit anti-SRBC IgG. FcR were not detected on uninfected endothelial cells, but were demonstrated on S. aureus infected cells using IgG, but not IgM labeled SRBC. FcR expression was dependent on the initial bacterial density (greater than or equal to 8 x 10(7) cfu/ml) and on phagocytosis of the staphylococci, but not on new protein synthesis. IgG labeled SRBC binding was blocked by aggregated IgG but not IgM. SRBC coated with the F(ab')2 portion of IgG did not bind, thus confirming that FcR were specifically involved in this interaction. FcR are expressed after S. aureus invasion of human endothelial cells and may contribute to the vasculitis which often accompanies S. aureus-endovascular infections.
Hepatitis B virus nucleocapsid/pre-S2 fusion proteins expressed in attenuated Salmonella for oral vaccination.
Authors: Schödel F , Milich D R , Will H
Published: December 2022

Hybrid HBV nucleocapsid-pre-S(2) fusion proteins were stably expressed in several aromatic-dependent attenuated Salmonella typhimurium and Salmonella dublin strains. When these live recombinant bacteria were administered i.p. to BALB/c mice they induced high titer anti-hepatitis B virus core Ag (HBc) and detectable anti-pre-S2 serum antibodies. Upon oral feeding of the recombinant salmonellae to mice, the rate of seroconversion to anti-HBc was dependent on the salmonella strain used. With the best carrier strain high titer anti-HBc antibodies and lower titer anti-pre-S2 serum IgG antibodies were observed two weeks after a single oral immunization. The Ig class and IgG subclass distribution of anti-HBc antibodies after i.p. and oral immunization is consistent with the induction of functional T cell help.
Characterization of monocyte adherence to human macrovascular and microvascular endothelial cells.
Authors: Beekhuizen H , Corsèl-van Tilburg A J , van Furth R
Published: December 2022

The interaction of monocytes with cultured large vessel venous and arterial endothelial cells (EC) and with cultured microvascular EC was studied. Analysis of time-lapse microcinematographic video recordings showed that monocytes adhere rapidly to the surface of EC and subsequently remain spherical and fixed to the initial site of adherence. Some monocytes adherent to EC stretch out within 30 to 90 min and migrate over the EC surface or become stretched for about 10 to 30 min and then detach from the EC surface and move rapidly over the EC monolayer. It was shown that the interaction of monocytes with EC is dynamic, that the morphology of monocytes adherent to EC changes constantly, and that stretching of the monocytes over the surface of the EC is not an inevitable and irreversible consequence of binding. A quantitative adherence assay was developed in which both the morphology and the number of monocytes bound to EC were determined. For each type of EC the number of monocytes bound to a single EC was found to be linearly related to the number of monocytes added and was lower for smaller EC. The adherence of monocytes to venous and arterial EC followed a different time course than the adherence to capillary EC and adherence to both types of macrovascular EC was higher than adherence to microvascular EC was higher than adherence to microvascular EC. The percentage of adherent monocytes with a stretched morphology was lower when these cells were adherent to capillary EC than to both types of macrovascular EC and increased upon addition of serum. Adherence of monocytes to venous, arterial, and capillary EC was partially inhibited by mAb directed against the alpha-chain of lymphocyte function-associated Ag-1 or C3bi receptor (with mAb LM2/1, but not with mAb OKM1) and by mAb against the common beta-chain of the three leukocyte adhesion molecules. The degree of inhibition of monocyte adherence to EC by mAb against lymphocyte function-associated Ag-1 alpha and the common beta-chain was dependent on the type of EC and was higher for venous EC (57 to 70% inhibition) than for arterial (40 to 44% inhibition) and capillary (44 to 49% inhibition) EC. Inhibition of monocyte adherence obtained with anti-C3bi receptor-alpha mAb was similar for each EC type. mAb against p150, 95 did not affect adherence. None of the mAb could block binding completely; combinations of the mAb also did not result in increased inhibition of monocyte adherence to EC.
T cell regulation of collagen-induced arthritis in mice. I. Isolation of Type II collagen-reactive T cell hybridomas with specific cytotoxic function.
Authors: Chiocchia G , Boissier M C , Ronziere M C , Herbage D , Fournier C
Published: December 2022

After immunization with native type II collagen (CII), susceptible strains of mice (H-2q) develop a polyarthritis that mimics rheumatoid arthritis. Although the underlying mechanisms are still undefined, T cells and particularly CD4+ lymphocytes seem to play a crucial role in the initiation of collagen-induced arthritis. To investigate whether CD8+ cells may participate in the pathogenesis of the disease, we have generated lines and clones of cytotoxic T cell hybridomas reactive to CII by fusion of lymph node and spleen cells from bovine native CII-primed C3H.Q (H-2q) mice and the AKR-derived thymoma cell line BW 5147. Clones were selected for their ability to lyse syngeneic macrophages pulsed with bovine native CII in an Ag-dependent manner. The two hybrid clones that were characterized, exhibited cell surface phenotypes of cytotoxic cells and reacted with CII purified from various species. However, each of them recognized different determinants on the CII molecule. P3G8 clone was specific for an epitope shared by CII and type XI collagen, whereas P2D9 clone reacted with CII and type IX collagen. Both hybridomas recognized CII-pulsed targets in association with H-2Kq molecules. These data indicate that the two CII-specific cytotoxic clones recognize different epitopes that are shared by other articular collagens and will allow us to test their influence on the development of arthritis in vivo.
Effector mechanisms in cyclosporine A-induced syngeneic graft-versus-host disease. Role of CD4+ and CD8+ T lymphocyte subsets.
Authors: Hess A D , Fischer A C , Beschorner W E
Published: December 2022

Syngeneic graft-versus-host disease (SGVHD) is a T cell-mediated autoimmune disease occurring postsyngeneic bone marrow transplantation and the administration of the potent immunosuppressive agent, cyclosporine A. Paradoxically, cyclosporine A disrupts the immunologic homeostasis governing self-tolerance. Our studies using an adoptive transfer model attempted to identify effector mechanisms associated with the autoimmune disease. Both CD4+ and CD8+ splenic T cells isolated from autoimmune donors were required for the adoptive transfer of active disease into lethally irradiated secondary recipients reconstituted with normal bone marrow. Doses of more than 5 x 10(6) of nylon wool depleted splenocytes from autoimmune donors effectively transferred disease into lethally irradiated secondary recipients. Splenocytes that are T cell depleted or CD4(+)-enriched cells did not elicit disease upon adoptive transfer. Nylon wool fractionated CD8+ splenocytes also failed to adoptively transfer disease unless high doses (greater than or equal to 30 x 10(6)) were used. The disease transferred with the CD8+ subset presented as acute type SGVHD and was self-limiting. The recombination of the individually isolated T cell subsets not only restored but also enhanced immune reactivity upon adoptive transfer. Moreover, use of the recombined subsets resulted in progressive disease with the development of chronic type SGVHD. The titration of each subset to the other suggested that a minimal number of CD4+ T cells was required to potentiate the CD8+ autoreactive cells in vivo. Further analysis of the helper cell involved demonstrated that it had a CD4+ CD45r- phenotype, characteristic of an amplifying helper cell population. Administration of IL-2 did not substitute for CD4+ Th cells but yet amplified the activity of unfractionated cells or recombined subsets implicating the role of other factors in the pathogenesis of SGVHD. Delineation of the effector mechanisms involved in SGVHD is critical in determining the underlying events that trigger either the production of autoreactive cells or the perturbation of the regulation of these autoreactive cells, culminating in autoimmunity.
Human granulocyte-macrophage colony-stimulating factor plus phorbol myristate acetate stimulate a promyelocytic cell line to produce an IL-1 inhibitor.
Authors: Mazzei G J , Bernasconi L M , Lewis C , Mermod J J , Kindler V , Shaw A R
Published: December 2022

We recently described an IL-1 inhibitor found in urine of febrile patients. It is a 26-kDa glycoprotein that acts by blocking the binding of IL-1 to its receptor. In a search for a cell source for the urinary IL-1 inhibitor, we tested three promyelocytic cell lines, H-161, AML-193, and HL-60, for their ability to produce this protein. Under normal culture conditions none of these cell lines produce detectable IL-1 inhibitory activity. The H-161 cells were treated with differentiation-inducing agents, i.e., sodium butyrate, hemin, retinoic acid, DMSO, vitamin D3, and PMA alone or in combination with IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, TNF-alpha, IFN-gamma, granulocyte-CSF, macrophage-CSF, granulocyte/macrophage-CSF (GM-CSF), and Con A and tested for the production of IL-1 inhibitor. Production of IL-1 inhibitor was detected in cell supernatant, when H-161 cells were differentiated to adherent macrophage-like cells under the influence of PMA followed by a second signal provided by GM-CSF. Treatment of the other two cell lines, AML-193 and HL-60, with PMA plus GM-CSF also yielded similar IL-1 inhibitor protein. Partial purified H-161-derived IL-1 inhibitor showed specific binding to IL-1R-bearing cells and blocked the binding of IL-1 to its receptor and is thus similar to the urinary-derived molecule. We conclude the GM-CSF provides a signal to adherent macrophage-like cells to become "inhibitory macrophages" and to produce a competitive inhibitor of IL-1.
Epitope mapping of recombinant HeLa SmB and B' peptides obtained by the polymerase chain reaction.
Authors: Elkon K B , Hines J J , Chu J L , Parnassa A
Published: December 2022

SmB and SmB' are the major antigenic proteins contained within small nuclear RNP particles that are recognized by both human SLE and MRL mouse anti-Sm sera. We amplified cDNA obtained from HeLa cells by using the polymerase chain reaction and identified two clones, U2 and L13, that encode SmB and SmB', respectively. The nucleotide sequences of these two clones were identical except for the insertion of a 145-bp sequence in U2 that contained an early in frame termination codon and a potential 3' consensus splice site. The predicted amino acid sequences of HeLa SmB and B' proteins were therefore identical except for the COOH terminal 2 (U2) and 11 (L13) amino acids. U2, L13, and four subclones of U2 (F-B, B-R, F-X, and X-B) were ligated to pATH vectors and expressed as trpE fusion proteins. Epitope mapping with 12 human SLE and 12 MRL/lpr mouse anti-SmB/B' sera revealed that antibodies directed against the X-B peptide accounted for most (65.5 +/- 15.4 and 63.2 +/- 25.3%), B-R intermediate levels (51.5 +/- 30.8 and 18 +/- 19.6%), and F-X none of the anti-SmB activity in human and mouse sera, respectively. Ten human and two mouse sera contained antibodies that cross-reacted with epitopes located within the proline-rich, COOH-terminal, 27-residue peptide encoded by B-R and the NH2-proximal F-B peptide. These observations suggest that a) the polymerase chain reaction is a powerful ancillary method to synthesize autoantigens, b) SmB and B' in HeLa cells are derived from alternative splicing of a common RNA transcript, and c) both SLE and MRL anti-SmB/B' sera recognize multiple epitopes (some shared and some unique) on these proteins.
Functional properties of the asialo-fifth component of human complement.
Authors: Schultz D R , Arnold P I
Published: December 2022

Removal of exposed, terminal sialic acid (SA) from carbohydrate chains N-glycosidically linked to asparagine residues of highly pure human C5 with bacterial sialidase increased C-mediated hemolysis of antibody-sensitized sheep E maximally 2.77-fold. Sialidase-treated C5 used as a reagent for the titration of C6, C7, C8, and C9 resulted in increased titers of all these components compared to buffer-treated C5. As determined by a fluorometric method, ca. 65% of the SA was enzymically hydrolyzed under optimal conditions. Endoglycosidase F incubated with C5 followed by monosaccharide analyses by anion exchange chromatography with pulsed amperometric detection revealed both high mannose and complex (terminate in SA) oligosaccharides were hydrolyzed; no effect was found on the functional activity of C5. Approximately 4% of the complex oligosaccharides were hydrolyzed from C5. Comparison of sialidase- and buffer-treated C5 decay rates from EAC1gp(4b,oxy2a,3b)hu resulted in two linear components of the decay curve with sialidase-treated C5, but one linear component with buffer-treated C5. Of the sialidase-treated 125I-C5 15% was bound to EAC1gp(4b,oxy2a,3b)hu compared to 9.3% of buffer-treated 125I-C5. Furthermore, 27% of sialidase-treated 125I-C5 was bound to EAC1gp,4bhu compared to 16.6% of buffer-treated 125I-C5, but no lysis occurred after the addition of C6-C9. The mechanism of increased hemolytic activity after removal of SA from C5 is: the Tmax is prolonged at 30 degrees C (ca. 15 min vs 9 min), and a higher percentage of C5 binds to cellular intermediates compared to buffer-treated C5.
High concentrations of bacterial lipopolysaccharide, but not microbial infection-induced inflammation, activate macrophage C3 receptors for phagocytosis.
Authors: Griffin F M , Mullinax P J
Published: December 2022

Macrophage C3 receptors are normally immobilized in the plane of the cells' plasma membrane and are unable to promote phagocytosis even though they promote avid particle binding. We have previously identified a lymphokine that activates macrophage C3 receptors for phagocytosis both in vitro and in vivo, and others have found that certain types of nonimmunologically mediated inflammation are also able to activate mononuclear phagocyte C3 receptors. These findings raised the possibility that macrophage C3 receptor activation is a universal consequence of inflammation. We sought in the present experiments to determine whether or not inflammation induced by microbial infection in a nonimmune host resulted in activation of macrophage C3 receptors. We injected mice i.p. with either viable microorganisms, microbe-containing immune complexes, or bacterial LPS. Macrophages were harvested by peritoneal lavage 4 days later; nearly all lavage fluids grew the microorganism with which the mouse had been injected, indicating that an infection had been established. Monolayers of macrophages were established and their interaction with sheep E coated with C3 (EIgMC) was determined. All macrophages bound EIgMC, but only macrophages from mice injected with either very high concentrations of LPS or microbe-containing immune complexes ingested them. C3 receptors of macrophages that ingested EIgMC were mobile; others were not. Thus, inflammation induced by microbial infection does not commonly, if at all, activate macrophage C3 receptors; microbe-containing immune complexes and high concentrations of LPS do. The mechanism of receptor activation in each case is C3 receptor mobilization, which is probably mediated by a lymphokine.
Herpes simplex virus glycoprotein D is recognized as antigen by CD4+ and CD8+ T lymphocytes from infected mice. Characterization of T cell clones.
Authors: Johnson R M , Lancki D W , Fitch F W , Spear P G
Published: December 2022

Several previous reports have described the surprising inability to detect murine CTL specific for glycoprotein D (gD), one of the important protective immunogens of HSV. Using slight variations of published procedures, we were able to show that the immune response to HSV in infected mice includes the generation of CTL specific for gD. C3H/OuJ (H-2k) mice were infected by injection in the hind footpads with purified HSV-1. Lymphocytes from draining lymph nodes were then isolated and shown to proliferate in response to, and to kill, transformed fibroblasts (H-2k) expressing HSV-1 gD. Two gD-specific T cell clones were isolated. One clone, designated CGD1, was shwon to be CD8+. This clone recognizes HSV-1 gD, but not HSV-2 gD, in the context of class I MHC molecules and kills the appropriate MHC-matched fibroblasts expressing HSV-1 gD. Unusual features of this cytolytic clone include augmentation by IL-4 of proliferative responses to Ag, inhibition of its lytic activity by a mAb specific for Thy-1 and recognition of infected fibroblasts in preference to infected lymphoblasts. The other clone, designated CGD3, was shown to be CD4+. This clone recognizes both HSV-1 gD and HSV-2 gD in the context of class II MHC molecules and has cytolytic potential.
Nucleotide sequence and genomic organization of a human T lymphocyte serine protease gene.
Authors: Caputo A , Sauer D E , Rowe P B
Published: December 2022

We have determined the nucleotide sequence of 4508 base pairs of human genomic DNA which contain the human serine esterase gene from cytotoxic T lymphocytes (SECT) (equivalent to the 1-3E cDNA clone) and include 879 bp of 5' flanking DNA and 393 bp of 3' flanking DNA. The gene consists of five exons of 88, 148, 136, 261, and 257 nucleotides separated by four introns of 1043, 455, 205, and 643 nucleotides. The location of introns with respect to protein coding sequences in the SECT gene is identical to that of the human cathepsin G and murine granzyme B genes. Comparison of SECT gene exonic sequences to murine granzyme B-F cDNA sequences indicates similarities of 75 and 72% for granzymes B and C and 61, 59, and 61% for granzymes D, E, and F, respectively. The 5' flanking sequence of the SECT gene showed similarity only to the 5' flanking sequence of the murine granzyme B gene, indicating that these genes are homologous. Comparison of the SECT gene sequence to the human cathepsin G sequence indicated no similarity in the 5' flanking DNA although the exonic sequences show 64% sequence similarity overall and 45% sequence similarity in the respective 3' untranslated regions. These similarities suggest that the SECT and cathepsin G genes are members of the same family of serine protease genes. Evidence from high and low stringency Southern transfer analysis of human genomic DNA indicates the presence of another gene of at least 85% sequence similarity to the SECT gene.
Novel recombinations of the IG kappa-locus that result in allelic exclusion.
Authors: Feddersen R M , Martin D J , Van Ness B G
Published: December 2022

Allelic exclusion of Ig H and L chain gene loci serves to ensure that a B cell expresses a single specificity antibody. The analysis of Abelson murine leukemia virus transformed cells that rearrange the kappa-locus during growth in cell culture has provided the opportunity to characterize intermediate steps in Ig gene rearrangement. By sequential cloning of an Abelson murine leukemia virus transformed cell line we have observed a novel two-step pathway that results in a rearrangement of a V kappa gene segment into the J-C kappa intron. This type of rearrangement effectively excludes functional kappa expression from that allele. A truncated mRNA product resulting from the V kappa signal exon splicing to the C kappa exon is diagnostic of these unique rearrangements. In addition to demonstrating a novel mechanism for allelic exclusion, the two-step pathway described serves to explain how V-intron recombination products were generated in previously described cell lines.
Post-transcriptional regulation of granulocyte-macrophage colony-stimulating factor synthesis in murine T cells.
Authors: Bickel M , Cohen R B , Pluznik D H
Published: December 2022

The granulocyte-macrophage CSF (GM-CSF) gene is known to be controlled at a variety of levels in different cell types. We showed previously that GM-CSF production by lectin or phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate (TPA]-treated T cells was unaffected by cyclosporin A whereas IL-2 and IL-3 expression were. Cyclosporin A is thought to inhibit transcription that suggests that IL-2 and IL-3 are regulated primarily at the transcriptional level while GM-CSF is not. The lack of coordinate gene expression is of particular interest because all three mRNA share the presence of adenosine uridine-rich sequences in the 3' untranslated region and these sequences are believed to act by modulating mRNA stability. We measured the level of GM-CSF mRNA in untreated cells and found it to be extremely low. GM-CSF mRNA levels increased approximately 60-fold within 6 h of TPA-treatment. Nuclear run-on transcription analysis of the same cells showed readily detectable GM-CSF transcription in unstimulated cells that increased less than twofold after TPA treatment. However, IL-2 transcription was insignificant before TPA addition. Actinomycin D chase experiments showed that GM-CSF transcripts in untreated cells have a very short half-life (approximately 45 min) although transcripts in TPA-treated cells have a half-life exceeding 3 h. These findings indicate that GM-CSF production in EL-4 cells treated with TPA is regulated predominantly by modulation of cytoplasmic mRNA half-life.
Differentiation of the IL-3-dependent NFS-60 cell line and adaption to growth in macrophage colony-stimulating factor.
Authors: Nakoinz I , Lee M T , Weaver J F , Ralph P
Published: December 2022

Macrophage CSF (M-CSF) induces responsive bone marrow precursors into rapid growth and differentiation to mature macrophages. Available cell lines that depend on M-CSF for growth are well differentiated and rather adherent. We investigated the effects of M-CSF on immature myeloid cell lines as models of the marrow precursors. The murine line NFS-60 requires IL-3 for growth and also responds to granulocyte-CSF and granulocyte-macrophage-CSF. Cultures of one NFS-60 subline, when switched from IL-3 to 10% L cell conditioned media, a source of M-CSF, or purified M-CSF, frequently acquired large, adherent cells. The adherent cells grew slowly in the presence of M-CSF, in contrast to the majority population of small, round, rapidly growing cells. The large cells had properties of differentiated macrophages that were absent in the nonadherent cells. Cells with macrophage phenotype were not observed in IL-3-supported cultures over many passages. A subline was derived from NFS-60 that grew rapidly and continuously in human or murine M-CSF as round, nonadherent cells. The line, called M-NFS-60, responded well to M-CSF and IL-3, weakly to granulocyte-CSF and not at all to murine granulocyte-macrophage-CSF, IL-4, or human IL-1. A mAb to human M-CSF specifically inhibited only M-NFS-60 proliferation induced by the human growth factor, whether produced by mammalian or bacterial cells. This study shows two effects of M-CSF on the IL-3-dependent NFS-60 line. Upon first exposure to M-CSF, cells may undergo global differentiation to slowly replicating macrophages in conditions we have not been able to define. The more common effect is rapid growth of immature myeloid cells like the bone marrow precursors, but with a block to differentiation. Thus, these cells may be useful as models of M-CSF-induced differentiation, and of permanently maintained macrophage precursors.
Role of IL-5 in IL-2-induced eosinophilia. In vivo and in vitro expression of IL-5 mRNA by IL-2.
Authors: Yamaguchi Y , Suda T , Shiozaki H , Miura Y , Hitoshi Y , Tominaga A , Takatsu K , Kasahara T
Published: December 2022

We recently demonstrated in vivo that IL-5 was an important mediator of eosinophilia in mice with parasite infections. In this study, we examined whether or not IL-5 was actually responsible for the eosinophilia induced by injection of human rIL-2. Mice administered hIL-2 developed eosinophilia during the course of the series of injections. This eosinophilia could be suppressed by a single injection of mAb against murine IL-5. The number of eosinophilic precursors increased more in the spleen cells of the IL-2-treated mice in comparison to the control mice, although in bone marrow precursors showed little change. Similarly, the number of granulocytic precursors increased markedly in the spleen cells of IL-2-treated mice. In vitro polymerase chain reaction amplification of cDNA subfragments corresponding to IL-5 mRNA (reverse transcription-polymerase chain reaction), followed by Southern blot analysis, revealed enhancement of IL-5 mRNA expression in spleen cells 3 days after starting the human IL-2 injections. We also observed enhanced murine IL-5 mRNA expression in spleen cells stimulated with IL-2 in vitro. The level of murine IL-5 mRNA expression by IL-2-stimulated spleen cells from normal mice increased at 24 h, reached a plateau after 48 h, and decreased again within 72 h of starting culture. These results indicate that the eosinophilia induced by IL-2 in vivo is presumably mediated by IL-5 released from IL-2-stimulated lymphocytes.
Homophilic binding of mouse monoclonal antibodies against GD3 ganglioside.
Authors: Chapman P B , Yuasa H , Houghton A N
Published: December 2022

R24, a mouse IgG3 mAb against GD3 ganglioside, was shown to bind to itself in a homophilic manner. This was demonstrated by augmented binding of 125I-labeled R24 to the cell surface of GD3+ cells by unlabeled R24 and by direct binding of biotinylated R24 to R24 adsorbed on solid phase. Although homophilic binding was evident when R24 was bound to solid phase, R24-R24 aggregates could not be detected in solution under otherwise identical conditions. R24 bound to four other mAb (two IgG3, one IgG2a, one IgM) directed against GD3 but did not bind to a panel of 21 other mAb including other IgG3 mAb and mAb directed against non-GD3 ganglioside. Evidence implicating the GD3-binding site of R24 in homophilic binding included the following observations: 1) F(ab')2 fragments of R24 could bind to R24, 2) an antiidiotypic mAb against the GD3-binding site of R24 inhibited R24 homophilic binding, 3) an IgM anti-GD3 mAb also demonstrated homophilic binding to R24, and 4) homophilic binding was a function of immunoreactivity and avidity for GD3. R24 variants with 40-fold lower avidity for GD3 demonstrated a similar decrease in homophilic binding. Inasmuch as R24 bound to R24 F(ab')2 fragments and specifically to anti-GD3 mAb, it appeared that the target for homophilic binding was an epitope within the V region of anti-GD3 mAb. It is likely that homophilic interactions result in increased affinity of R24 for GD3 through increased effective valency of antibody-Ag complexes.
Cytolytic T lymphocytes from the BALB/c-H-2dm2 mouse recognize the vesicular stomatitis virus glycoprotein and are restricted by class II MHC antigens.
Authors: Browning M J , Huang A S , Reiss C S
Published: December 2022

BALB/c-H-2dm2 mice (H-2KdI-AdI-EdDd), a congenic strain of BALB/c mice, have a deletion of the class I MHC Ag, H-2Ld. This gene encodes the exclusive class I MHC-restricting gene product for vesicular stomatitis virus-specific cytolytic T lymphocytes. When dm2 mice were immunized with infectious vesicular stomatitis virus, a specific CTL response was generated. These CTL lysed VSV-infected targets that expressed Iad gene products, but not VSV-infected Iad- targets. The CTL were used initially as long term cytolytic lines; 13 CTL clones were derived by limit dilution. All of the clones expressed the phenotype CD3+, CD4+, CD8-; some clones expressed TCR that are members of the V beta 8 family, others did not. The clones were restricted by class II MHC Ag, both I-Ad and I-Ed serving as restricting elements for individual clones of the panel. All of the clones derived from dm2 mice were specific for the immunizing serotype, Indiana, of VSV and did not lyse syngeneic cells infected with VSV of the New Jersey serotype. Studies using defective interfering virus particles, UV light-inactivated virus, and purified micelles of the viral glycoprotein indicated that infectious virus was not required for sensitization of target cells for immune recognition by the class II MHC-restricted CTL clones. Additional studies using recombinant vaccinia virus vectors to sensitize targets confirmed the specificity of the clones for the viral glycoprotein. These studies also demonstrated a cryptic population of class II-restricted CTL in BALB/c lines specific for VSV G. Naturally occurring variant viruses and mutant viruses, selected for escape from neutralization by mAb, were used in an effort to map the determinant(s) recognized; on the basis of patterns of target cell lysis, three groups of epitopes recognized by the clones were defined. Therefore, in the absence of the class I MHC Ag required for a CTL response to VSV, dm2 mice generated CTL with the CD4+ phenotype that recognized different epitopes on the viral glycoprotein, and lysed cells in a class II-MHC restricted, Ag-specific manner.
Lymphocyte-mediated activation of cultured endothelial cells (EC). CD4+ T cells inhibit EC class II MHC expression despite secreting IFN-gamma and increasing EC class I MHC and intercellular adhesion molecule-1 expression.
Authors: Doukas J , Pober J S
Published: December 2022

Endothelial cells (EC) were cocultured with allogeneic PBL, CD4+ T cells, or CD8+ T cells, and the degrees of EC activation induced examined by determining patterns of endothelial class I and class II MHC and intercellular adhesion molecule-1 (ICAM-1) expression. Coculture with PBL or CD8+ T cells uniformly increases class I MHC and ICAM-1 expression on all EC within a culture, but induces class II MHC expression on only a subpopulation(s) of EC. This heterogeneous EC response to coculture contrasts with the uniform class II expression on all EC induced by IFN-gamma in replicate wells. CD4+ T cells, when compared to equal numbers of unfractionated PBL or CD8+ T cells, are more effective at increasing class I MHC and ICAM-1 but are unable to induce class II MHC expression. The failure of CD4+ T cells to induce EC class II MHC Ag is not due to insufficient activation of the T cells, as PHA-activated CD4+ T cells also do not induce significant class II expression. In addition, conditioned media (CM) from CD4+ T cell/EC contain greater levels of immunoreactive IFN-gamma than do CM from PBL/EC cocultures. Rather, CD4+ T cells appear to actively inhibit the induction of EC class II Ag but not class I or ICAM-1 by IFN-gamma. Inhibition occurs at the time of induction, as CD4+ T cells are not capable of down-regulating previously induced class II Ag. CM from CD4+/EC (but not PBL/EC) cocultures also inhibits IFN-gamma induction of EC class II MHC expression. The inhibitory activity is generated during CD4+ T cell-EC cell contact, and is enhanced by PHA. The inhibitory activity(ies) of the CD4+/EC-CM is as yet unidentified, and is only minimally reversible by cocktails of neutralizing antibodies directed against TNF-alpha, TNF-beta (lymphotoxin), IFN-alpha and IFN-beta. In conclusion, CD4+ and CD8+ T cells are each effective activators of EC, but the patterns of activation produced by these subsets are quite distinct, largely due to generation of a soluble inhibitor(s) of class II MHC induction during coculture of CD4+ T cells with EC.
Protein F. A novel F(ab)-binding factor, present in normal liver, and largely released in the digestive tract during hepatitis.
Authors: Bouvet J P , Pirès R , Lunel-Fabiani F , Crescenzo-Chaigne B , Maillard P , Valla D , Opolon P , Pillot J
Published: December 2022

Significant percentages of patients suffering from non-A non-B hepatitis (43%) and B hepatitis (35%) were found to release an Ig-binding factor in their stools. This factor, which we called "protein F" was less frequently observed (20%) in patients suffering from other liver disorders, and was found in only 6.7% of healthy subjects (p less than 10(-7), less than 10(-4), and less than 0.03, respectively). The specificity of the detection test (a nonimmune ELISA-like assay) was confirmed by inhibition experiments. Binding was located on the F(ab) fragment of Ig, irrespectively of their isotype. Protein F was inactivated by pepsin, neuraminidase, and high concentrations of subtilisin, whereas it was resistant to trypsin and chymotrypsin. Molecular sieving by HPLC indicated an apparent molecular mass of 175 kDa. In preparative SDS-PAGE, the molecular mass was 85 kDa in favor of a dimer disrupted under dissociating conditions. Preparative IEF showed the isoelectric charge to lie between 3.9 and 4.1. Analysis of liver extracts from two patients suffering fron non-A non-B hepatitis, and from a transplant donor, revealed the presence of the factor in the three cases.
On the species specificity of the interaction of LFA-1 with intercellular adhesion molecules.
Authors: Johnston S C , Dustin M L , Hibbs M L , Springer T A
Published: December 2022

Species restrictions in immune cell interactions have been demonstrated both in Ag-specific responses of T lymphocytes and the phenomenon of natural attachment. To determine the possible contribution of adhesion receptors to these restrictions, we have studied binding between the murine and human homologues of LFA-1 (CD11a/CD18) and ICAM employing purified human LFA-1 and ICAM-1 (CD54) bound to solid substrates. Murine cell lines bind to purified human LFA-1 through ICAM-1 and at least one other counter-receptor. This provides evidence for multiple counter-receptors for LFA-1 in the mouse as well as in the human. In contrast to binding of murine ICAM-1 to human LFA-1, murine LFA-1 does not bind to human ICAM-1. The species specificity maps to the LFA-1 alpha subunit, because mouse x human hybrid cells expressing the human alpha subunit associated with a mouse beta subunit bind to human ICAM-1, whereas those with a human beta subunit associated with a murine alpha subunit do not. Increased adhesiveness for ICAM-1 stimulated by phorbol esters could be demonstrated for hybrid LFA-1 molecules with human alpha and murine beta subunits.
Affinity chromatography of catalytic autoantibody to vasoactive intestinal peptide.
Authors: Paul S , Volle D J , Mei S
Published: December 2022

An autoantibody in human IgG that hydrolyzes vasoactive intestinal peptide (VIP) was identified. The IgG did not hydrolyze VIP unless an unidentified inhibitor was removed by dialysis. The VIP antibody was fractionated by affinity chromatography on immobilized VIP by using IgG without VIP-hydrolytic activity as the starting material. The affinity-purified antibody catalyzed the hydrolysis of VIP (nominal kcat/Km: 1.1 X 10(6) M-1 min-1). The values of Km for the affinity-purified antibody preparation and unfractionated IgG (110 nM and 112 nM) suggested relatively tight antibody-VIP binding. A comparison of the reverse phase HPLC profiles of antibody-treated [Tyr10-125I]VIP with that of synthetic [125I]VIP(1-16) suggested that unfractionated IgG and the affinity-purified antibody cleaved the same peptide bond in VIP (Gln16-Met17).
Agents that elevate cAMP stimulate DNA fragmentation in thymocytes.
Authors: McConkey D J , Orrenius S , Jondal M
Published: December 2022

Increases in the cAMP level are often inhibitory in mature T lymphocytes and may be involved in the development of tolerance to self Ag. In this report, agents inducing an increase in the cAMP level by independent mechanisms were found to stimulate DNA fragmentation, characteristic of a suicide program known as apoptosis, in isolated thymocytes. Data obtained with cAMP analogs known to act synergistically to stimulate protein kinase A suggested that the latter directly mediated endonuclease activation. Agents previously shown to stimulate protein kinase C and to inhibit Ca2(+)-dependent, TCR-mediated thymocyte apoptosis, including IL-1, also blocked both DNA fragmentation and cell death in response to cAMP, suggesting interactions ("cross-talk") between the two protein kinase systems. As it has been proposed that apoptosis mediates negative cell selection in the thymus, our results indicate that cAMP may play a role in the development of functional mature T lymphocytes.
Identification and expression of the Tla region gene T11b and its Qa-like product.
Authors: Oudshoorn-Snoek M , Demant P
Published: December 2022

In spite of the large number of class I genes in the Qa-Tla region of the H-2 complex, only few membrane-bound Qa and TL Ag have been identified. We show that one of the Qa-Tla region genes, the T11b gene, is transcribed in lymphoid cells, lymphoma cell lines, teratocarcinoma cell lines, and L cells transfected with the cloned T11b gene. The T11b gene potentially encodes a polypeptide with normal class I characteristics. The product as present at the cell surface of L cells transfected with the cloned T11b gene, is a sialylated protein of m.w. 41,000, associated with beta 2-microglobulin. This T11b Ag shares epitopes with H-2K and H-2D molecules of various haplotypes and with Qa-2 molecules, but has distinct biochemical properties. RFLP analysis revealed that the T11b gene is found in mice of the Tlab and Tlaf haplotype. Genes homologous to, but distinct from, T11b (allelic or duplicated) are present in all Tla haplotypes tested.