bluuebunny/crossref_metadata_2025 huggingface dataset:
Lateral diffusion of antigen receptors artificially incorporated onto B lymphocytes.
Authors: Londo T R , Peacock J S , Roess D A , Barisas B G
Published: December 2022

In the companion paper, we have shown that palmitate conjugates of a monoclonal anti-DNP IgA (protein 315) incorporated onto B lymphocytes can bind DNP antigens and that this binding causes polyclonal B cell activation. In this study we use fluorescence photobleaching recovery (FPR) techniques to examine the lateral diffusion and mobile fractions of antigen-receptor complexes on receptor-decorated B cells as functions of antigen concentration and epitope density. Antigens used in this study are DNP conjugates of polymerized flagellin (DNP-POL) and linear dextran of 2 X 10(6) m.w. (DNP-DEX). The diffusion coefficient observed for antigen bound to artificial receptors decreases monotonically with increased antigen dose and epitope density. When the artificial receptor-bearing cells are labeled with either relatively high concentrations of medium epitope density antigen or high epitope density antigen, a large fraction of antigen-receptor complexes become immobile in the time scale of the experiment. We attribute this behavior to extensive receptor cross-linking by antigen. In parallel with these FPR experiments, we examined the effects of antigen concentration and epitope density on the polyclonal humoral response of receptor-decorated B cells. We found that the response is a function of both antigen concentration and epitope density similar to that seen in natural B cells. The combined results of these experiments show that cell activation results when the diffusion coefficient of the antigen-receptor complex ranges between 10 X 10(-11) cm2 sec-1 and 5 X 10(-11) cm2 sec-1. These values represent threefold and sixfold decreases from the diffusion coefficient of antigen-free receptors, respectively. However, when either a high antigen concentration or epitope density causes a large fraction of antigen-receptor complexes to become immobile, B cells become unresponsive not only to the bound antigen, but also to LPS. Results obtained in this study are very similar to those obtained in a study performed with natural antigen-specific B cells. Therefore, for the responding population of receptor-decorated B cells, it is possible that antigens activate and paralyze these B cells by mechanisms similar to those by which antigens regulate normal B cell responses.
Subcellular distribution of leukotriene C4 binding units in cultured bovine aortic endothelial cells.
Authors: Chau L Y , Hoover R L , Austen K F , Lewis R A
Published: December 2022

The subcellular distribution of specific binding sites for [3H]leukotriene C4 ([3H]LTC4) was analyzed after sedimentation of organelles from disrupted bovine aortic endothelial cells on sucrose density gradients and was shown to be in membrane fractions I (20% sucrose) and IV (35% sucrose). Saturation binding studies of [3H]LTC4 on endothelial cell monolayers at 4 degrees C demonstrated high-affinity binding sites with a dissociation constant (Kd) of 6.8 +/- 2.2 nM (mean +/- SD) and a density of 0.12 +/- 0.02 pmol/10(6) cells. At 4 degrees C, the specific binding of [3H]LTC4 by each of the subcellular fractions reached equilibrium at 30 min and remained stable for an additional 60 min. After 30 min of incubation with [3H]LTC4, the addition of excess unlabeled LTC4 to each subcellular fraction reversed more than 70% of [3H]LTC4 binding in 10 min. The [3H]LTC4 binding activities of subcellular fractions were enhanced approximately twofold to fourfold in the presence of Ca2+, Mg2+, and Mn2+, whereas Na+, K+, and Li+ were without effect. As measured by saturation experiments, the Kd and density of LTC4 binding sites in fraction I were 4.8 +/- 1.6 nM and 16.5 +/- 1.9 pmol/mg of protein, respectively, and in fraction IV were 4.7 +/- 1.5 nM and 81.4 +/- 19 pmol/mg of protein, respectively. Inhibition of [3H]LTC4 binding in membrane-enriched subcellular fractions I and IV by LTC4 occurred with molar inhibition constant (Ki) values of 4.5 +/- 0.1 nM and 4.7 +/- 1.2 nM, respectively, whereas Ki values for LTD4 were 570 +/- 330 nM and 62.5 +/- 32.8 nM, respectively, and for LTE4 were greater than 1000 nM for each fraction; LTB4 and reduced glutathione were even less active. FPL55712, a putative antagonist of the sulfidopeptide LT components of slow reacting substance of anaphylaxis, had Ki values of 1520 +/- 800 nM and 1180 +/- 720 nM for [3H]LTC4 binding sites on membrane-enriched subcellular fractions I and IV, respectively. Thus as defined by Kd, Ki, and specificity, the LTC4 binding units that are distributed to the plasma membrane and the binding units in the subcellular fraction of greater density were similar to each other. Pretreatment of the isolated subcellular membrane fractions with trypsin abolished [3H]LTC4 binding by fraction I, enriched for the plasma membrane marker 5' nucleotidase, and that by fraction IV, enriched for the mitochondrial membrane marker succinate-cytochrome C reductase.(ABSTRACT TRUNCATED AT 400 WORDS)
Growth inhibition and cytotoxicity of tumor necrosis factor in L929 cells is enhanced by high cell density and inhibition of mRNA synthesis.
Authors: Kirstein M , Fiers W , Baglioni C
Published: December 2022

L929 cells were growth-inhibited after 1 to 2 days of treatment with human recombinant tumor necrosis factor (rTNF). This effect of rTNF was largely reversible, and L929 cells resumed normal growth when rTNF was removed. The rTNF showed growth inhibitory and cytotoxic activity when L929 cells approached a high cell density and grew slowly. This was shown in experiments in which L929 cells approached confluency at different times after being seeded at increasing initial densities. The rTNF had little effect on the growth of cells seeded at the lowest density tested. L929 cells cultured to high density synthesized RNA at a reduced rate. This suggested that a reduced rate of RNA synthesis may be at least in part responsible for the growth inhibitory and cytotoxic activities of rTNF on cells grown to high density. Treatment with inhibitors of RNA synthesis potentiated the cytotoxic activity of rTNF. Inhibition of mRNA synthesis was apparently responsible for the enhanced sensitivity to rTNF, as shown by experiments with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an inhibitor of the synthesis of poly(A)-containing RNA.
Cells containing IgE in the intestinal mucosa of mice infected with the nematode parasite Trichinella spiralis are predominantly of a mast cell lineage.
Authors: Alizadeh H , Urban J F , Katona I M , Finkelman F D
Published: December 2022

To determine whether IgE+ cells in the intestinal mucosa of nematode-infected mice were of a mast cell or a lymphocyte lineage, the intestinal mucosae of mast cell-deficient w/wv mice were examined for IgE+ cells after inoculation with Trichinella spiralis muscle-stage larvae. Immunofluorescence staining techniques were used to detect IgE associated with cells in the intestinal mucosa. Comparisons were made among four strains of mice, w/wv (mast cell-deficient), +/+ (normal congenic littermates of w/wv), BALB/c, and SJL, that were either uninfected controls or inoculated with T. spiralis. Tissue sections from the small intestine of T. spiralis-infected BALB/c, SJL, and +/+ mice were fixed in ethanol and were stained with an affinity-purified F(ab')2 rabbit anti-mouse IgE followed by FITC goat anti-rabbit IgG. Large numbers of cells in the intestinal mucosa exhibited bright fluorescence. When other sections of intestines from these mice were processed in Carnoy's fixative and were stained with alcian blue at low pH (a metachromatic stain for mast cells) or alcian blue followed by immunofluorescence staining for IgE, large numbers of mast cells were observed in the intestinal mucosa, and 70 to 90% stained positively for IgE. There was a considerable number of cells in the intestinal mucosa which were IgE+ but which did not stain with alcian blue. Few alcian blue-positive cells and no IgE+ staining cells were present in the intestinal mucosa of control, uninfected +/+, BALB/c, and SJL mice. To determine whether these IgE+ alcian blue-negative cells were of a lymphocyte or a mast cell lineage, the mast cell-deficient w/wv mouse strain was examined after infection with T. spiralis. In contrast to BALB/c, SJL, or +/+ mice, few cells in the intestinal mucosa of T. spiralis-infected w/wv mice stained with alcian blue or were positive for IgE. However, when the IgE response in the MLN of the w/wv mice was compared to the IgE response of BALB/c, SJL, and +/+ mice, numerous IgE+ cells, but no alcian blue-positive cells, were observed in the parenchyma of the MLN from all four strains of T. spiralis-infected mice. In addition, flow microfluorometric analysis of MLN cells stained for surface IgE in suspension showed a comparable proportion of IgE-bearing cells, which were mostly B lymphocytes, among all four strains of T. spiralis-infected mice.(ABSTRACT TRUNCATED AT 400 WORDS)
Molecular cloning of the cDNA encoding the T3 gamma subunit of the mouse T3/T cell antigen receptor complex.
Authors: Krissansen G W , Owen M J , Fink P J , Crumpton M J
Published: December 2022

Complementary DNA (cDNA) clones encoding the T3 gamma subunit of the mouse T3/T cell antigen receptor complex were isolated from a cDNA library prepared from a cytochrome C-specific, I-E (class II antigen)-restricted murine T cell clone. The cDNA hybridized to a single mRNA transcript of 1 kb in size the expression of which was restricted to T lymphocytes. The 182-amino acid sequence deduced from the cDNA revealed a signal peptide, a 89-amino acid extracellular domain with one N-glycosylation site, a hydrophobic domain with a centrally located glutamic acid residue, and a 44-amino acid intracellular domain. Comparative analysis of the mouse T3 gamma sequence with those of the human T3 gamma chain and the related human and mouse T3 delta chains revealed that the regions incorporating the extracellular cysteine residues were highly conserved. More importantly, the mouse T3 gamma subunit retained the potential serine phosphorylation site (Ser 126) unique to the human T3 gamma subunit, in the membrane-proximal region of the intracellular domain. The 16-amino acid region flanking Ser 126 was totally conserved (100%) in the gamma subunits, yet showed little homology (38 to 44%) with the corresponding region in the delta subunits, in spite of the otherwise high degree of conservation (61 to 68%) between the remainder of the cytoplasmic domains of the gamma and delta chains.
Role of interleukin 2 (IL 2) and hemopoietin-1 (H-1) in the generation of mouse natural killer (NK) cells from primitive bone marrow precursors.
Authors: Migliorati G , Cannarile L , Herberman R B , Bartocci A , Stanley E R , Riccardi C
Published: December 2022

The development of natural killer (NK) cells from bone marrow (BM) precursors was studied. Recombinant interleukin 2 (IL 2) was able to induce the in vitro development of NK cells when added to cultures of mouse BM cells. Treatment of donor mice with 5-fluorouracil (150 mg/kg i.v.), which eliminates more differentiated cells but spares less differentiated cells, appears to augment NK cell development. The "NK stem cell" was found to be asialo GM1-, Thy-1+, Lyt-2-, and Lyt-1-. The cells generated in vitro had a typical phenotype of NK cells, being asialo GM1+, Lyt-5+, Thy-1+, Lyt-2-, and Lyt-1-. These effector cells also had specificity characteristics of NK cells lysing the NK-susceptible YAC-1 and K562 targets, but not the NK-resistant EL/4 or allogeneic and syngeneic blasts. Hemopoietin-1 (H-1), a factor which acts on very primitive multipotent BM cells, was able to cooperate with IL 2, increasing the development of NK cells. In contrast, other factors such as interleukin 3 or colony-stimulating factor did not cause induction of NK activity when added to cultures of BM cells, indicating that this effect, i.e., induction of NK cell development, is peculiar to IL 2. These results indicate that IL 2 can act as a differentiation as well as growth factor for NK cells, and that H-1 can promote the development of functional activity in a lymphocyte subpopulation as well as affect the differentiation of myelomonocytic and other cell lineages. This experimental system appears quite useful for characterization of BM precursors for NK cells, and should help to better understand the relationship of the NK cell lineage to the T cell or other lineages.
Cytokine activity after human bone marrow transplantation. Production of interferons by peripheral blood mononuclear cells from recipients of HLA-identical sibling bone marrow transplants.
Authors: Cooley M A
Published: December 2022

Peripheral blood mononuclear cells (PBMC) from 43 recipients of HLA-identical sibling bone marrow transplants were tested for their capacity to produce IFN after stimulation in vitro with PHA. Total IFN production, as measured in a bioassay, and production of IFN-gamma, measured by radioimmunoassay, was within or above the normal range (mean production by PBMC from normal persons +/- 2 SD) for 29 of 39 and 31 of 43 recipients, respectively. Production did not correlate with time after transplant, or with the presence or type of immunosuppressive chemotherapy used, although there was a tendency for lower production in recipients with chronic graft-vs-host disease. This near normal production of IFN contrasts with our previous observation that IL 2 production in bone marrow transplant recipients is severely impaired for at least 6 mo post-transplant.
Class II-restricted T cell responses in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. I. Cross-specificity among TMEV substrains and related picornaviruses, but not myelin proteins.
Authors: Miller S D , Clatch R J , Pevear D C , Trotter J L , Lipton H L
Published: December 2022

Following intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV), susceptible mouse strains develop a chronic demyelinating disease characterized by mononuclear cell-rich infiltrates in the central nervous system. Current evidence strongly supports an immune-mediated basis for myelin breakdown, with an effector role proposed for TMEV-specific, major histocompatibility class II-restricted delayed-type hypersensitivity, which temporally correlates with disease onset and remains chronically elevated in susceptible mice. This study examined the fine specificity of class II-restricted T cell responses in TMEV-infected mice to better define the relevant virus-encoded T cell determinant(s) responsible for triggering the demyelinating process, and to determine if class II-restricted neuroantigen-specific autoimmune responses could be detected in mice with TMEV-induced demyelination. The data clearly show that T cell responses in TMEV-infected mice are directed against determinants shared by closely related TMEV strains and are cross-reactive with related picornaviruses, such as encephalomyocarditis virus. In contrast, class II-restricted autoimmune responses against syngeneic mouse spinal cord homogenate and the two major protein components of myelin, myelin basic protein and proteolipid protein, are not demonstrable in susceptible SJL/J mice undergoing chronic TMEV-induced demyelinating disease, but are readily seen in SJL/J mice displaying chronic, relapsing experimental allergic encephalomyelitis. Cross-reactivity (or lack thereof), as determined by functional T cell analyses, was found to correlate with the extent of exact amino acid homology between the TMEV capsid proteins, the two neuroantigens, and related picornaviruses. The data thus do not support a major role for autoimmune responses against myelin proteins in TMEV-induced demyelinating disease, but are consistent with our previously proposed hypothesis that TMEV-specific T cell responses constitute a major effector mechanism of myelin breakdown.
Macrophage C1q: characterization of a membrane form of C1q and of multimers of C1q subunits.
Authors: Martin H , Heinz H P , Reske K , Loos M
Published: December 2022

It has been shown recently that C1q, a subcomponent of the first component of the classical complement pathway, is synthesized by macrophages and that endogenous C1q is detectable on the macrophage membrane. In this report, we demonstrate that membrane-associated C1q, which contains the A, B, and C chains of C1q, is structurally distinct from fluid-phase C1q in that the B chain of the membrane species is approximately 1000 m.w. less than its fluid-phase counterpart. By using biosynthetically ([3H]proline) labeled C1q from guinea pig peritoneal macrophages, we found that the membrane form of C1q is derived from already secreted C1q. The demonstration of a distinct membrane form of C1q supports earlier functional studies which implicated C1q as a membrane-associated molecule with receptor functions for those molecules which also interact with fluid-phase C1q, such as polyanions, immune complexes, and bacteria. Furthermore, we show that, in the vicinity of macrophages, C1q is very susceptible to oxidation manifested by the formation of disulfide bonds. By SDS-PAGE (nonreduced and reduced), we demonstrate the existence of disulfide-linked multimers (180,000 m.w., 360,000 m.w.) which are composed of the A, B, and C chains of C1q.
Antigen-driven T cell clones can proliferate in vivo, eradicate disseminated leukemia, and provide specific immunologic memory.
Authors: Klarnet J P , Matis L A , Kern D E , Mizuno M T , Peace D J , Thompson J A , Greenberg P D , Cheever M A
Published: December 2022

The aim of the current study was to determine the ability of antigen-driven cloned helper cell independent cytotoxic T lymphocytes (HITc) to proliferate and to survive in vivo and to mediate tumor therapy. The HITc clone utilized (denoted 1.B6) was specifically cytolytic to FBL-3, a syngeneic Friend virus-induced murine leukemia. Activation in vitro (48 hr) with FBL-3 induced secretion of interleukin 2 (IL 2), expression of IL 2 receptors (IL 2R), and in vitro proliferation. These cells could be "rested" for several weeks without stimulation, which resulted in reduced expression of IL 2R; however, restimulation with antigen resulted in reinduction of IL 2R and proliferation. The ability of cloned HITc to proliferate and to survive in vivo was examined in cyclophosphamide (CY) pretreated donor mice congenic for the Thy-1 gene. Adoptively transferred cloned HITc could be found in large numbers, and were widely distributed in vivo 1 wk after transfer. In tumor therapy, 1.B6 cells when injected into a site of tumor (i.p.) and used as an adjunct to CY were effective against disseminated FBL-3. In this circumstance, cloned 1.B6 cells could be recovered from cured mice 125 days after transfer and were shown to specifically lyse tumor and proliferate in vitro in response to FBL-3. Thus as an adjunct to CY, tumor-specific cloned HITc are capable of eradicating disseminated leukemia, persisting long-term in vivo, and providing specific immunologic memory.
Leukocyte-endothelial cell recognition: evidence of a common molecular mechanism shared by neutrophils, lymphocytes, and other leukocytes.
Authors: Lewinsohn D M , Bargatze R F , Butcher E C
Published: December 2022

The interaction of leukocytes with endothelial cells is intrinsic to the process of leukocyte extravasation, whether during the entry of blood polymorphonuclear leukocytes and monocytes into sites of acute and chronic inflammation, or during the homing of lymphocytes to lymphoid organs. A lymphocyte surface glycoprotein, defined by monoclonal antibody MEL-14, has been described that appears to mediate lymphocyte recognition of postcapillary venules in peripheral lymph nodes, and to control the migration of lymphocytes from the blood into these lymphoid organs. We now report that the antigenic determinant recognized by MEL-14 is present at high levels on other leukocytes as well, including neutrophils, monocytes, and eosinophils; and we demonstrate involvement of the MEL-14 antigen in neutrophil-endothelial cell interactions. MEL-14 immunoprecipitates a neutrophil surface protein of Mr approximately 100,000, similar in m.w. to the 80,000 to 90,000 dalton lymphocyte surface MEL-14 antigen, and it blocks the interaction of neutrophils with endothelial cells in an in vitro model of adhesion to postcapillary venules in lymph node frozen sections. Neutrophil binding to lymph node venules is also inhibited by PPME, a mannose-6-phosphate-rich yeast polysaccharide that is thought to mimic the endothelial cell ligand for the MEL-14-defined lymphocyte receptor. Interestingly, neither MEL-14 nor PPME exhibit a major effect on neutrophil binding to postcapillary venules in Peyer's patches, suggesting that as for lymphocytes, the neutrophil MEL-14 antigen is involved in recognition of tissue-specific endothelial determinants. Finally, we show that MEL-14 inhibits the capacity of neutrophils to migrate from the blood into sites of acute inflammation in the skin. These observations lead us to propose that receptors for tissue-specific endothelial determinants are utilized by neutrophils and lymphocytes and probably other leukocytes during the physiologic process of leukocyte extravasation in vivo.
In vivo and in vitro activation of alveolar macrophages by recombinant interferon-gamma.
Authors: Black C M , Catterall J R , Remington J S
Published: December 2022

In vivo administration of recombinant interferon-gamma (rIFN-gamma) was previously shown to result in activation of the microbicidal activities of peritoneal macrophages (PM phi). Because macrophages at different anatomical sites vary in their functional capacities, we considered it of interest to determine whether administration of murine rIFN-gamma, either in vitro or in vivo, can enhance the microbicidal activity of resident alveolar macrophages (AM phi) and to compare the effects of rIFN-gamma on AM phi and PM phi. After incubation in vitro with rIFN-gamma, the antimicrobial activities of both murine AM phi and PM phi were enhanced, as assessed by their ability to inhibit replication of the intracellular parasite, Toxoplasma gondii. This effect was dose dependent for AM phi over a range of 0.1 to 1 U/ml and for PM phi over a range of 0.5 to 1000 U/ml. In this assay, the minimum dosage required for in vitro activation of AM phi was one-half that required for activation of PM phi, suggesting a greater sensitivity of AM phi to the in vitro activity of rIFN-gamma. Macrophages from both anatomical sites were also activated when rIFN-gamma was administered in vivo. This effect was dose dependent over a range of 10(3) to 10(5) U/mouse. Freshly harvested AM phi and PM phi from mice injected 24 hr earlier with 10(4) U rIFN-gamma by either the i.v. or i.p. routes markedly inhibited intracellular multiplication of Toxoplasma. In contrast, AM phi and PM phi from control mice permitted fourfold to ninefold increases in numbers of intracellular Toxoplasma. The anti-toxoplasma activity of AM phi and PM phi gradually diminished over a period of 3 days when assayed at successive 24 hr periods after a single i.v. injection of rIFN-gamma. At 3 days after injection, a substantial loss of anti-toxoplasma activity was observed with PM phi as compared with controls; residual anti-toxoplasma activity was still demonstrable in AM phi at 3 days. These results demonstrate that in vitro as well as in vivo treatment with rIFN-gamma confers on AM phi an enhanced antimicrobial activity. These findings provide a rationale for evaluating rIFN-gamma in the treatment of pulmonary infections, especially those due to opportunistic pathogens against which AM phi play a major role in host defense.
Identification of a lymphocyte-activating pentapeptide sequence in the Fc region of human IgG1.
Authors: Hobbs M V , Morgan E L , Houghten R A , Thoman M L , Weigle W O
Published: December 2022

The synthetic peptide p23, representing residues 335 to 357 in the Fc region of human IgG1, was previously shown to induce Ig secretion in murine spleen cell cultures. In this report, overlapping peptides based on the sequence of p23 were synthesized to further map the active site in this molecule. The results from these studies indicate that leu-pro-pro-ser-arg (residues 351 to 355) retained the B cell differentiation-inducing properties of p23; however, expression of activity by this sequence was markedly influenced by N-flanking sequences. By using T cell-depleted spleen cell cultures, it was determined that at least two signals are required for p23-induced Ig secretion: one supplied by p23 directly and one supplied by a T cell-replacing factor present in p23-conditioned spleen cell supernatants. Both signals were mapped into the sequence leu-pro-pro-ser-arg. However, the latter signal, but not the former signal, again appeared to be influenced by sequences proximal to the active site. These data indicate that although the leu-pro-pro-ser-arg sequence is able to provide both required signals for p23-induced Ig secretion in spleen cell cultures, there may be subtle differences in how the cell types involved in this response interact with and/or are activated by this sequence.
The chemotherapeutic effect of praziquantel against Schistosoma mansoni is dependent on host antibody response.
Authors: Brindley P J , Sher A
Published: December 2022

To assess the role of host humoral immune responses in the mechanism of action of praziquantel (PZQ) against Schistosoma mansoni, the efficacy of the drug was compared in infected B cell-depleted (mu-suppressed) vs immunologically intact C3H/HeN mice. We found that PZQ was on the average only 20% as effective in eliminating adult schistosomes from mu-suppressed as compared with control animals. Indeed, in three of four experiments performed, the drug failed to significantly reduce adult worm burdens in the mu-suppressed mice. These results were not due to a delay in parasite death in the infected B cell-depleted animals, because adult worms recovered from these mice as late as 7 wk after chemotherapy were indistinguishable in number and appearance from those recovered from non-drug-treated animals. The efficacy of PZQ against schistosomes in mu-suppressed mice was completely restored by passive transfer of immune serum from donor mice infected for 6 wk and partially restored with IgG purified from the same sera. Moreover, IgG as well as IgM antibodies were detected by immunofluorescence on the surface of adult worms recovered from intact mice as early as 1 hr after administration of the drug in vivo. The tubercles of the male worms appeared to be a major site for antibody binding. These results formally demonstrate that the mechanism of action of PZQ, the most important anti-schistosomal compound in current use, involves a synergy between the drug and the humoral immune response of the host, and suggest that the relevant effector antibodies act directly against parasite antigens which become exposed on the surface of the worms as a consequence of interaction with the drug.
Suppressed expression of surface Ia on macrophages by lipopolysaccharide: evidence for regulation at the level of accumulation of mRNA.
Authors: Koerner T J , Hamilton T A , Adams D O
Published: December 2022

The surface expression of class II major histocompatibility molecules (immune associated or Ia antigens) is an acquired property of macrophages, essential to their ability to interact effectively with T lymphocytes. Surface expression of Ia is induced by stimulants such as interferon-gamma and is suppressed by agents such as lipopolysaccharide (LPS). Recent studies on several cultured cell lines indicate that interferon-gamma can heighten cellular levels of mRNA encoding Ia, and the level of such mRNA may represent an important regulatory focus for controlling expression of surface Ia. Murine peritoneal macrophages were treated with interferon-gamma and/or LPS and expression of Ia mRNA determined by Northern blot analysis with a probe specific for the murine beta-chain of I-A. mRNA specific for I-A beta was not detectable in explanted macrophages obtained from sites of sterile inflammation but was induced by treatment of purified recombinant interferon-gamma. This effect was dose dependent and was optimal by 24 hr after stimulation. Ia-specific mRNA preceded the surface expression of Ia as monitored by a radioimmunoassay using a monoclonal antibody specific for I-A beta. When a physiologic dose of LPS was added concomitantly with the interferon-gamma, the time course of induction if Ia-specific mRNA was not altered, but the amount of such mRNA detected was suppressed 40 to 80%. This effect was dependent on the dose of LPS, and the levels of mRNA correlated closely with subsequent surface expression of Ia. The ability of LPS to suppress both mRNA and cell surface Ia expression required that the suppressive agent be added within 12 hr of the inducing stimulus. This is the time frame during which accumulation of mRNA occurs. Thus the data demonstrates that accumulation of specific mRNA is a major regulatory focus governing expression of Ia both by interferon-gamma and LPS.
A peptide secreted by human alveolar macrophages releases neutrophil granule contents.
Authors: MacArthur C K , Miller E J , Cohen A B
Published: December 2022

A monoclonal antibody was developed against an 8,000-kDa enzyme-releasing peptide (ERP) released from human alveolar macrophages. ERP was isolated on an immunoaffinity column containing the antibody bound to staphylococcal protein A-Sepharose. Release of ERP from the macrophages is not changed by plastic adherence, phagocytosis, calcium ionophore, or phorbol esters. The peptide was not antigenically similar to interferon-gamma, tumor necrosis factor, or interleukin 1 alpha or 1 beta. The release of constituents from azurophilic and specific granules was the main identified biologic function of ERP. ERP was a more effective secretagogue in the untreated neutrophils and f-met-leu-phe was more effective in the cytochalasin B-treated neutrophils. Absorption of ERP from macrophage-conditioned medium removed a small amount of the chemotactic activity; however, the immunopurified peptide was not chemotactic or chemokinetic for neutrophils, and at high concentrations, it suppressed base line chemokinesis. Treatment of washed macrophages with trypsin released active ERP of approximately the same m.w. of spontaneously secreted ERP. These studies showed that human alveolar macrophages release a peptide which is a secretagogue for human neutrophils under conditions which may be encountered in the lungs during certain disease states. Proteolytic enzymes which are free in the lungs may release the peptide and lead to the secretion of neutrophil enzymes.
Combined oral/nasal immunization protects mice from Sendai virus infection.
Authors: Nedrud J G , Liang X P , Hague N , Lamm M E
Published: December 2022

Based on the concept of a common mucosal immune system wherein mucosal associated lymphocytes traffic among the various mucous membranes, the murine gastrointestinal tract was immunized with Sendai virus antigens in order to elicit a virus-specific immune response in the respiratory tract. Multiple intragastric (oral) administration of live or killed Sendai virus induced IgA and IgG antiviral antibodies in both gastrointestinal secretions and serum. When cholera toxin as an adjuvant was included along with virus, gut IgA and IgG as well as serum IgA responses were enhanced. Antiviral antibodies induced in respiratory secretions by oral killed virus plus cholera toxin, however, were variable and protection from virus challenge was not demonstrated. Significantly higher levels of respiratory antiviral antibodies were induced if immunization with oral killed Sendai virus/cholera toxin was combined with intranasal administration of small amounts of killed virus. The combined immunization also resulted in protection of both the upper and lower respiratory tracts from virus infection. Protection of the upper respiratory tract was correlated with the presence of IgA antiviral antibodies in nasal washings. On the other hand, protection of the lower respiratory tract was correlated with IgG antiviral antibodies in bronchoalveolar lavage fluids. Immunization with intranasal killed virus alone conferred partial protection to the lower respiratory tract and no protection to the upper respiratory tract. Thus, oral immunization with killed virus antigen could prime for a protective immune response in the murine respiratory tract and this protective response included IgA antibodies.
Insulin-specific human T cells. Epitope specificity, major histocompatibility complex restriction, and alloreactivity to a diabetes-associated haplotype.
Authors: Miller G G , Pollack M S , Nell L J , Thomas J W
Published: December 2022

T cells from an insulin-treated diabetic (ML, HLA DR1, w6) were stimulated in vitro with insulin, cloned at limiting dilution, and examined for their fine specificity and genetic restriction. T cell lines (TCL) derived from beef insulin stimulation were highly specific for epitopes on beef insulin, whereas pork insulin stimulation generated T cells that recognized determinants shared with beef insulin. Included among TCL reactive with pork insulin is one line (P2/9) that is autoreactive with human insulin. Antigen-presenting cells of known HLA type and monoclonal antibodies directed at class II major histocompatibility complex antigens were used to confirm the role of HLA-DR in restricting the response of insulin immune T cells. No preference or determinant selection within the donor's haplotypes was identified because either DR1 or DRw6 antigen-presenting cells could present the A loop of beef insulin. A TCL that recognized the A loop of beef insulin in association with DR1 was also alloreactive to HLA DR3, or a molecule closely linked to it, in the absence of insulin. A second T cell clone with insulin specificity and alloreactivity was also derived by allo stimulation of the donor's cells with DR3+ cells. When tested with a series of DR3+ stimulator cells, the alloreactivity was directed at diabetes-associated haplotypes. These data show that the T cell repertoire for insulin of a single diabetic donor encompasses that of multiple inbred animal strains and includes fine specificity for one to two amino acids, recognition of autologous insulin, and cross-reactivity with an allogeneic major histocompatibility complex antigen.
Human tumor necrosis factor potentiates human interleukin 1-mediated rat pancreatic beta-cell cytotoxicity.
Authors: Mandrup-Poulsen T , Bendtzen K , Dinarello C A , Nerup J
Published: December 2022

Previous studies have established that the cytokine interleukin 1 (IL-1) is selectively cytotoxic for isolated human and rat pancreatic beta-cells. This observation raises the possibility that insulin-dependent diabetes mellitus is in part due to immunologically mediated mechanisms involving IL-1. However, other cytokines are produced during immunologic responses. To study possible modulatory effects of other cytokines on IL-1-mediated beta-cell cytotoxicity, we added human recombinant IL-1 alpha and beta (rIL-1 alpha, rIL-1 beta), tumor necrosis factor (rTNF), lymphotoxin (rLT), and interferon-gamma (rIFN-gamma) separately or in combinations to the culture medium of isolated rat islets of Langerhans. A half-maximal inhibition of glucose-stimulated insulin release after 7 days of culture was obtained with 100 pg/ml of rIL-1 beta, whereas 1000 pg/ml of rIL-1 alpha were necessary to obtain an equivalent effect. While ineffective in causing inhibition of beta-cell function or morphologic damage to islets alone 2.5 to 25 ng/ml of rTNF, but not 40 ng/ml of rLT, or 25 ng/ml of rIFN-gamma markedly potentiated the inhibition of beta-cell secretory response and dissolution of islet integrity caused by rIL-1 alpha and beta. The potentiating effect of rTNF was more pronounced if the rTNF was added after 60 min of preincubation of the islets with rIL-1 beta, than if rIL-1 beta was added after 60 min of preincubation with rTNF. rTNF did not interfere with the activity of rIL-1 alpha or beta on lymphocytes. Combinations of rIFN-gamma and rTNF or rLT did not affect beta-cell function. In conclusion, rTNF strongly potentiates the functional inhibition of beta-cells and the morphologic disintegration of islets caused by rIL-1 in vitro. These data, seen in context with previous observations of rIL-1-mediated beta-cell cytotoxicity, suggest that macrophages present in the intra-islet mononuclear cell infiltrate in insulin-dependent diabetes mellitus may secrete monokines that could be important effector molecules in beta-cell destruction.
Cholera toxin and pertussis toxin regulate the Fc receptor-mediated phagocytic response of human neutrophils in a manner analogous to regulation by monoclonal antibody 1C2.
Authors: Gresham H D , Clement L T , Volanakis J E , Brown E J
Published: December 2022

Data presented in this paper indicate that polymorphonuclear leukocyte (PMN) Fc receptor-mediated phagocytosis can be markedly augmented and that this augmentation is under regulatory control. Stimulation of PMN with either a low m.w., heat-labile cytokine(s) (the culture supernatant effluent from a YM-10 Centricon unit, YM-10E), phorbol esters (phorbol dibutyrate), or the polyene antibiotic, amphotericin B, enhances Fc-mediated ingestion in a dose-dependent manner. YM-10 effluent- and amphotericin B-stimulated ingestion is completely abrogated by treating the PMN with either pertussis toxin (PT), cholera toxin (CT), or a monoclonal antibody (mAb), 1C2. However, neither toxin nor mAb 1C2 affects nonstimulated ingestion or phagocytosis stimulated by phorbol esters or synthetic diacylglycerol. Increasing intracellular cyclic adenosine monophosphate levels by stimulation with prostaglandin E1 and the phosphodiesterase inhibitor, isobutylmethylxanthine, does not mimic the effect of either toxin or mAb 1C2. In addition, toxin-mediated inhibition is not due to loss of either the Fc receptor recognized by mAb 3G8 or the antigen recognized by mAb 1C2. These data indicate that both CT and PT regulate the phagocytic response of PMN, in a manner like mAb 1C2, probably by affecting a guanosine 5'-triphosphate-binding protein distinct from those that regulate adenylate cyclase. Since phorbol ester-stimulated ingestion is not inhibited by either PT, CT, or mAb 1C2 and phorbol esters activate protein kinase C directly, phagocytosis amplification regulated by PT, CT, and mAb 1C2 may involve protein kinase C activation.
Isolation and characterization of a protective antigen for adjuvant-free immunization against Schistosoma mansoni.
Authors: King C H , Lett R R , Nanduri J , el Ibiary S , Peters P A , Olds G R , Mahmoud A A
Published: December 2022

A single, 68,000 m.w. glycoprotein antigen from adult Schistosoma mansoni was purified by immunoaffinity chromatography with the use of a newly developed, protective, anti-schistosome murine monoclonal antibody. Immunization with two doses of 0.5 microgram or 1 microgram of purified antigen, without adjuvants, afforded a mean 28% reduction in parasite recovery in CF1 mice, and 2-% reduction in parasite BALB/c mice. On immunoblotting, the 68,000 m.w. antigen was common to S. mansoni adults and schistosomula, whereas parasite eggs contained only cross-reacting low m.w. antigens of 19,100 and 16,000. Immunization resulted in the development of anti-antigen antibody and enhanced immediate cutaneous hypersensitivity to the 31-3B6 antigen. By contrast, delayed-type hypersensitivity and sensitization to circumoval granuloma formation were not observed in immunized mice. It was concluded that the 68,000 m.w. 31-3B6 antigen represents a candidate vaccine for adjuvant-free immunization against S. mansoni.
Differential effects of antibodies to Lyt-2 and L3T4 on cytolysis by cloned, Ia-restricted T cells expressing both proteins.
Authors: Jones B , Khavari P A , Conrad P J , Janeway C A
Published: December 2022

Cloned T cell lines specific for the antigen ovalbumin (OVA) in the context of self I-A or I-E class II MHC-encoded molecules were found to express equivalent levels of the Lyt-2 and L3T4 surface molecules by FACS analysis. Functionally, these cloned T cell lines will kill OVA-pulsed, class II MHC-bearing B lymphoma cells. This system allowed us to examine the relative contribution of the Lyt-2 and L3T4 molecules to recognition of antigen in the context of self class II MHC molecules. We find that anti-L3T4 is 25- to 100-fold more potent at inhibiting cytotoxicity by these cloned lines than is anti-Lyt-2, where potency is calculated as the ratio of the concentration needed for inhibition divided by the concentration needed for binding. Both antibodies can completely inhibit cytotoxicity. These results suggest that the accessory molecules Lyt-2 and L3T4 can play two different roles in antigen:MHC recognition. In class II MHC recognition, L3T4 plays a dominant role, highly sensitive to inhibition by anti-L3T4. By contrast, Lyt-2 plays a minor yet important role in the cell interaction, perhaps by adhesion strengthening. Previous analysis in several laboratories supports the concept that the ligand for L3T4 is class II MHC, whereas the ligand for Lyt-2 is class I MHC. The present results are consistent with the hypothesis that these molecules are actually part of the T cell antigen:MHC recognition complex, and play an important role in the class of MHC molecule recognized by a T cell alpha:beta heterodimeric receptor. As such, they are not accessory proteins, but a direct part of the recognition apparatus, and the term accessory protein may apply only in those cases in which the specificity of the T cell receptor is for a different class of MHC than that normally associated with the L3T4 or Lyt-2 molecule.
Unresponsiveness of MRL/MP-lpr/lpr mice to antigen given subcutaneously in adjuvant: partial restoration of response after local injection of B cells.
Authors: Ron Y , Isakov N , Sprent J
Published: December 2022

MRL/lpr mice were used as a model for seeking information on the role of B cells as antigen-presenting cells in vivo. In confirmation of the finding of other workers, MRL/lpr mice with pronounced lymphadenopathy failed to undergo T cell priming in lymph nodes (LN) after s.c. injection of keyhole limpet hemocyanin (KLH) in complete Freund's adjuvant (CFA): thus, in contrast to normal mice or young MRL/lpr mice, the LN cells from older MRL/lpr mice failed to give secondary T proliferative responses to the injected antigen in vitro. Because previous work from this laboratory has shown that B cells play a major role in priming T cells in LN of normal mice, we tested the possibility that the lack of T cell priming to KLH seen in older MRL/lpr LN reflected the relative paucity of B cells. To test this idea, MRL/LPR mice were injected s.c. with B cells taken from normal or young MRL/lpr mice 1 day before priming with KLH/CFA. In the case of old (20 to 24 wk) MRL/lpr mice with massive lymphadenopathy, prior injection of B cells had virtually no effect in promoting LN priming. In marked contrast, injection of B cells into mice exhibiting less-pronounced LN enlargement (mice tested at 14 to 16 wk) was highly effective in restoring LN priming; before B cell injection, the LN of these mice contained only 2 to 5% B cells. These findings add to the evidence that T cell priming in LN is heavily dependent on the presence of B cells.
Co-culture of human lung-derived mast cells with mouse 3T3 fibroblasts: morphology and IgE-mediated release of histamine, prostaglandin D2, and leukotrienes.
Authors: Levi-Schaffer F , Austen K F , Caulfield J P , Hein A , Gravallese P M , Stevens R L
Published: December 2022

Human mast cells, dispersed from lung tissue by proteolytic treatment and enriched to a purity of 23 to 68% by density-gradient centrifugation, were maintained ex vivo for up to 13 days when co-cultured with mouse skin-derived 3T3 fibroblasts in RPMI 1640 containing 10% fetal calf serum. The human mast cells were adherent to the fibroblast cultures within 2 to 4 hr after seeding, and after 7 days of co-culture were localized between the layers of fibroblasts. The cell surfaces of the mast cells and the fibroblasts did not form tight junctions, but rather approached within 20 nm of each other. The co-cultured mast cells did not divide; they maintained their cellular content of histamine and TAMe esterase and resembled in vivo mast cells in that their secretory granules exhibited scroll patterns and their nuclei were oval. Both the freshly isolated and the co-cultured mast cells responded to activation with anti-human IgE by exocytosing histamine and generating and releasing arachidonic acid metabolites. When freshly isolated mast cells were activated immunologically, they exocytosed 38 +/- 8% of their total histamine content and released 28 +/- 1.9 ng (mean +/- range, n = 2) of immunoreactive equivalents of prostaglandin D2 (PGD2) per microgram of total cellular histamine, but did not generate significant amounts of either leukotriene C4 (LTC4) or leukotriene B4 (LTB4). The 1-wk co-cultured mast cells, on the other hand, exocytosed 43 +/- 2.4% of their total histamine content, and released 86 +/- 10, 43 +/- 20, and 5.2 +/- 5.2 ng (mean +/- SD, n = 4) of immunoreactive equivalents of PGD2, LTC4, and LTB4, respectively, per microgram of histamine. Thus, human lung-derived mast cells can be maintained ex vivo when co-cultured with fibroblasts, and, when treated with anti-IgE, they metabolize arachidonic acid via both the cyclooxygenase and the 5-lipoxygenase pathways.
Autoimmune syndrome after induction of neonatal tolerance to alloantigens: effects of in vivo treatment with anti-T cell subset monoclonal antibodies.
Authors: Merino J , Schurmans S , Luzuy S , Izui S , Vassalli P , Lambert P H
Published: December 2022

BALB/c (H-2d) mice rendered tolerant to h-2b alloantigens by neonatal injection of semiallogeneic (C57BL/6 X BALB/c)F1 spleen cells develop autoimmune features due to an abnormal activation of persisting F1 donor B cells. The role of T cells in this autoimmune syndrome was studied by in vivo treatment of tolerant mice with anti-L3T4(GK-1.5) or anti-Ly-2 (H-35-17.2) monoclonal antibodies. The treatment of tolerant mice from day 2 to day 21 of life with anti-L3T4 MAb completely prevented the occurrence of circulating immune complexes of anti-ssDNA anti-Sm and anti-hapten (FITC) IgG antibodies as well as the glomerular deposition of Ig that were usually seen in untreated tolerant mice. This effect persisted for at least 6 wk after stopping this treatment. When the injections of anti-L3T4 MAb were delayed until day 15 of life, a very significant decrease of the autoimmune manifestations was still observed. Treatment of tolerant mice with anti-Ly-2 MAb during the same period had no effects on the autoimmune disease as compared with untreated tolerant mice. No effects on the maintenance of tolerance vs H-2b alloantigens were observed after treatment with anti-L3T4 MAb, as followed by the decrease of CTL and CTL-p alloreactivity and by the persistence of F1 donor B cells, indicated by the presence of Ig bearing the Ighb donor allotype. These results suggest the existence of interactions between L3T4+ T cells and persisting autoreactive B cells from F1 donor origin in the development of the autoimmune syndrome after neonatal induction of transplantation tolerance.