Showing papers in "Journal of Immunology in 1975"

Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A.

Abstract: The Cowan I strain of the bacterium Staphylococcus aureus has been used as an adsorbent for antibodies complexed with radiolabeled antigens from cell lysates. This application is advanced as a superior alternative to other methods of immune precipitation for the isolation of antigens. It exploits the high adsorption capacity for IgG molecules by protein A molecules on the cell walls of certain strains of staphylococci, along with the advantageous sedimentation properties of the bacteria. The interaction of immune complexes with the adsorbent was defined initially using a model system of bovine serum albumin with a high excess of rabbit anti-bovine serum albumin antibodies (IgG). The uptake of immune complexes under these conditions was extremely rapid, occurring within seconds, whereas maximum binding of free IgG was much slower. In addition, once bound the complexed antigen could not be displaced from the adsorbent either by large amounts of normal IgG or by extra free antibody. Antigen could be eluted almost completely from the inert adsorbent for analytic or preparative purposes with a variety of solvent systems, such as the detergent SDS in combination with urea and high temperature, and neutral salts with strong lyotropic salting in properties. The efficacy of the protein A-antibody adsorption technique was tested in direct comparisons with a conventional double antibody precipitation method for the isolation of mouse lymphocyte IgM. The bacterial adsorbent not only had a distinct advantage in speed of antigen isolation, but analyses by polyacrylamide gel electrophoresis in SDS also revealed consistently higher antigen recoveries, lower levels of background radioactivity, and an absence of other cell components which may nonspecifically bind to and complicate analyses using conventional immune precipitates.

Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes.

Abstract: A variety of methods have been devised for the study of spontaneous and directed cell migration. Among these, the membrane filter method introduced by Boyden in 1962, with its more recent modifications, has become the technique of choice for studies of leukocyte migration in vitro. This method, however, cannot be applied without alteration to studies of chemotaxis and spontaneous migration of cells of different types. We describe in this report a new and simple method for studying human leukocyte chemotaxis, in vitro, which is based upon migration of cells under agarose gel. This method has application to both polymorphonuclear leukocytes and monocytes, permits measurement of both chemotaxis and spontaneous migration, requires fewer cells per test, and is rapid, simple, reproducible, and inexpensive to set up.

Reticulum cell sarcoma: an effector cell in antibody-dependent cell-mediated immunity.

Abstract: A transplantable, murine reticulum cell sarcoma is described which exhibits the cytologic, adherence, and phagocytic properties of macrophages. It forms specific rosettes with erythrocytes in the presence of the corresponding anti-serum. The ascites cells mediate antibody-dependent cellular immunity as assayed by release of radioactivity from 51Cr-labeled erythrocytes. The contribution of contaminating host cells in the cytotoxic reaction was ruled out by growing the tumor in F1 mice and removing the host cells by anti-H2 serum and complement. The tumor cells have receptors for IgG2a and IgG2b immunoglobulins. The availability of a pure population of effector cells in the immune system allows study of the biochemical processes pursuant to lysis of foreign cells.

IgG on Lymphocyte Surfaces; Technical Problems and the Significance of a Third Cell Population

Abstract: Direct immunofluorescence performed with the F(ab)2 fragment of rabbit antibodies to IgG revealed that membrane bound IgG was only rarely found on the surface of small peripheral blood lymphocytes (PBL). In contrast whole antibodies to IgG used in fluorescence gave much higher levels of cells with IgG surface staining. This staining resulted from secondary IgG binding, in part due to the uptake of newly formed immune complexes. IgM-and IgD-bearing cells were brightly stained in relatively similar percentages by both the whole and F(ab)2 forms of the class-specific antibodies; they constitute the principal membrane Ig of PBL. Evidence was obtained indicating that a special population of cells with Fc receptors but lacking membrane Ig was primarily involved in the high IgG binding. This population also formed sheep erythrocyte rosettes when optimal conditions were utilized.

Identification of macrophage-like characteristics in a cultured murine tumor line.

Abstract: A mouse tumor line P388D 1 passaged in tissue culture for many years has been characterized morphologically and functionally as a macrophage like cell. P388D 1 cells phagocytize latex particles and firmly adhere to glass and plastics. In addition they have been shown to carry cell-bound receptors for immunoglobulin (Fc) and complement (C3). They fail to stain with fluorescent anti-mouse Ig or heterologous anti-mouse. Functionally, these cells exhibited high effector activity in an antibody-dependent, cell-mediated cytotoxic system. The accessibility of well characterized and pure macrophage-like cell lines, such as P388D 1 , will facilitate studies where cell purity is essential.

The Role of Cyclic AMP in the Chemotactic Responsiveness and Spontaneous Motility of Rabbit Peritoneal Neutrophils The Inhibition of Neutrophil Movement and the Elevation of Cyclic AMP Levels by Catecholamines, Prostaglandins, Theophylline and Cholera Toxin

Abstract: Agents known to affect intracellular levels of cyclic AMP in many diverse systems have been tested for their effect on the chemotaxis induced by Escherichia coli culture filtrates, spontaneous motility and cyclic AMP levels of rabbit peritoneal neutrophils. Prostaglandin E1 and A1 but not prostaglandin F2α increased neutrophil cyclic AMP levels and, correspondingly, only the former two prostaglandins inhibited chemotaxis. Nevertheless, a quantitative relationship between prostaglandin stimulation of cyclic AMP and inhibition of chemotaxis could not be found. Epinephrine, isoproterenol, and, to a much lesser extent, norepinephrine increased neutrophil cyclic AMP through beta adrenergic stimulation. Only epinephrine and isoproterenol inhibited chemotaxis, but the inhibition was variable and not related to the ability of these catecholamines to increase intracellular cyclic AMP. Cholera toxin increased neutrophil cyclic AMP after a 30-min lag period which paralleled its inhibitory effect on chemotaxis and spontaneous motility. However, the effect on chemotaxis required 50 ng/ml of toxin whereas the effect on cyclic AMP was manifested at 2 ng/ml of toxin. Prior to 30-min preincubation there was no effect of even 1250 ng/ml of toxin on either cyclic AMP or chemotaxis. Choleragenoid prevented the effects of toxin on both cyclic AMP and chemotaxis. The bacterial chemotactic factor obtained from E. coli culture filtrates did not effect a measurable change in levels of neutrophil cyclic AMP. The data indicate that even though cyclic AMP is not, in the main sequence of events, triggering the chemotactic response, increases in neutrophil cyclic AMP may modulate the movement and thus the chemotactic responsiveness of the neutrophil.

The isolation and partial characterization of neutrophil chemotactic factors from Escherichia coli.

Abstract: Heat-stable, chemotactically active peptides have been obtained from Escherichia coli culture filtrates. They range in size between 150 and 1500 daltons and are anionic at neutral pH. Free carboxyl groups but not free amino groups appear to be required for activity. The N-terminal group may be blocked. There do not appear to be internal aromatic or basic residues in the chemotactically active fractions. A highly purified, not completely characterized, fraction was found to contain aspartic acid, serine, glutamic acid, alanine, and glycine.

Activated Macrophages in Congenitally Athymic “Nude” Mice and in Lethally Irradiated Mice

Abstract: Resistance to the facultative intracellular bacteria, Brucella abortus and Listeria monocytogenes, is principally the result of acquisition of enhanced antibacterial activity by host macrophages, probably in response to lymphokines released by T lymphocytes. However, the present paper describes a surprisingly high resistance on the part of both congenitally athymic “nude” mice and of lethally irradiated mice compared with normal controls. This enhanced bactericidal activity was evident 24 hr after infection, and could also be demonstrated in macrophages from nude mice cultured in vitro. It was concluded that the macrophages of these animals had been activated before infection.

Macrophages Activated in Vitro with Lymphocyte Mediators Kill Neoplastic but not Normal Cells

Abstract: Normal guinea pig macrophages incubated for 3 days in vitro with mediator-rich lymphocyte supernatants become cytotoxic for the syngeneic tumors, Line 1 hepatoma and MCA-25 fibrosarcoma. Under identical experimental conditions the survival of two normal syngeneic cell types, fibroblasts and kidney cells, was not affected. The activating supernatants were prepared by stimulating sensitized lymphocyte cultures with an antigen unrelated to the target cells. Therefore, this type of macrophage-mediated cytotoxicity appears to be nonspecific but restricted to cells with malignant growth capacities.

Increased risistance of immunoglobulin A dimers to proteolytic degradation after binding of secretory component.

Abstract: The contribution of secretory component to the stability of secretory IgA against proteolysis has been studied by a new approach, i.e., by comparing the proteolytic degradation of the complexes formed in vitro between these proteins and secretory component. The results show that attachment of secretory components to the dimeric backbone of the secretory IgA molecule is accompanied by a significantly increased resistance of this backbone against digestion by both trypsin and pepsin. This protective effect may be a physiologic function of secretory component or may be due merely to unspecific blocking by secretory component of one or more sensitive peptide bonds in the IgA backbone.

Identification of Two Populations of Immunoglobulin-Bearing Lymphocytes in Man

Abstract: Human peripheral blood lymphocytes bearing easily detectable surface immunoglobulin were quantitated with routine immunofluorescence procedures. The mean value in 17 healthy adults was 18% (range 9 to 30%). When the cells were preincubated at 37°C and washed at the same temperature before staining, the mean value decreased to 9% (range 4.5 to 18%) (p These temperature-related effects were explained by IgG that remained bound to the cell membrane at 4°C, but eluted at 37°C in serum-free medium. The total number of Ig-bearing lymphocytes and T lymphocytes determined by rosette formation with sheep erythrocytes approached 100%. These studies indicate two populations of Ig-bearing lymphocytes in healthy subjects: one with surface-stable Ig determinants, and another that lacks these markers, but has receptors capable of binding IgG.

B lymphocyte differentiation induced by lipopolysaccharide. I. Generation of cells synthesizing four major immunoglobulin classes.

Abstract: Cultured cells from mouse thoracic duct, spleen, lymph node, Peyer9s patches, and bone marrow gave rise to cells containing large amounts of cytoplasmic IgM, IgG1, and IgG2 when stimulated by bacterial lipopolysaccharide (LPS). Differentiation of IgA producers occurred in bone marrow only, and to a lesser extent than other classes. Significant IgM responses preceded development of cells containing other classes. The differentiation of IgG and IgA producers did not appear to depend on T cells, since cultures from nu/nu or thymectomized-irradiated, bone marrow-protected mice responded as well as normals. Cultures from mice rendered deficient in B cells by anti-µ treatment responded normally to T cell mitogens, but did not proliferate or give rise to immunoglobulin-secreting cells when stimulated with LPS. Bone marrow cultures gave relatively meager proliferative responses to LPS, but generated as many or more immunoglobulin-secreting cells as did other tissues.

Thymus-Independent Antigens: The Preparation of Covalent, Hapten-Ficoll Conjugates

Abstract: A general approach is presented for covalently attaching carboxyl and primary amino groups to Ficoll and other high molecular weight polysaccharides in a readily controlled manner. The polysaccharides are omicron-carboxymethylated with chloroacetate in NaOH solutions. Amino groups are next introduced by monoamide formation with ethylenediamine using a water-soluble carbodiimide. Reactions are carried out in aqueous solutions; excess reactants and by-products are removed by dialysis. Three different ways for coupling the 2,4-dinitrophenyl hapten to the amino derivative of Ficoll are described. Preliminary evidence indicates that 2,4-dinitrophenyl-Ficolls, prepared by procedures developed in this study, are potent thymus-independent antigens which can specifically stimulate B cells both in vivo and in vitro. Hapten-Ficoll conjugates should be exceptionally useful for studies of B lymphocyte activation because of the ready availability of the starting material and the ease with which a wide variety of hapten derivatives may be prepared.

Modulation of Cyclic AMP in Purified Rat Mast Cells I. Responses to Pharmacologic, Metabolic, and Physical Stimuli

Abstract: The cyclic adenosine 3′,5′-monophosphate (cAMP) content of isolated unstimulated mast cells and the changes induced by a variety of pharmacologic, metabolic, and physical stimuli were studied. A modified bovine serum albumin density gradient purification method consistently provided mast cell preparations which were 95% or more pure, without apparent damage, and a 73% recovery of the mast cells applied to the gradients. The measured cAMP in unstimulated mast cells was high, a mean of 16 picomoles per million cells. Moderate agitation or contact with glass increased cAMP content about 2-fold. When calcium was omitted from the medium mast cell cAMP was markedly elevated; incremental increases in added calcium ion concentration from 1 µM to 1 mM caused a linear decrease in cAMP content. Theophylline (3 to 20 mM) caused a dose-related increase in mast cell cAMP content, approximately 2-fold at 20 mM theophylline. Epinephrine (0.01 to 1 mM) caused a modest, dose-related increase in cAMP. In the presence of theophylline, epinephrine increased cAMP levels equal to or greater than the sum of the effects of the agents used individually. The increase in cAMP induced by epinephrine was completely inhibited by 100 µM propranolol and partially inhibited by 10 µM propranolol, thus suggesting that a β adrenergic receptor is involved. Prostaglandin E 1 (PGE 1 ) and histamine (in the presence of theophylline) also raised cAMP. Carbamylcholine (1 nM) lowered cAMP 38%. Atropine (0.1 mM) inhibited the decrease in cAMP induced by 1 nM carbamylcholine by 83% indicating that a muscarinic receptor is involved. In these homogeneous single cell suspensions, therefore, cholinergic and β adrenergic agents have opposing effects on cAMP levels. Diazoxide (10 µM) and adenine (1 µM) caused 37 and 32% decreases in cAMP, respectively. The availability of highly purified mast cells and the identification of agents which either decrease or increase cAMP content allows a direct examination of the role of cAMP in histamine release.

Repopulation with IgA-containing cells of bronchial and intestinal lamina propria after transfer of homologous Peyer's patch and bronchial lymphocytes.

Abstract: These results suggest that there may be a common mucosal immunologic system, and that repopulation of gut and lung lamina propria may be through the organized lymphoid tissue therein.

Immune and Antibody Responses to an Isolated Capsid Protein of Foot-and-Mouth Disease Virus

Abstract: The purified capsid proteins VP1, VP2, and VP3 of foot-and-mouth disease virus type A12 strain 119 emulsified with incomplete Freund's adjuvant were studied in swine and guinea pigs. Swine inoculated on days 0, 28, and 60 with 100-μg doses of VP3 were protected by day 82 against exposure to infected swine. Serums from animals inoculated with VP3 contained viral precipitating and neutralizing antibodies, but such serums recognized fewer viral antigenic determinants than did antiviral serums. Capsid proteins VP1 and VP2 did not produce detectable antiviral antibody in guinea pigs, and antiviral antibody responses in swine to a mixture of VP1, VP2, and VP3 were lower than the responses to VP3 alone. However, when swine were inoculated with VP1, VP2, and VP3 separately at different body sites, no interference with the response to VP3 was observed. Vaccine containing VP3 isolated from acetylethylenimine-treated virus appeared less protective for swine than vaccine containing VP3 from nontreated virus. Trypsinized virus, which contains the cleaved peptides VP3a and VP3b rather than intact VP3, produced approximately the same levels of antiviral antibody responses in guinea pigs as did virus. Conversely, an isolated mixture of VP3a and VP3b did not produce detectable antiviral antibody responses in guinea pigs. The VP3a-VP3b mixture did, however, sensitize guinea pigs to elicit such responses following reinoculation with a marginally effective dose of trypsinized virus.

Studies of surface immunoglobulins on human B lymphocytes. I. Dissociation of cell-bound immunoglobulins with acid pH or at 37 degrees C.

Abstract: Lymphocyte preparations isolated from the human peripheral blood were exposed to different acid pH or incubated at 37°C and the presence of immunoglobulin (Ig) on the cell surface was examined by immunofluorescence (IF) tests. Subsequently, such treated cells were incubated in the autologous serum or in the purified IgG, IgA or IgM proteins and their ability to bind each class of Ig was examined. The results showed that IgG molecules dissociated from large proportions of IgG-positive cells upon exposure to pH 4 at 1°C for 1 min or upon incubation at 37°C for 20 min. The cells from which IgG had been dissociated could again combine with IgG, whereupon the number of positive cells increased, being restored to the number equivalent to or higher than those before acid or 37°C treatment. These results indicated that the treatment could elute the cell-bound IgG present on the cell and that the receptor sites were not degraded by the treatment and could combine with IgG. These cell-bound IgG were observed not only on the monocytes, but also on the small lymphocytes. It was also found that certain proportions of mononuclear cells carried the cell-bound IgA that could be dissociated with acid pH or 37°C. No cell-bound IgM was observed on any mononuclear cells. Microscopic observations before and after acid or 37°C treatment revealed that the staining distribution of the cell-bound IgG and IgA on the cell was granular, appearing as a discontinuous fluorescence ring and forming multiple aggregates but no typical polar caps on warming. In contrast, IgG, IgA, and IgM stable to acid or 37°C treatment were found on the lymphocytes but not on the monocytes, and their staining distribution was uniformly diffuse, appearing as a continuous ring and forming a typical cap on warming. Exposure of the cells to pH 4 or to 37°C could also elute the cell-bound IgG passively adsorbed to the human lymphoid cells in a culture, but did not affect the intrinsic S.Ig on the lymphoid cells in a culture or on the lymphoma cells. These results indicate that the exposure of the cells to acid pH or to 37°C may enable us to detect unfailingly S.Ig lymphocytes by removing the cell-bound IgG and IgA present on the monocytes and/or lymphocytes. Thus, an average value of approximately 10% was obtained for the S.Ig lymphocyte in the lymphocyte preparations from 11 healthy individuals. In addition, the results provided the evidence that, even in normal peripheral blood lymphocytes, there may be a population of B lymphocytes which lack the S.Ig but carry the cell-bound Ig.

Immunologic Properties of Bacterial Lipopolysaccharide (LPS): Correlation between the Mitogenic, Adjuvant, and Immunogenic Activities

Abstract: Bacterial lipopolysaccharide (LPS) was demonstrated to have the capacity in mice to enhance the response to soluble bovine serum albumin (BSA) and to interfere with the induction of tolerance to human gamma-globulin (HGG) These adjuvant activities were shown to occur under conditions in which LPS could also function as a B cell mitogen This positive correlation was established by utilizing two experimental situations in which LPS was non-mitogenic for spleen cells Thus, on the one hand, it was found that LPS did not function as an adjuvant in C3H/HeJ mice, a unique strain whose spleen cells were also unresponsive to LPS-induced mitogenesis On the other hand, in strains which did respond to LPS mitogenically, LPS failed to function as an adjuvant when it was chemically altered to reduce its in vitro mitogenic activity A correlation was also observed between mitogenesis and the capacity of LPS to function as a specific immunogen i mice In contrast to the sustained and prolonged plaque-forming cell response that was observed in mice whose spleen cells were also responsive to LPS-induced mitogenesis, the response was relatively transient in the C3H/HeJ strain These results are discussed in view of the possible in vivo modes of action of LPS

Cytotoxic Effects of Antigen- and Mitogen-Induced T Cells on Various Targets

Abstract: Mitogen-induced cytotoxicity was studied by culturing mouse spleen cells with an optimum mitogenic dose of concanavalin A (Con A) for 2 days and, after washing, assessing their ability to lyse tumor targets. We show that rapid lysis occurs only when the correct concentration of an agglutinating mitogen (phytohemagglutinin P (PHA) or Con A) is present in the assay. PHA is more efficient than Con A in revealing the cytotoxic effect. The Con A-induced effector cytotoxic cell is shown to be sensitive to anti-theta serum and complement. Cytotoxic T cells were also induced by H-2 different allo-immunization. These effector cells specifically lyse targets which bear the immunizing H-2 antigens in the absence of PHA. When PHA is present in the assay, they will also lyse syngeneic tumor targets nonspecifically. Since not all dividing T cells (e.g., T lymphomas, and T blasts induced by M locus different, H-2 similar mixed lymphocyte culture) lyse PHA-P815, we propose that this assay measures only a subset of effector T cells, namely, cytotoxic T cells, regardless of their antigen specificity. The tumor cells, P815 and EL4, are sensitive both to antigen-specific T cell-mediated lysis in the absence of PHA and to nonspecific, PHA-revealed lysis. Small lymphocytes, T cell blasts, and B cell blasts are sensitive to lysis by T cells which are directed against H-2 antigens on their surface, but are not very susceptible to nonspecific T cell lysis in the presence of PHA. The reason for this difference in susceptibility of tumor and normal cells to PHA-revealed nonspecific T cell lysis is not known but may have some relevance to in vivo tumor rejection.

Complement activation by interaction of polyanions and polycations. I. Heparin-protamine induced consumption of complement.

Abstract: Interactions of heparin and protamine in fresh human serum, in amounts far below those required for complement depletion by either agent alone, were found to induce virtually complete depletion of total hemolytic complement activity This depletion was dependent on time, temperature, pH, divalent cations, and serum concentration The predominant complement component hemolytic activity depleted was C1; under appropriate reaction conditions C4 and C2 were depleted as well Equivalent amounts of heparin along induced lesser but substantial depletion of C1, potentiation of C4 and C2, and minimal depletion of C3-9, whereas equivalent amounts of protamine had no effect upon complement component activities We conclude that interaction of heparin with protamine, like interaction of antibody with antigen, markedly enhances its ability to interact with the first component of complement and activate the classical complement pathway It is suggested that complement activation by interactions between certain polyanions and polycations, like interactions between antigens and antibodies, may have a role in the initiation of inflammatory reactions

Spontaneous Modulation of Granulomatous Hypersensitivity in Schistosomiasis Mansoni

Abstract: Spontaneous diminution of granuloma formation around schistosome eggs in chronic schistosomiasis mansoni has been demonstrated previously. In the present study these findings were confirmed by injecting schistosome eggs into the pulmonary microvasculature and removing the lungs 8 days later from mice infected for 4, 8, 12, 16, and 20 weeks; the mean area of inflammation around the eggs was then measured. At 4 weeks a primary reaction was seen, by 8 weeks a massive secondary reaction occurred, but by 12 weeks the lesion was considerably reduced in size, and at 20 weeks it was smaller than the primary reaction. Concomitant measurements of humoral hemagglutinins to soluble egg antigens (SEA) revealed no detectable antibodies at 4 and 6 weeks, relatively low levels at 8 weeks, and an exponential increase in hemagglutinins at 12 weeks and beyond. Immunodiffusion analysis revealed no precipitins at 8 weeks, 1 major band and 2 minor bands at 12 and 16 weeks, and 2 major bands and 1 minor band at 20 weeks. Spleen cells from 8-week-infected mice showed peak migration inhibitory factor (MIF) output at a concentration of 1 µg/ml of soluble egg antigens and suppression of lymphokine secretion at higher concentrations. At 12 and 16 weeks, exponentially lower antigen concentrations both stimulated and suppressed peak MIF output; at 20 weeks MIF was not detectable after stimulation over a wide range of antigen concentrations. Delayed footpad swelling to SEA reached its peak at 10 weeks and declined thereafter, but the response to PPD in tuberculin-sensitized schistosome infected mice remained constant over the period of time studied.

Cell-mediate cytotoxicity in vitro of human lymphocytes against a tissue culture melanoma cell line (igr3).

Abstract: Purified peripheral blood lymphocytes from 13 healthy donors, 6 melanoma patients and 1 halo nevus patient were tested for cytotoxic activity against an allogeneic melanoma cell line (IGR3) in, at least, one of the following assays: cell-mediated cytotoxicity (CMC), antibody-dependent cellular cytotoxicity (ADCC) and microcytotoxicity assays (MA). The lymphocytes were isolated by Ficoll-Triosil gradient centrifugation (fraction F) followed by removal of iron-phagocytosing and adherent cells (fraction FFF) and by subsequent passage through anti-IgG columns (fraction FFF-C). Leukocytes of each fraction were identified by different methods including morphology, rosetteformation, phagocytic activity, and membrane fluorescence. CMC activity paralled ADCC activity at a log lower level of sensitivity. In both assays lymphocytes of fractions F and FFF had the highest activity, whereas in fraction FFF-C cytotoxicity was strongly reduced. In all three lymphocyte fractions CMC and ADCC activity could be blocked by preincubation of the effector cells in aggregated IgG. Furthermore, depletion of E rosette-forming lymphocytes slightly increased ADCC and CMC activity, whereas depletion of EA and EAC rosette-forming lymphocytes strongly decreased it. Our results therefore indicate that in both CMC and ADCC assays, non-adherent, non-phagocytic Fc receptor-bearing lymphocytes (“K” cells) were the active cytotoxic cells. In MA, on the other hand, mononuclear phagocytes seemed to be the most active cell population. So far no significant difference was observed in CMC, ADCC, and MA between control persons and melanoma patients.

Inhibition of proliferation of lymphoma cells and T lymphocytes by suppressor cells from spleens of tumor-bearing mice.

Abstract: We have recently demonstrated suppressor cells in spleens of mice bearing tumors induced by Moloney murine sarcoma virus (MSV) which were non-T cells and inhibited phytohemagglutinin Moloney (PHA)-induced DNA synthesis of syngeneic normal spleen cells. From the present study, the suppressor cells appeared to be macrophages since they were radioresistant, inactivated by carrageenan, and removed by adherence columns and an iron/magnet technique. We have also found that suppressor cells were still fully active when added 16 hr after the mitogen, thus indicating that early mitogen-induced changes were not the target of suppressive action. It appeared that suppressor cells inhibited metabolic events related to the initiation of DNA synthesis and that they had a selective effect on proliferation-dependent lymphocyte effector functions. PHA-induced cytotoxic reactivity which in our system is largely independent of DNA synthesis was not depressed but actually enhanced in MSV spleens. Cytotoxicity of MSV spleen cells against syngeneic lymphoma cells was unaffected by suppressor cells whereas lymphocyte stimulation by mitomycin C-treated syngeneic lymphoma cells was inhibited. MSV spleen cells also inhibited DNA synthesis of cultured murine lymphoma cells. This function was only slightly diminished after treatment with anti-θ serum plus guinea pig complement. Furthermore, spleen cells from MSV tumor-bearing nude mice were as effective as spleen cells from their heterozygous littermates, thus suggesting that T lymphocytes are not the main effector cells of inhibition of lymphoma cell DNA synthesis. The inhibitor cells were radioresistant, inactivated by carrageenan, and removed by adherence columns and the iron/magnet technique. These data strongly suggest that the inhibitor cells of lymphoma cell DNA synthesis are macrophages and that they belong to the same group of cells as the suppressor cells of PHA-induced lymphocyte proliferation.

Receptors for immunoglobulin and complement on human alveolar macrophages.

Abstract: Specific cell membrane surface receptors for immunoglobulin and complement components were identified by rosette fromation on in vitro cultured alveolar macrophages obtained from 24 humans and from rabbits. Respiratory macrophages have easily identified receptors for IgG and the C3b fragment of the third component of complement. A macrophage receptor for the C3d fragment was detected only when purified human complement components were used to form erythrocyte-antibody-C3 immune complexes but was not detected when whole human serum was used as the source of complement. No IgM cell receptor was identified. Thus, with respect to membrane receptors, alveolar macrophages resemble peripheral blood monocytes. These studies in addition emphasize the importance of using a variety of immune reagents, expecially the use of human reagents, to determine correctly these cell receptors.

Host Defenses Against Influenza Virus: The Role of Anti-Hemagglutinin Antibody

Abstract: In CBA mice the protection provided by transferred immune spleen cells or by antibody has been investigated in immunologically intact, cyclophosphamide-treated and thymus-deprived animals infected with A/PR8 virus. The degree of protection was more closely related to serum antibody levels than to the presence of immune lymphocytes in recipients. Comparison of the protective efficiency of various anti-influenza antisera with different specificities within an influenza A subtype indicated that antibodies recognizing the strain-specific determinants of the influenza hemagglutinin have an important role in protection. Physiologic amounts of transferred antibodies were shown to protect immunodepressed mice, suggesting that, provided a sufficient amount of specific antibodies is secreted, the participation of effectors of cell-mediated immunity is not essential. However, our results suggest that thymus-derived lymphocytes have an indirect role in protection by enhancing, through their helper effect, the secretion of anti-influenza antibodies.

Cationic proteins of human granulocytes. VI. Effects on the complement system and mediation of chemotactic activity.

Abstract: The chymotrypsin-like cationic proteins of human granulocytes are shown to possess the ability to produce conversion of the complement components C1s, C4, C3, and C5 as detected by crossed immuno-electrophoresis. This ability seems to be a direct proteolytic effect. Incubation of cationic proteins with serum or functionally pure preparations of C3 and C5 is shown to generate the formation of chemotactic activity which is abolished by prolonged incubation. Also, the chemotactic activity of porcine C5a or spontaneously activated C5 is abolished by incubation with cationic proteins. It is suggested that the chymotrypsin-like cationic proteins of human granulocytes after extrusion from the phagocytic cell play an important role for generation of inflammatory mediators.

Thymic involution: effect on T cell differentiation.

Abstract: The thymus of long-lived BC3F mice involutes progressively throughout life, beginning at 6 weeks of age. This is manifested by the loss of cortical lymphoid mass and by the degenerative changes in epithelial cells. The purpose of this study was to determine to what extent age-related degenerative changes of the thymus affect its capacity to influence the maturation of thymic-derived (T) cells. Accordingly, thymic lobes of mice ranging in age from 1 day to 33 months were implanted under the kidney capsule of T cell-deprived syngeneic young adult TXB mice, and the emergence of T cells was assessed kinetically by various morphologic and functional indices which may be reflective of different T cell subpopulations. They are: a) histology of the thymsu graft, b) lymphocyte repopulation of the T cell-dependent areas of lymph nodes, c) total number of splenic lymphocytes carrying theta antigens (theta-+), d) T cell-dependent humoral immune response and e) proliferative response of splenic cells to plant lectins, phytohemagglutinin (PHA) and succinyl-concanavalin A (s-Con A), and allogeneic lymphocytes. The results revealed that the T cell-transforming influence of thymic tissues generally decreases with increasing age. The difference in the patterns of recovery of the various indices of thymus-grafted TXB mice suggests that the extent to which T cells can mature is dependent upon the degree of involution the thymic tissue has undergone with age. In particular thymic tissues lose the capacity to influence the following functions with advancing age: 1) lymphocyte repopulation of the T cell-dependent areas of lymph nodes; 2) mitogenic reactivity of splenic cells to T cell-specific mitogens (PHA and s-Con A): 3) number of splenic theta-+ lymphocytes and splenic T cell helper function; and 4) mitogenic reactivity of splenic T cells to allogeneic lymphocytes.

Similar Idiotypic Specificity for the Membrane IgD and IgM of Human B Lymphocytes

Abstract: Certain cases of chronic lymphocytic leukemia possess monoclonal bands in the serum. Idiotypic antibodies to the isolated IgM protein of one such case demonstrated that the leukemic lymphocytes carried the identical specificity on their lymphocytes. Both the lymphocyte IgM and the IgD possessed this same specificity. This was demonstrated best through the use of rhodamine-conjugated Fab fragments of IgM- and IgD-specific antisera which were both capped by the idiotypic antiserum.

The Mechanism of Basophil Histamine Release Induced by Antigen and by the Calcium Ionophore A23187

Abstract: The mechasism of human basophil histamine release by the calcium ionophore A23187 has been compared to that induced by the interaction of antigen with cell bound IgE antibody. Ionophore induced histamine release (Ion. H.R.) occurs with the leukocytes of both normal and allergic donors. It is completely calcium dependent; LaCl3 inhibits both Ion. H.R. and antigen induced histamine release (Ag. H.R.) at about 10-minus 7 M. The kinetics of Ion. H.R. suggest that this process has no "desensitization" phase as does Ag. H.R. and the ionophore is fully active on antigen-desensitized cells. Pharmacologic studies indicate that dibutyryl cyclic AMP and agents which increase endogenous cyclic AMP levels do not inhibit Ion. H.R. as they inhibit the early stages of Ag. H.R. Of the agents which affect microtubules, colchicine inhibits and D2O enhances Ion. H.R. in a manner which is qualitatively similar but quantitatively less marked than their effects on Ag. H.R. The metabolic antagonist 2-deoxyglucose inhibits both Ion. H.R. and Ag. H.R. in a similar fashion. Based on these data and the observation that cells pretreated with ionophore show a marked (synergistic) enhancement of Ag. H.R. we conclude that Ion. H.R. has a similar or identical mechanism to the later stages if Ag. H.R. but "short circuits" the cyclic AMP-associated events of Ag. H.R.

The structure and function of immunoglobulin domains. II. The importance of interchain disulfide bonds and the possible role of molecular flexibility in the interaction between immunoglobulin G and complement.

Abstract: The observation that reduction of the inter-chain disulfides in rabbit antibody destroys its ability to interact with complement was confirmed and shown to be true also of human meyloma IgG1 subclass proteins In the latter case a C1-binding assay was used Further studies indicated that it was the interheavy chain disulfides which were essential for complement-binding activity: Non-covalently reassembled IgG (LHHL) was devoid of C1-fixing activity whereas molecules formed from covalently linked heavy chain dimers, and reduced and alkylated light chains (ie, LH-HL) were as active as the parent intact IgG Fc fragments from IgG1 bound C1 and this activity was insensitive to the presence or absence of intact interchain disulfides These bonds therefore are neither directly involved in C1 binding nor essential for the integrity of the binding site We have also shown that although IgG4 does not bind C1, Fc fragments derived from this subclass fix C1 with an affinity comparable to that of the corresponding fragment from IgG1 These data suggest that quaternary interaction with other regions of the molecule (ie, Fab) may modulate the activity of the C1-binding site