bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced production of human gamma-interferon in nylon column-fractionated cell cultures.
Authors: van Reis R , Lubiniecki A S , Olson R A , Stromberg R R , Madsen J A , Friedman L I
Published: December 2022

The production of human gamma-interferon (HuIFN-gamma) in unfractionated and nylon wool column-fractionated leukocyte cell cultures stimulated with PMA and PHA was investigated. Production was studied with normal and reduced autologous serum protein levels in 96-hr spinner cultures. A 10- to 15-fold enhancement of production and a 50-fold increase in specific activity of crude HuIFN-gamma was demonstrated in nylon column-fractionated/reduced serum cell cultures. Kinetic analysis revealed a production rate maximum within 6 hr of induction in unprocessed cell cultures, whereas production occurred at an essentially constant rate for 48 hr in fractionated cell cultures.
Glucocorticoid inhibition of lymphokine secretion by alloreactive T lymphocyte clones.
Authors: Kelso A , Munck A
Published: December 2022

The effect of glucocorticoids on lymphokine production by T lymphocytes was examined by using long-term alloreactive T cell clones that secreted one or more of the lymphokines interleukin 2 (IL 2), interferon-gamma, macrophage-activating factor (MAF), and colony-stimulating factor when stimulated by an antigen or a mitogen. Production of all of these four lymphokines was inhibited when glucocorticoids were added at physiologic concentrations (10(-8) to 10(-6) M) to clones stimulated with concanavalin A (Con A). Clones were heterogeneous with respect to their sensitivity to glucocorticoid inhibition of MAF production; cytolytic clones were generally more resistant than noncytolytic clones. The glucocorticoid dexamethasone (Dex) and an IL 2-containing supernatant exerted opposing effects on clonal MAF production. Kinetics experiments showed that Dex inhibited MAF production by reducing the rate of secretion without causing a compensatory increase in the duration of secretion, whereas the IL 2 source increased the rate and the total amount of MAF secretion. Dex abrogated the effect of IL 2. Inhibition by Dex was apparent from the earliest time of detectable MAF production (about 4 hr after stimulation) and increased with longer exposure until production ceased (12 to 24 hr). Pre-exposure and removal of Dex before Con A stimulation also inhibited MAF release. Effects of Dex on lymphokine secretion by clones could be dissociated from effects on their growth in response to stimulator cells and IL 2. Factor production by the 16 clones tested was inhibited to some degree. Proliferation, however, by two of these clones (both cytolytic) was unaffected by Dex, whereas proliferation of two noncytolytic clones was strongly inhibited even in the presence of a saturating dose of IL 2.
Effect of glucocorticosteroids on epidermal cell-induced immune responses.
Authors: Aberer W , Stingl L , Pogantsch S , Stingl G
Published: December 2022

Recent reports indicate that pharmacologic doses of glucocorticosteroids induce structural alterations in epidermal Langerhans cells. In this study we hoped to determine whether steroid-induced changes in Langerhans cell surface characteristics are paralleled by alterations in Langerhans cell-dependent immunologic functions of epidermal cells. We found that both topically and systemically administered steroids led to a dose-dependent reduction in the number of Ia-bearing epidermal cells. This numerical decrease was paralleled by a substantial impairment of Langerhans cell-dependent immunologic functions of epidermal cells in that their capacity to induce antigen-specific, syngeneic, and allogeneic proliferation of T cells from non-steroid-treated animals was substantially reduced. The capacity of epidermal cells to generate ETAF activity, however, was not adversely affected by the steroid treatment. After cessation of treatment, Langerhans cell numbers and Langerhans cell-dependent in vitro functions slowly and gradually returned to normal values. We propose that the ability of glucocorticosteroids to interfere with the generation of T cell-dependent immune responses may be due, at least in part, to their interference with antigen-presenting cell function.
Suppression of IgE synthesis in mouse plasma cells and B cells by rat IgE-suppressive factor.
Authors: Uede T , Huff T F , Ishizaka K
Published: December 2022

Rat IgE-binding factors were assessed for the ability to regulate the IgE response of mouse spleen cells. A rat-mouse T cell hybridoma, 23B6, produced two species, i.e., 15,000 (15K) daltons and 30K daltons, of rat IgE-binding factors upon incubation with rat IgE. The 15K IgE-binding factor selectively suppressed the in vitro IgE response of DNP-KLH-primed BALB/c spleen cells to homologous antigen without affecting the IgG1 response, whereas the 30K IgE-binding factor failed to affect both the IgE and IgG1 responses. Incubation of IgE-producing hybridoma cells with the 15K IgE-binding factor resulted in a parallel decrease in the proportion of IgE-secreting cells, cytoplasmic IgE-containing cells, and sIgE+ cells. The same IgE-binding factor suppressed IgE formation by mouse plasma cells in DNP-KLH-primed or KLH-primed BDF1 mice without affecting the IgG1-forming cells. In contrast, the 30K IgE-binding factor failed to affect the formation of IgE by the hybridoma and the plasma cells. It was also found that incubation of the splenic lymphocytes with the 15K IgE-binding factor resulted in a decrease in sIgE+ B cells; the 30K IgE-binding factor failed to do so. The results indicate that the binding of the 15K IgE-binding factor to plasma cells and to sIgE+ B cells results in suppression of IgE synthesis in these cells.
Human neutrophils and eosinophils have structurally distinct Fc gamma receptors.
Authors: Kulczycki A
Published: December 2022

Human Fc gamma receptors were isolated from surface radioiodinated granulocytes and eosinophils by using repetitive affinity chromatography on human IgG-Sepharose columns. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated that cell preparations containing eosinophils possessed a 43,000 Mr Fc gamma-binding macromolecule. Nylon wool-filtered cells from patients with eosinophilia and cell cultures derived from normal donors provided highly purified eosinophil preparations that expressed only the 43,000 Mr Fc gamma receptor. Granulocyte populations yielded the 52,000 to 68,000 Mr Fc gamma receptor characteristic of neutrophils as well as the Fc gamma-binding macromolecules apparently derived from eosinophils. The 43,000 Mr Fc gamma receptor of the eosinophil and the 31,000 and 34,000 Mr fragments that appear to be derived from it were able to rebind selectively to human IgG1-Sepharose, Fc gamma 1-Sepharose, IgG3-Sepharose, and Fc gamma 3-Sepharose. In contrast, the 52,000 to 68,000 Mr Fc gamma receptor from neutrophils could rebind only to IgG1-Sepharose and Fc gamma 1-Sepharose. The results demonstrate that the Fc gamma receptor of human eosinophils is distinct in structure from the neutrophil Fc gamma receptor and that these Fc gamma receptors, at least in their solubilized states, differ in specificity for human IgG3.
Immunochemical studies of the purified human platelet receptor for the alpha 1(I)-chain of chick skin collagen.
Authors: Chiang T M , Kang A H , Dale J B , Beachey E H
Published: December 2022

The membrane receptor of human platelets that binds the alpha 1(I)-chain of chick skin collagen was purified to homogeneity by molecular sieve chromatography, affinity chromatography, and preparative gel-electrophoresis. Rabbits that were immunized with the purified receptor emulsified in complete Freund's adjuvant produced antibody, as measured by an enzyme-linked immunosorbent assay. The antiserum was shown to be highly receptor-specific by immunoprecipitation of solubilized platelet membrane protein that had been labeled with (gamma-32P)ATP and protein kinase. Platelet aggregation induced by alpha 1(I) as well as native chick skin collagen was inhibited by both the purified receptor and antibody against the receptor in a dose-dependent fashion. In contrast, neither the receptor nor the antiserum inhibited ADP-induced aggregation. These studies provide the most definitive evidence that alpha 1(I) and native collagen interact specifically with a cell surface receptor on human platelets to induce aggregation.
C-reactive protein inhibits pneumococcal activation of the alternative pathway by increasing the interaction between factor H and C3b.
Authors: Mold C , Kingzette M , Gewurz H
Published: December 2022

We previously studied two alternative pathway activators, Streptococcus pneumoniae and positively charged liposomes, which react with C-reactive protein (CRP). Binding of CRP to these surfaces initiates classical pathway but blocks alternative pathway activation. In this study, we investigated the mechanism of this inhibition using S. pneumoniae, R36a. R36a were pretreated with CRP (CRP-R36a) or buffer and incubated with C2-deficient human serum to which 125I-labeled C3 had been added. The amount of specific 125I-C3 binding was decreased from 8200 mol/CFU on R36a to 2200 mol/CFU on CRP-R36a. In contrast, when the same experiment was performed with purified factors B, D, P, and C3, in the absence of regulatory proteins, specific 125I-C3 uptake was slightly lower on R36a (6100 mol/CFU) than on CRP-R36a (8100 mol/CFU). The ability of the fixed C3b to inactivate factor B in the presence of factor D was equivalent on the two surfaces. The binding of the regulatory factor H to C3b fixed to R36a and CRP-R36a was compared by using purified 125I-labeled factor H. The ratio of factor H bound to C3 bound was twofold greater on CRP-R36a than on R36a. This increase was found by using C2-deficient serum or purified factors B, D, P, and C3 to fix C3b to the surfaces. The ability of CRP to inhibit C3 binding to R36a was restored by the addition of factors H and I to factors B, D, P, and C3. These results indicate that CRP inhibits alternative pathway activation by increasing regulation of bound C3.
Activation of murine macrophage cell lines. Possible involvement of protein kinases in stimulation of superoxide production.
Authors: Kiyotaki C , Bloom B R
Published: December 2022

The mechanism by which phorbol myristate acetate (PMA) activates mononuclear phagocytes to produce O2- was investigated in two cloned murine macrophage-like cell lines. J774.16 cells produce O2- upon stimulation by PMA and aggregated IgG, but a variant clone derived from it, J774.C3C cells, do not, probably because of a functionally defective cytochrome b. 4-O-methyl 12-O-tetradecanoyl-phorbol-13-acetate (4-O-methyl TPA) did not stimulate O2- production in either clone. Studies on the ionic requirements for production of O2- indicated that the extracellular Ca2+ and Mg2+ concentrations did not affect O2- production of J774.16 cells. Furthermore, calcium flux studies with the use of 45Ca2+ indicated that PMA caused no detectable influx of Ca2+, produced a modest release of presumed loosely cell-associated 45Ca2+, but did not detectably change the rate of Ca2+ efflux. By using quin 2 acetoxymethyl ester, no evidence could be adduced that PMA caused intracellular Ca2+ redistribution. Because membrane potential changes are seen in other systems of activation, membrane potential changes in the macrophages stimulated by PMA were measured both by means of the fluorescence ionophore, dis-S-C3-(5), and by direct measurement by using a K+-electrode. Under conditions that stimulated O2- production, no membrane depolarization was observed. Under the conditions for stimulating O2- production, PMA also did not induce increased turnover of membrane phospholipids or arachidonic acid. Although J774.16 and J774.C3C produce prostaglandin D2 at resting state, PMA neither released arachidonic acid from membrane lipids, nor induced release of prostaglandins and thromboxane at least for 10 min after stimulation. Similarly, PMA failed to increase transmethylation of phospholipids or break down the methylated phospholipids. PMA and aggregated IgG, however, induced protein phosphorylation in both J774.16 and J774.C3C cells. Among these, mainly two sets of phosphorylated proteins with m.w. of 42,000 and pI 6.85 to 7.3 and m.w. of 58,000 and pI 8.15 to 9.3 are common in both stimuli. Biologically inactive 4-0-methyl TPA did not induce phosphorylation of these proteins. From the above results, we conclude that PMA induces O2- production in a macrophage line without detectable changes in membrane potential; Ca2+ flux, intracellular Ca2+ increase or redistribution, or increase of membrane lipid turnover. Because two stimuli that induce O2- production in J774.16 cells induced phosphorylation of common proteins, whereas a biologically inactive analog did not, protein kinases, possibly protein kinase C, may be involved in the activation for O2- production.
Cellular immunity induced by avirulent Salmonella in LPS-defective C3H/HeJ mice.
Authors: Eisenstein T K , Killar L M , Stocker B A , Sultzer B M
Published: December 2022

An avirulent strain of Salmonella, SL3235, has been shown to confer high levels of immunity on lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice. Immunized mice were also protected against challenge with Listeria monocytogenes, indicating that the Salmonella vaccine activates macrophages. It was shown that protection and macrophage activation occurred without correction of the LPS defect, as assessed by in vivo endotoxin toxicity, in vitro spleen cell mitogenicity, and the ability of in vivo treatment with LPS to enhance in vitro macrophage ingestion of C3b-coated erythrocytes. It is concluded that LPS responsiveness is neither a necessary nor a sufficient condition for Salmonella immunity, and that macrophage activation can apparently occur in C3H/HeJ mice in the face of a sustained LPS defect.
In vitro T cell-mediated killing of Pseudomonas aeruginosa. I. Evidence that a lymphokine mediates killing.
Authors: Markham R B , Goellner J , Pier G B
Published: December 2022

Previous studies have demonstrated in vivo that T cells can provide protective immunity, in the absence of antibody, against infection with the extracellular Gram-negative bacterium Immunotype 1 (IT-1) Pseudomonas aeruginosa. We established an in vitro system in which immune T cells, after reexposure to bacterial antigens and to macrophages, secrete a product that kills the bacteria. Although macrophages are required for in vitro killing, they function neither as antigen-presenting nor as phagocytic cells in this system. T cells from animals immunized against a different P. aeruginosa immunotype will not kill IT-1 organisms; but the supernatants produced by IT-1 immune T cells after exposure to macrophages and IT-1 P. aeruginosa organisms are nonspecifically effective in killing unrelated bacteria. Because the supernatants from immune T cells lose their bactericidal properties upon minimal dilution, we conclude that if this mechanism is active in vivo, it must play a role in local immunity.
Junctional diversity is essential to antibody activity.
Authors: Jeske D J , Jarvis J , Milstein C , Capra J D
Published: December 2022

Many mechanisms of antibody diversification have been shown to exist, including combinatorial pairings of heavy and light chains, the use of multiple gene segments (combinatorial diversity), and the imprecise joining of these gene segments (junctional diversity). The contributions of each of these mechanisms to functional antibody activity has not been fully explored, especially in the case of junctional diversity. A chain recombination experiment between an anti-arsonate monoclonal antibody and an anti-oxazolone molecule in which light chains differ essentially only at the V/J junctional position show that junctional diversity may play an important role in antigen binding.
Antigen-specific T contrasuppressor factor in cell-mediated immunity: interactions leading to eradication of the tolerant state.
Authors: Ptak W , Bereta M , Ptak M , Gershon R K , Green D R
Published: December 2022

Interactions between a T cell-derived, antigen-specific, contrasuppressor factor (TcsF) and immune T cells that block the action of T suppressor factors and allow the transfer of cellular immunity into tolerant recipients are described. Immune T cells from contact-sensitized donors are capable of transferring specific immunity into normal recipients but not into animals rendered tolerant to the specific antigen. Brief exposure of the immune cells to the TcsF enables the effective transfer of immunity into such tolerant recipients. In addition, treated immune cells become resistant to subsequent exposure to T suppressor factor (capable of inhibiting transfer of immunity to normal recipients). A cyclophosphamide-sensitive, I-J+, Ly-2 T transducer cell is required in the immune donor cell population for contrasuppression to be induced by the TcsF plus specific antigen. These cells release an antigen-non-specific contrasuppressive factor capable of rendering immune targets, depleted of transducer cells, resistant to suppression (either by suppressor factor or in the tolerant recipient). The results indicate that contrasuppression in contact sensitivity is antigen specific and that the balance of suppression and contrasuppression determines tolerance vs responsiveness in this system. The symmetrical resemblance of the contrasuppressive interactions to those of suppression in contact sensitivity are discussed.
Antigen-specific xenogeneic CTL activity in the Syrian hamster.
Authors: O'Hara R M , Duncan W R
Published: December 2022

Previous studies of the Syrian hamster have demonstrated a lack of cytotoxic T lymphocyte (CTL) activity in vaccinia virus-infected animals. Our laboratory has reexamined CTL activity in both the classical inbred strains, MHA, CB, and LSH, as well as the recently inbred strain MIT. Primary and secondary CTL specific for the immunizing antigen have been detected after in vitro culture in MIT but were not demonstrable in the classical strains. Only lymph node cells of the responding animal demonstrated this activity, spleen cells being phenotypically devoid of such a response. Identification of the cell responsible for cytolysis as a T cell was demonstrated by nylon wool nonadherence, specificity on Con A blasts, and the lack of surface immunoglobulin, as demonstrated by cell-sorter analysis.
Regulation of antibody release by naturally occurring anti-immunoglobulins in cultures of pokeweed mitogen-stimulated human peripheral blood lymphocytes.
Authors: Birdsall H H , Rossen R D
Published: December 2022

Immunoregulatory influences of human anti-immunoglobulins (anti-Ig) were studied in cultures of peripheral blood lymphocytes (PBL) from 11 normal donors. Pokeweed mitogen (PWM)-stimulated PBL released anti-Ig specific for Fab or Fc fragments of IgG, often within the first 24 to 72 hr in vitro. PBL that released more than 1 ng/ml IgM anti-Fab during the first 72 hr in vitro ultimately produced significantly less antibody (Ab) by the 12th day than PBL that released no detectable IgM anti-Fab during the first 3 days in culture. Adding affinity-purified human anti-Fab to PWM-stimulated PBL also suppressed the later Ab release by these cells. Suppression was polyclonal, affecting IgM anti-Fc, IgM anti-Fab, and IgM anti-tetanus toxoid Ab, and was directly dependent on the quantity of anti-Fab added. Anti-Fab Ab, isolated from single donor sera, were more suppressive, nanogram for nanogram, than were equal quantities of IgG anti-Fab obtained from Cohn Fraction II, when added to autologous donor PBL in vitro. Affinity-purified IgM anti-Fc, from pooled rheumatoid arthritis patient sera, also suppressed Ab release by PWM-stimulated PBL in a dose-dependent manner. These observations suggest that anti-Ig may exert a significant immunoregulatory role in man that can override to some extent the T cell-dependent stimulus for polyclonal B cell activation provided by PWM.
The effect of in vitro UV irradiation on the production of IL 1 by murine macrophages and P388D1 cells.
Authors: Ansel J , Luger T A , Kock A , Hochstein D , Green I
Published: December 2022

Ultraviolet irradiation (UV) exposure may lead to the development of multiple immunologic defects. One such defect is a dysfunction of normal antigen-presenting cell (APC) activation of T lymphocytes after whole body or in vitro UV. Although the mechanism of this interaction is not clearly defined, several possibilities have been suggested. One proposal is that UV may inhibit or abrogate the APC IL 1 signal and thus prevent normal T cell activation. To investigate this possibility further, we examined the functional consequences of UV on murine peritoneal adherent cell (PAC) activation of a cloned antigen-specific T cell hybridoma (A2.2.E10). In agreement with previous reports, we found a marked UV-induced inhibition of PAC activation of A2.2.E10 after sublethal UV. To correlate this UV-APC dysfunction with UV alterations of IL 1 production, both the IL 1-producing murine macrophage cell line P388D1 and normal murine PAC were exposed to various amounts of in vitro UV and the 24-hr post-UV IL 1 activity production of these cells was determined. The results surprisingly indicated that certain amounts of sublethal UV may actually augment the production of IL 1 activity, by using a dose range that clearly inhibits antigen presentation. This UV-induced activity was cycloheximide-sensitive, suggesting that de novo protein synthesis rather than release from cells was responsible for the increased IL 1 activity. In addition, the UV-induced IL 1 activity had a m.w. of 14K, consistent with previous reports, and demonstrated pyrogen activity when tested in the rabbit pyrogen assay. Thus UV clearly inhibits normal APC function; however, this may not be due to abrogation of IL 1 production, but rather the result of UV toxicity for other complex events involved in antigen presentation.
Localization of macrophage agglutination factor activity to the gelatin-binding domain of fibronectin.
Authors: Godfrey H P , Angadi C V , Wolstencroft R A , Bianco C
Published: December 2022

We previously proposed that macrophage agglutination factor (MAggF, a T cell-derived guinea pig lymphokine) is a fibronectin (FN). We now show MAggF binding to gelatin and to peritoneal macrophages is mediated by domains similar to corresponding domains of plasma FN. MAggF activity in lymphokine concentrates prepared by two different methods differed nearly 10-fold in m.w. on gel filtration chromatography. Despite this difference, MAggF dose-activity curves of both preparations were parallel, and MAggF in both preparations bound reversibly to gelatin and to monoclonal anti-guinea pig FN immunoadsorbents. MAggF activity in one preparation was inhibited by the addition of soluble monoclonal antibody specific for the gelatin-binding domain of human FN; inhibitory activity of this antibody was blocked by purified guinea pig plasma FN or partially purified MAggF from the other preparation. Measured MAggF activity of both preparations was reduced in a dose-dependent manner by pretreatment of indicator macrophages with monoclonal anti-human monocyte FN receptor antibody or F(ab')2 fragments or with guinea pig plasma FN. Neither anti-FN receptor antibody nor plasma FN interacted directly with MAggF. Indirect immunofluorescence studies confirmed the presence of uncomplexed plasma membrane receptors for FN on indicator macrophages in MAggF-responsive populations that were able to bind added FN. Our identification of MAggF as lymphokine FN provides a basis for future biochemical analysis of delayed hypersensitivity inflammatory reactions.
Isolation and characterization of the third component of complement in the serum of the clawed frog, Xenopus laevis.
Authors: Sekizawa A , Fujii T , Katagiri C
Published: December 2022

The hemolytic activity against SRBC in the serum of normal Xenopus is dependent on specific antibody and both Ca++ and Mg++, whereas the activity against RRBC is dependent on Mg++ alone. Both of these hemolytic activities disappeared after treatment of the serum with zymosan or with the specific rabbit antiserum against one of the zymosan-binding proteins in Xenopus serum. By using this antiserum as a probe, a complement component (XC) was purified as a single entity from the Xenopus plasma after polyethylene glycol precipitation, DEAE-Sepharose CL-6B, Sepharose CL-6B, and Sephadex G-200 column chromatographies. The XC, contained at 2.3 mg/ml in normal serum, showed an electrophoretic mobility of beta-globulin, with a m.w. of 204,000 (204K) comprising two distinct subunits of 125K and 85K, which are linked with each other by disulfide bonds. The 204K protein exhibited a strong hemolytic activity in association with other components in Xenopus serum. Digestion of 204K protein by trypsin resulted in a specific cleavage of the 125K subunit and a conversion of its immunoelectrophoretic mobility to the anodal side, leaving the 85K subunits intact. The treatment of XC with SDS and urea resulted in the splitting of 125K subunits into 78K and 40K, but this splitting was inhibited upon pretreatment with methylamine, suggesting the presence of a thiol ester bond in the XC. The amino acid composition of the XC revealed a striking resemblance to that of mammalian C3. In all aspects, the 204K protein (XC) is regarded as representing the C3 of Xenopus laevis, which plays a key role in both the classical and alternative hemolytic pathways.
Inhibition of immune precipitation by complement.
Authors: Hong K , Takata Y , Sayama K , Kozono H , Takeda J , Nakano Y , Kinoshita T , Inoue K
Published: December 2022

Normal human complement serum (NHS) inhibited precipitin reactions between tetanus toxoid and human or rabbit anti-tetanus toxoid IgG antibody, between bovine serum albumin (BSA) and rabbit anti-BSA IgG antibody, and between hen egg albumin and rabbit anti-egg albumin IgG antibody. Ethylene-diaminetetraacetic acid (EDTA) prevented this inhibition. Mg-ethyleneglycol-bis(aminoethyl)-tetra-acetic acid-(EGTA) also prevented the inhibition except with lower concentrations of antibody and antigen. Therefore, the inhibition of immune precipitation seemed to occur mainly through the classical pathway of complement activation. The alternative pathway was usually dispensable, but it augmented the inhibition. Guinea pig complement serum (NGS) was less effective than NHS in inhibiting immune precipitation. Guinea pig serum deficient in C4 (C4DGS) did not inhibit the immune precipitation. Mouse complement serum was effective for inhibiting precipitation, and C5-deficient serum was as effective as normal serum. Therefore, the inhibition of immune precipitation is considered to occur by activation of complement up to the step of C3. The size of the soluble immune complexes formed in the presence of NHS varied depending on the concentrations of antibody and antigen, even when the ratio of antigen to antibody was constant. On incubation at 37 degrees C immune precipitation was inhibited by 1/2 dilution of NHS for 2 to 3 hr and then gradually increased to the level in the absence of complement. When the immune complexes were formed in the presence of serum containing complement, fragments of C4 and C3 were incorporated into the soluble immune complexes. The C3 fragments incorporated into the soluble complexes were C3b, iC3b, C3c, and C3d, some of which were bound covalently with heavy chains of IgG antibody molecules. Some of the covalent linkages between C3 fragments and IgG seemed to be destroyed by alkali treatment, but not by hydroxylamine treatment. The formation of covalent bonds between IgG and C3 and probably C4 was essential for inhibition of immune precipitation, because inhibitors of their formation, such as putrescine, cadaverine, and salicylhydroxamic acid, effectively prevented the inhibition of precipitation. When antigen and antibody reacted in the presence of mixtures of various combinations of isolated complement components, C1, C4, C2, and C3 showed maximal inhibition of immune precipitation, whereas factors I and H had little effect.
Stereospecificity of leukotriene B4 and structure-function relationships for chemotaxis of human neutrophils.
Authors: Dahinden C A , Clancy R M , Hugli T E
Published: December 2022

The chemotactic activity of leukotriene B4 (5S, 12R Dihydroxy 6, 14 cis, 8, 10 trans eicosatetraenoic acid) (LTB4) was examined by using a sensitive Boyden-chamber assay. The activity of LTB4 was compared to other biosynthetic stereoisomers: 5S, 12R Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (6-trans LTB4); 5S, 12S Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (12-epi-6-trans LTB4), 5S, 12S DiHETE; the metabolic product 20-Hydroxy LTB4 (20-OH LTB4); methylated LTB4 (Methyl-LTB4), and the related monoHETE 5-HETE and 12-HETE. The compounds were purified by several steps of reverse phase and straight phase HPLC. The LTB4 exhibits measurable chemotactic activity at 10(-9) M with maximal activity at 10(-7) M and an ED50 of 10(-8) M. The LTB4 isomers and monoHETE were less chemotactic than previously reported. The monoHETE (5-HETE and 12-HETE), the isomer 12-epi-6-trans LTB4, and 5S, 12S DiHETE fail to attract neutrophils at levels between 10(-6) and 10(-5) M. If these compounds are chemotactic, then activity is at least four orders of magnitude less than that of LTB4. The isomer 6-trans LTB4 at 10(-6) to 10(-5) M induced chemotaxis with an extrapolated ED50 value of 10(-5) M, indicating that a trans for cis change in configuration at position 6 reduces the chemotactic activity of LTB4 by 1000-fold. Conversely, the metabolic product 20-OH LTB4 is at least as active as the native compound LTB4. Methylation of the carboxyl group of LTB4 reduces its chemotactic activity by two orders of magnitude. These results indicate a high degree of stereospecificity for the LTB4 receptor with strict dependence on hydroxyl group, and triene configuration and considerable dependence on the carboxyl group. Modification at the aliphatic omega end of the LTB4 molecule has a minimal effect on function, suggesting that the hydrophobicity of this portion of the molecule is not important for optimal activity. Furthermore, we propose that metabolic products of LTB4 may be of greater importance than LTB4 as physiologic inflammatory mediators in vivo.
Neutrophil degranulation detected by right angle light scattering: spectroscopic methods suitable for simultaneous analyses of degranulation or shape change, elastase release, and cell aggregation.
Authors: Sklar L A , Oades Z G , Finney D A
Published: December 2022

We have used simultaneous spectrometric analysis of right angle scattering and elastase release from human neutrophils to demonstrate the similarity of these two measures of degranulation. Both responses depend on the presence of cytochalasin B, and are similar in kinetics, dose-response, and dependence on receptor occupancy at the formyl peptide receptor. This scattering response is shown to be largely independent of cell aggregation. In the absence of cytochalasin B, a rapid and transient right angle scatter response of a different character, probably associated with cell ruffling, is detected. Either right angle response can be detected by flow cytometry.
Exacerbation of murine cutaneous leishmaniasis by adoptive transfer of parasite-specific helper T cell populations capable of mediating Leishmania major-specific delayed-type hypersensitivity.
Authors: Titus R G , Lima G C , Engers H D , Louis J A
Published: December 2022

The effect of adoptive transfer of in vitro-propagated Leishmania major-specific T cell populations on the course of experimentally induced cutaneous leishmaniasis was studied in mice. The L. major-specific T cells expressed the T helper/inducer phenotype and were able in vitro to a) mount a specific proliferative response, b) provide specific helper activity for antibody responses, c) activate parasitized macrophages resulting in L. major destruction, and d) secrete macrophage-activating factors as tested in a tumoricidal assay. These T cells were also found capable of transferring parasite-specific delayed-type hypersensitivity responses to normal syngeneic mice. Results indicated that the i.v. transfer of these L. major-specific T cell populations into normal syngeneic mice exacerbated cutaneous lesions induced by infection with L. major. This effect on the disease process appeared to be dependent upon recognition of parasite antigens by the injected T cells because no exacerbation of the disease process was seen after the transfer of similar T cell populations specific for an antigen unrelated to the parasite, namely ovalbumin. However, the inclusion of ovalbumin in the L. major infecting inoculum resulted in an exacerbating effect of ovalbumin-specific T cells on cutaneous leishmaniasis. These unexpected results were supported by observations showing that immunization of mice with L. major antigens in complete Freund's adjuvant 7 days before infection with L. major led to exacerbated lesions. A similar aggravation of L. major-induced cutaneous lesions was also observed in mice previously immunized with an unrelated antigen provided that this antigen was included in the L. major infecting inoculum.
Interaction of reovirus with cell surface receptors. IV. The reovirus type 3 receptor is expressed predominantly on murine Lyt-2,3+ and human T8+ cells.
Authors: Epstein R L , Finberg R , Powers M L , Weiner H L
Published: December 2022

Reovirus type 3 binds to approximately 20% of murine and human T cells via the viral hemagglutinin, a small outer capsid polypeptide. By using purified viral particles as a ligand in a standard plate separation technique, we have been able to enrich human peripheral blood and murine splenic T cells for reovirus receptor-positive cells (reovirus 3+) to levels of 88 to 92%. Analysis of reovirus 3+ T cells with monoclonal antibodies that identify inducer and suppressor/cytotoxic cells demonstrated that in the mouse, 68% of reovirus 3+ cells were Lyt-2+, and in the human, 60% were T8+. In reciprocal experiments, when subpopulations of murine and human T cells were prepared with the use of monoclonal anti-T cell reagents, 16% of Lyt-1+ and 81% of Lyt-2+ cells bound reovirus, whereas 30% of T4+ and 65% of T8+ cells bound reovirus. To determine whether reovirus type 3 identified a functional as well as a phenotypic category of cells, an antigen-specific cytotoxic T cell assay was employed. There was complete loss of cytotoxic activity in the reovirus 3+ cell population and slight enhancement of cytotoxic activity in the cell population from which reovirus 3+ cells were removed. This suggested that reovirus was binding to functionally active suppressor cells. Furthermore, adoptive transfer of antigen-specific T cells that were enriched for reovirus 3+ cells demonstrated suppression of cytoxic T cell activity. These results suggest that reovirus type 3 may identify a structure common to a subclass of murine and human T cells and that by using the virus as a natural biologic probe for cell surface receptors, one may be able to functionally segregate murine cytotoxic from suppressor T cells.
Molecular regulation of MHC class III (C4 and factor B) gene expression in mouse peritoneal macrophages.
Authors: Sackstein R , Colten H R
Published: December 2022

To study the molecular regulation of C4 and factor B synthesis in mouse peritoneal macrophages, mouse C4 cDNA clones isolated from an H-2d haplotype liver cDNA library, and a previously described mouse factor B cDNA clone, pBmB2 (9), were used to assess quantitative and qualitative differences in C4 and factor B mRNA in resident and elicited cells. The C4 clones that were isolated, pBmS2 (1 Kb) and pBmS10 (0.9 Kb), overlap and together span a 1.5 Kb coding region of mouse pro-C4, extending from the alpha-chain through the gamma-chain; four nucleotide substitutions are evident in comparing 316 bp of the sequence of clone pBmS10 to that of a previously described mouse C4 clone, pMLC4/w7-2 (23). By using these probes, Northern blot analysis of total cellular RNA revealed similar C4 mRNA levels in resident peritoneal macrophages from high-C4 (B10.A) and low-C4 (C3HeB) strains. Pulse and pulse-chase studies of C4 and factor B synthesis were performed on resident, starch-elicited, and thioglycollate-elicited peritoneal macrophages at two culture time periods, 0 to 9 and 24 to 33 hr, and total cellular RNA was isolated from each population at 4.5 and 28.5 hr of culture for Northern blot analysis of C4 and factor B mRNA content. The data demonstrate that as previously reported, C4 production decreases in elicited compared with resident macrophages and decreases with time in culture; however, factor B synthesis does not differ among resident and elicited cells and it increases with time in culture. The variations in C4 and factor B production by mouse peritoneal macrophages are not associated with alterations in C4 and factor B protein processing, catabolism, or secretion; rather, they are a function of differences in net amounts of C4 and factor B mRNA. These data provide direct evidence that the regulation of expression of these class III MHC genes in mouse peritoneal macrophages is a pretranslational event.
A glycopeptide extract can inhibit cytotoxic T lymphocyte-target cell conjugation in an H-2-restricted manner.
Authors: Pimlott N J , Miller R G
Published: December 2022

Glycopeptides extracted from P815, a DBA/2 (H-2d) mastocytoma, were able to inhibit conjugate formation between anti-H-2d CTL and an H-2d target, but were not able to inhibit conjugate formation between anti-H-2k or anti-H-2b CTL and an appropriate target. This genetically specific effect was seen for CTL generated in primary bulk cultures and for long-term CTL lines, and was observed regardless of the strain combinations used to generate CTL. The phenomenon was quantitatively reproducible between independent experiments and between three separate preparations of P815 glycopeptide. These results suggest that recognition by CTL involves cell surface carbohydrate groups, and that the structure of these groups is influenced by H-2.
Cloned cytolytic T cells can suppress primary cytotoxic responses directed against them.
Authors: Fink P J , Rammensee H G , Bevan M J
Published: December 2022

In vivo and in vitro, murine peripheral T cells can suppress or "veto" the activation of cytotoxic T lymphocytes directed against antigens presented by those T cells. This suppression is antigen-specific and H-2-restricted. The recognition event initiating this suppression appears to be unidirectional; precursors of cytotoxic T lymphocytes recognize the antigen-bearing veto cell and are thereby inactivated--the veto cell need not recognize the CTL precursor. We show here that 3/3 cytolytic T cell clones can exert veto activity in vitro on normal spleen cells which do not bear antigens the T cell clones can recognize. This suppression results in greatly diminished cytotoxic activity generated during a primary 5-day mixed lymphocyte culture against antigens which the veto cell expresses, but not against third-party antigens present in the same culture. In this same system, a noncytolytic T cell clone will not serve as a source of veto cells. Secondary cytotoxic responses are relatively resistant to the veto cell activity of cloned cytolytic T cells. The cloned veto cells do not suppress the generation of cytotoxic activity directed against antigens they recognize (and presumably carry over via antigen-specific receptors). Cold target competition during the cytotoxic assay has been eliminated as a possible mechanism for T cell clone-induced suppression, and suppression cannot be reversed by the addition to the mixed lymphocyte cultures of supernatants from concanavalin A-activated spleen cells. It is suggested that this mechanism of inactivating primary cytotoxic T lymphocyte responses could play an important role in the maintenance of self-tolerance and in the induction and maintenance of tolerance to allografts.
Sequential substages of natural killer cell-derived cytolytic factor (NKCF)-mediated cytolysis as defined by glutaraldehyde modulation of the target cell.
Authors: Deem R L , Targan S R
Published: December 2022

The substages of natural killer cell-derived cytolytic factor (NKCF)-mediated cytolysis have been examined by using the bifunctional protein cross-linking reagent glutaraldehyde. Glutaraldehyde (2 X 10(-4%)) enhanced both the level and rate of lysis mediated by NKCF when the factor was prebound to the L929 target cell. However, glutaraldehyde enhanced lysis only when added at specific time points (2 to 4 hr after addition of NKCF, which was 6 to 8 hr before any lysis occurred), indicating that NKCF-mediated lysis is a dynamic process. When 10(-3%) glutaraldehyde was added at the same time as NKCF, inhibition of lysis was observed. Pretreatment of L929 target cells, but not of NKCF, with glutaraldehyde resulted in inhibition of NKCF-mediated cytolysis. In addition, it was shown that glutaraldehyde-pretreated L929 and K562 cold target cells were not as effective in competing for binding of NKCF in a cold target competition assay, or as effective in absorbing NKCF at 4 degrees C, indicating that glutaraldehyde modified the NKCF receptor to block binding of NKCF to the target cell. Thus, NKCF-mediated cytolysis can be divided into at least four substages based upon sequential modulation by glutaraldehyde: 1) NKCF binding stage (inhibited by glutaraldehyde), 2) glutaraldehyde-enhanceable nonlytic stage, 3) glutaraldehyde-nonenhanceable nonlytic stage, and 4) glutaraldehyde-nonenhanceable lytic stage.
Response of human B cells to Staphylococcus aureus Cowan I: T-independent proliferation and T-dependent differentiation to immunoglobulin secretion involve subsets separable by rosetting with mouse erythrocytes.
Authors: Ito S , Lawton A R
Published: December 2022

We separated T-depleted mononuclear cells into subsets by rosetting with mouse erythrocytes and studied proliferation and differentiation responses to Staphylococcus aureus Cowan I (SAC), pokeweed mitogen (PWM), and a combination of the two polyclonal activators. All of the T cell-independent proliferation of unfractionated B cells in response to SAC was attributable to mouse erythrocyte rosette-forming cells (BMR+). BMR- cells were not stimulated to proliferate by SAC in the presence or absence of T cells, but did proliferate to PWM plus irradiated T cells. Co-stimulation of BMR+ cells with SAC and PWM in the presence of autologous T cells did not lead to immunoglobulin secretion. The B cells stimulated to divide by SAC apparently do not become responsive to B cell differentiation factors and are distinct from those that undergo T cell-dependent differentiation.
Human IgE synthesis in vitro by pokeweed mitogen-stimulated human lymphoid cells: verification with a reconfirmed epsilon-specific radioimmunoassay.
Authors: Chen P P , Nonaka M , O'Hair C H , Cohen P A , Zuraw B L , Katz D H
Published: December 2022

This study was performed to address the controversy concerning human IgE biosynthesis in vitro induced by stimulation with pokeweed mitogen (PWM) or other agents. The controversy has focused on the specificity of reagents employed for quantitatively determining human IgE in culture supernatant fluids. Specifically, questions have been raised as to whether certain anti-human IgE antibody reagents possess anti-idiotypic reactivities, thereby resulting in reactions with Fab determinants of polyclonal immunoglobulins which would yield false-positive readings of IgE protein levels. We present a detailed analysis confirming that the goat anti-human IgE antibody designated GAHE(PS), which was initially isolated by affinity chromatography with the same IgE(PS) myeloma protein used for immunization, binds poorly, if at all, with IgG, IgA, or IgM immunoglobulins, even at excessive concentrations (100 micrograms/ml). Moreover, GAHE(PS) displayed no reactivity with Fab fragments of IgG or free L-chains prepared from pooled polyclonal IgG isolated from Cohn fraction II. A second GAHE reagent was prepared by purification by affinity chromatography on a second, completely unrelated IgE myeloma protein (DZA), which differed from IgE(PS) in light chain class, thereby resulting in a reagent, designated GAHE(DZA), which was completely devoid of any possible reactivity with L-chain or idiotypic determinants affiliated with IgE(PS). By utilizing both reagents, the studies presented here confirmed that PWM-stimulated human lymphoid cell cultures synthesize increased quantities of IgE, which can be detected in comparable amounts by both GAHE(DZA) and GAHE(PS) in supernatant fluids from such cultures. Because incorporation of the reversible protein synthesis inhibitor, cycloheximide, totally abolished the PWM-induced increases in IgE levels in such cultures, these results verify that such increases reflect de novo synthesis of human IgE as a result of PWM stimulation in vitro.
Dissociation in the production of B cell-stimulating factors (BCGF and BCDF) and interleukin 2 by T cells from a common variable immunodeficient patient.
Authors: Saiki O , Shimizu M , Saeki Y , Kishimoto S , Kishimoto T
Published: December 2022

In the course of an analysis of lymphocyte functions of common variable immunodeficiency patients, we found one patient whose T lymphocytes released B cell differentiation factor (BCDF) and B cell growth factor (BCGF) without IL 2 production upon stimulation with Con A. The patient was a 68-yr-old woman with hypo-gamma-globulinemia (IgM: 31 mg/dl, IgG: 223 mg/dl, IgA: 23 mg/dl); she suffered from cryptococcal meningitis and pulmonary tuberculosis with a negative result of skin tests to PPD and cryptococcal antigen. The number of T cells and the ratio of T cell subsets (Leu-2a and Leu-3a) was normal. T cells showed no proliferative response to Con A and a low response to PHA (one-tenth of normal response). The addition of patient T cells to normal T cells did not inhibit the proliferation of normal T cells when stimulated by Con A. The culture supernatant of Con A-stimulated T cells contained no IL 2 activity when assayed by an IL 2-dependent T cell line. Expression of Tac antigen was not impaired by Con A stimulation and the addition of partially purified IL 2 from the supernatant of Jurkat T cell line-induced proliferation of Con A-stimulated T cells, indicating that the defect observed was not in IL 2-responding cells but in IL 2-producing cells. In contrast, the culture supernatant of the T cells stimulated by Con A or PHA contained BCDF activity as much as that of normal T cells when assayed by Cowan I-stimulated normal B cells or the B lymphoblastoid cell line SKW6-CL4. The supernatant also contained BCGF activity. These results suggest that B cell-stimulating factor (BCGF, BCDF) and IL 2 may be synthesized by different subsets of T cells or that the synthesis of those lymphokines are independently regulated in the same cells.