Showing papers in "Journal of Experimental Medicine in 1974"

Identification of a novel cell type in peripheral lymphoid organs of mice

Abstract: Several properties of lymphoid dendritic cells in situ have been determined, and contrasted to information previously established for lymphocytes and mononuclear phagocytes. Dendritic cells are not found in newborn mice, and their concentration in both spleen and mesenteric lymph node does not reach adult levels until 3–4 wk of age. Dendritic cells largely disappear from adherent populations following administration of steroids (2.5 mg hydrocortisone acetate s.c.) and ionizing radiation (Do of 100 rads for Co60). Splenic dendritic cells can originate from precursors located in both bone marrow and spleen itself, probably the red pulp. The mature splenic population does not actively divide (pulse labeling index with [3H]thymidine of 1.5–2.5%), but does turnover at substantial rate, 10+% of the total pool per day. The influx of new cells appears to be derived from a proliferating precursor compartment, but the mechanism for efflux or turnover is not known. Dendritic cells in spleen and node undergo little or moderate increase in numbers during development of a primary immune response. These in vivo characteristics, taken together, further distinguish dendritic cells as a novel cell type, distinct from mononuclear phagocytes and lymphocytes.

The route of enteric infection in normal mice

Abstract: This study followed the early pathogenesis of orally induced murine typhoid fever. Intragastrically administered Salmonella enteritidis moves quickly through the normal undisturbed gut so that only a small residuum remains in the cecum and large intestine after the first few hours. Dye injection of the gut wall was used to show that lymph from discrete portions of the gastrointestinal tract drains to separate lymph nodes, probably via the regional Peyer's patches. Plating techniques capable of detecting a single colony-forming unit of S. enteritidis within the different Peyer's patches and draining lymph nodes indicate that, although the cecum and large intestine are exposed to large numbers of Salmonella for longer time periods than the small intestine, the primary site of bacterial penetration involves the distal ileum. This area of the small intestine as well as the cecum are both drained by the distal mesenteric lymph nodes, and were the only nodes which contained detectable numbers of viable Salmonella over the first 24 h of infection. Neither the pyloric nor the proximal mesenteric lymph nodes (which drain the stomach and duodenum) nor the pancreatic and caudal lymph nodes (which drain the transverse and descending colon) contained viable Salmonella. Salmonella were observed to infect the ileal mucosa and its Peyer's patches. With time, this infection progresses to the draining lymph node and ultimately reaches the liver and spleen. Some of the implications of these findings relative to the development of acquired resistance to enteric disease are discussed.

Secretion of plasminogen activator by stimulated macrophages

Abstract: Cultured thioglycollate-stimulated peritoneal macrophages synthesize, accumulate, and continuously release high levels of plasminogen activators for at least 4 days whereas cultures of unstimulated macrophages do not; the higher specific catalytic activity of released vs. cell-associated enzyme suggests that the plasminogen activators are actively secreted. The major macrophage plasminogen activator is a serine protease of mol wt 48,000, and thus resembles the comparable enzyme released by virally transformed fibroblasts. Macrophages release a second plasminogen activator of mol wt 28,000 that is also a serine enzyme. The secretion products released by stimulated and unstimulated macrophages have been compared by SDS-polyacrylamide gel electrophoresis after chemical labeling with 3H-DFP or biosynthetic labeling with 14C-amino acids. These procedures show that some proteins are formed in both cultures, whereas others are uniquely secreted by each type of macrophage. The serine enzymes released by the two kinds of macrophages differ in specificity and electrophoretic mobility.

Identification of a novel cell type in peripheral lymphoid organs of mice. II. Functional properties in vitro.

Abstract: Several properties of lymphoid dendritic cells in situ have been determined, and contrasted to information previously established for lymphocytes and mononuclear phagocytes. Dendritic cells are not found in newborn mice, and their concentration in both spleen and mesenteric lymph node does not reach adult levels until 3–4 wk of age. Dendritic cells largely disappear from adherent populations following administration of steroids (2.5 mg hydrocortisone acetate s.c.) and ionizing radiation (Do of 100 rads for Co60). Splenic dendritic cells can originate from precursors located in both bone marrow and spleen itself, probably the red pulp. The mature splenic population does not actively divide (pulse labeling index with [3H]thymidine of 1.5–2.5%), but does turnover at substantial rate, 10+% of the total pool per day. The influx of new cells appears to be derived from a proliferating precursor compartment, but the mechanism for efflux or turnover is not known. Dendritic cells in spleen and node undergo little or moderate increase in numbers during development of a primary immune response. These in vivo characteristics, taken together, further distinguish dendritic cells as a novel cell type, distinct from mononuclear phagocytes and lymphocytes.

The effect of cells transferred into the mouse blastocyst on subsequent development.

Abstract: Bone marrow cells from CBA T6T6 mice and testicular teratocarcinoma cells from 129 SvSl mice were transferred into blastocysts from random-bred Swiss albino mice. The blastocysts were allowed to develop in foster mothers and the adults resulting from these blastocysts were studied for evidence of an effect of the transferred cells. A total of 137 adults resulted from the experiments, and one of the mice that had received teratocarcinoma cells in the blastocyst stage showed several thin stripes of agouti hair. All the adult animals received grafts of skin from animals identical to those supplying the cells. In all cases the animals that resulted from blastocysts into which cells had been transferred maintained skin grafts for a significantly longer period than controls. In a number of cases the graft developed agouti hair and in two cases the graft was maintained for approximately 2 mo. These experiments indicate that the transferred cells were able to establish small colonies in the embryos and that some of these cells persisted into the adult.

In vitro synthesis and secretion of lysozyme by mononuclear phagocytes

Abstract: Pure cultures of three types of mononuclear phagocytes-mouse peritoneal macrophages, unstimulated or after thioglycollate stimulation, and human monocytes-synthesize and secrete large amounts of lysozyme in vitro. The macrophage lysozyme is indistinguishable from authentic lysozyme in its ability to lyse M. lysodeikticus, inhibition by specific antisera, a similar size of 14,000 and cationic charge. Lysozyme secretion in culture is characterized by a large net increase in total lysozyme, 4-20-fold in 3 h, 75-95% of which is in the medium, and its continued extracellular accumulation over at least 2 wk in culture. Lysozyme is the major (14)C-labeled protein secreted into the medium by both unstimulated and thioglycollate-stimulated macrophages and the 0.75-1 microg produced per 1 x 10(6) cells/day represents 0.5-2.5% of the total cell protein. Lysozyme is a cell-specific marker for mononuclear phagocytes and the PMN, which contains preformed enzyme, since it is absent in lymphoid cells and a variety of fibroblast and epithelioid cell lines. Lysozyme production is also a useful measure of mononuclear phagocyte cell number. The rate of lysozyme production and secretion is remarkably constant for all cell types under a variety of culture conditions. Production by the mouse macrophage increases threefold on the 2nd day in culture and then remains linear with time. Production is optimal at a relatively low serum concentration, but can be maintained, in the absence of serum, in lactalbumin hydrolysate or, at a reduced level in basal media. The production and secretion of lysozyme are independent of the production of macrophage acid hydrolases. Net increase and secretion of lysozyme occur under conditions where acid hydrolases like N-acetyl beta-glucosaminidase, beta-glucuronidase, beta-galactosidase, and cathepsin D are neither accumulated nor secreted. Massive phagocytosis of latex particles has no effect on lysozyme production and secretion. Lysozyme production can be rapidly inhibited by treatment with cycloheximide (0.4 microg/ml) whereas inhibition of its production by colchicine (10(-6) M) occurs only after a lag period of more than 8 h, and is probably due to a secondary effect. These results show that mouse macrophages provide a simple in vitro system to measure lysozyme secretion and its control. These studies also indicate the possible importance of mononuclear phagocytes in the secretion of a variety of biologically active products and in the modification of their environment.

Generation of cytotoxic t lymphocytes in vitro : i. response of normal and immune mouse spleen cells in mixed leukocyte cultures

Abstract: Mouse cytotoxic T lymphocytes (CTL) were generated in mixed leukocyte cultures (MLC) using spleen cells as responding cells and irradiated allogeneic spleen cells as stimulating cells. Cytotoxicity was assessed by a quantitative 51Cr assay system and the relative frequency of CTL in individual cell populations was estimated from dose-response curves. Inclusion of 2-mercaptoethanol in the MLC medium resulted in a 20–40-fold increase in the relative number of CTL generated at the peak of the response. Under these culture conditions, cell-mediated cytotoxic activity was detectable in MLC populations as early as 48 h after the onset of the cultures. When spleen cells from mice immunized with allogeneic tumor cells 2–4 mo previously were cultured with irradiated spleen cells of the same alloantigenic specificity (MLC-Imm), it was found that the cell-mediated cytotoxic response was detectable earlier and reached higher levels than that observed in a primary MLC. At the peak of the response, MLC-Imm populations were observed to lyse up to 50% of the target cells within 3 h at a lymphocyte: target cell ratio of 0.3:1. Immunological and physical characterization of the effector cells generated in MLC-Imm indicated that they were medium to large-sized T lymphocytes. Altogether, these studies suggested the existence of an anamnestic cell-mediated cytotoxic response in MLC-Imm.

GENETIC CONTROL OF RESPONSES TO BACTERIAL LIPOPOLYSACCHARIDES IN MICE I. Evidence for a Single Gene that Influences Mitogenic and Immunogenic Respones to Lipopolysaccharides

Abstract: In vivo immune responses and in vitro mitogenic responses to bacterial lipopolysaccharides (LPS) have been compared in strains of C3H mice. C3H/HeJ spleen cultures did not support mitogenic responses to LPS and in vivo these mice produce low IgM responses to LPS. On the basis of these two responses, C3H/HeJ mice have been termed low LPS responders. All other strains of C3H mice tested (C3HeB/FeJ, C3H/DiSn, C3H/Str, CWB, CSW, and C3H/Sf and its H-2 congenics) are high LPS responders supporting large in vitro mitogenic and in vivo immune responses to LPS. The immune response difference between low and high LPS responders is a quantitative one. IgM responses are observed in C3H/HeJ mice in the range of 1.0–10 µg LPS. At lower and higher LPS concentrations, immune responses are not observed. In contrast, high LPS responders elicit LPS immune responses over a much wider dose range (0.1–200 µg). The ability to respond well to LPS is dominant as shown by the response of F1 hybrid mice of low responder and high responder strains. The linkage relationships of mitogenic and immune responsiveness to LPS have been investigated in backcross (C3H/HeJ x CWB)F1 x CWB mice. All mice that gave in vivo immune responses to LPS also supported mitogenic responses to LPS. The defect in C3H/HeJ mice that limits mitogenic and immune responsiveness to be due to a single autosomal gene which is not linked to the H-2 histocompatibility or heavy-chain allotype loci.

Role of complement in induction of antibody production in vivo : effect of cobra factor and other c3-reactive agents on thymus-dependent and thymus-independent antibody responses

Abstract: In an in vivo study in mice, suppression by the C3-cleaving protein of cobra venom (CoF), and other C3-reactive agents (zymosan, aggregated IgG, anti-C3 antibodies, and type III pneumococcal polysaccharide) of the thymus-dependent antibody responses to sheep erythrocytes, ovalbumin, and human IgG was demonstrated. The thymus-independent antibody response to polyvinyl-pyrrolidone was however unaffected by CoF. These and other published observations suggest that there may be a requirement for functional C3 in induction of thymus-dependent but not thymus-independent antibody production. A model for the role of C3 in lymphocyte cooperation is proposed based on these data analyzed in the light of existing knowledge of this process. It is postulated that fixed C3 interacting with macrophage See PDF for Structure and B-cell C3 receptors might enhance or facilitate T-dependent presentation of antigen to B cells.

Influence of dose and route of antigen injection on the immunological induction of T cells.

Abstract: Delayed-type hypersensitivity (DTH) develops in the absence of an adjuvant when mice are injected intravenously or subcutaneously with an appropriate dose of sheep red blood cells (SRBC). The optimal intravenous dose of 105 SRBC (in CD-1 mice) produces maximum DTH which decays exponentially from its peak on day 4. Increasing the dose of SRBC reduces and eventually abolishes all evidence of DTH. DTH fails to reappear in respose to secondary stimulation except in splenectomized mice in whom the development of DTH is not suppressed, even by massive doses of SRBC. Hence the suppression cannot be due to antigen as such. The optimal dose of SRBC for sensitization by footpad inoculation is 100-fold higher (107 SRBC in CD-1 mice), but even 109 SRBC do not block the induction of DTH by this route of immunization. A blocking dose of SRBC, given intravenously 1 day before footpad inoculation, completely suppresses cell proliferation in the draining lymph node, prevents PFC production there, and blocks the induction of DTH by a sensitizing dose of SRBC. If given 1 day after footpad sensitization, intravenous antigen has little effect on the cellular response in the regional node but DTH is still completely suppressed. Blocking of induction and expression may depend, therefore, on different mechanisms.

Evidence of suppressor cell activity in spleens of mice bearing primary tumors induced by moloney sarcoma virus

Abstract: Spleens from Moloney sarcoma virus (MSV) tumor-bearing C57BL/6N mice contained four times the normal number of mononuclear cells and displayed a markedly elevated "spontaneous" (mitogen-independent) DNA synthesis on a per cell basis. The number of macrophages were increased three-fold while there was a slight reduction in the percentage of T lymphocytes. The phytohemagglutinin (PHA) response on a per cell basis of spleens from tumor-bearing mice was decreased about 90% when compared with normal control mice. The primary in vitro immune response to sheep red blood cells was also suppressed to levels of less than 10% of normals. The PHA response could be restored by purification of MSV spleen cells by rayon adherence columns and by removal of phagocytic cells by an iron/magnet technique. The activity of suppressor cells in MSV spleens was demonstrated in mixtures with syngeneic normal spleen cells where a marked impairment of the PHA response was observed. Spleen cells from tumor-free nude mice and normal spleen cells treated by anti-theta serum plus guinea pig complement (C'), both totally unreactive to PHA, had no such effect. The inhibitor cell in MSV spleens was shown to be insensitive to inactivation by anti-theta plus C', but could be removed by the adherence columns and the iron/magnet technique. These data suggest that this suppressor cell is a cell of the monocyte/macrophage series. Suggestive evidence was also presented that the suppressor cells belong to a proliferating population in MSV spleens. Similar suppressor cells have been previously demonstrated in spleens of mice during a variety of immune responses. Our data show, that a tumor, although stimulating the immune system, nevertheless may be suppressive on certain immune functions through the activation of suppressor cells.

INDUCTION OF MACROPHAGE PLASMINOGEN ACTIVATOR BY ENDOTOXIN STIMULATION AND PHAGOCYTOSIS : Evidence for A Two-Stage Process

Abstract: The injection of thioglycollate medium into the peritoneal cavity of the mouse induces high levels of macrophage fibrinolytic activity due to the production and secretion of a plasminogen activator, a trypsinlike serine protease, which is absent in unstimulated macrophages. Intraperitoneal injection of endotoxin or mineral oil can stimulate only a fraction (<10%) of the fibrinolytic activity of thioglycollate cells, similar to the partial stimulation (<10%) seen 1-2 days after phagocytosis of latex or SRBC by unstimulated macrophages. The endotoxin-stimulated macrophages contain and release relatively low levels of plasminogen activator, but these primed cells can be triggered to produce and secrete high levels of enzyme, by phagocytosis of latex. Under conditions where the plasminogen activator is induced and secreted, there are no effects on the production and/or release of lysozyme or intracellular acid hydrolases, Discovery of a two-stage procedure for inducing macrophage plasminogen activator made it possible to study the role of cell priming and phagocytosis separately. Endotoxin was a more effective priming agent, weight for weight, than lipid A:BSA complex. Secretion of the plasminogen activator was induced only by thioglycollate, or endotoxin and latex. In situ fibrinolysis was induced by these agents and mineral oil, BCG, and fetal calf serum, in decreasing order of effectiveness. Phagocytosis of latex in all cases except thioglycollate stimulation, increased fibrinolytic activity from three- to sixfold. Latex and a variety of other particles such as M. lysodeikticus, aggregated gamma-globulin and immune complexes showed dose-dependent stimulation of fibrinolysis by endotoxin-primed macrophages. Although the initial phagocytic trigger was not specific for the substance employed, the ability to induce a sustained response depended on the persistence of the phagocytized particle within the cell. Fibrinolysis and secretion of plasminogen activator continued at high levels for at least 9 days after uptake of latex, a nondigestible particle, whereas plasminogen activator was secreted only transiently after ingestion of rapidly digested M. lysodeikticus. The induction of plasminogen activator secretion provides a mechanism by which the activated macrophage can exert a selective effect on its extracellular environment.

THE INTERACTION OF IgE WITH RAT BASOPHILIC LEUKEMIA CELLS : II. Quantitative Aspects of the Binding Reaction

Abstract: Previous studies indicated that cultured rat basophilic leukemia cells have surface receptors which bind IgE with high specificity. In this paper we describe some quantitative aspects of the phenomenon. The reaction mechanism appears to consist of a simple reversible binding reaction with k1 = 9.6 x 104 M–1 sec–1 and k–1 ⩽ 1.6 x 10–5 sec–1 at 37°C. The calculated KA is therefore ⩾ 6 x 109 M–1. The activation energy of binding was found to be 7.8 kcal/mol. The number of binding sites/cell varied between 3 x 105 to over 1 x 106. The binding was insensitive to pH's between 6–8 but at pH 3.0 complete dissociation of bound IgE occurred in ∼ 1 min at 0°C leaving the receptors for IgE intact. Ca++ plus EDTA and Mg++ plus EDTA produce a fairly marked reduction in binding capacity though these reagents alone produce much smaller effects.

Potentiation of t-cell-mediated immunity by selective suppression of antibody formation with cyclophosphamide

Abstract: Delayed-type hypersensitivity (DTH) appears in mice immunized with less than an optimal immunogenic dose of sheep red blood cells (SRBC), but is blocked progressively as antibody production increases in response to larger doses of SRBC. Treatment with cyclophosphamide (CY) was shown to release T cells from this inhibitory influence of the humoral response, and cause enhancement of DTH. The magnitude of this enhancing effect on T-cell activity was markedly dependent on the time of treatment relative to the time of immunization, and on the time chosen for measuring DTH. The reasons for these pronounced effects of timing are threefold: ( a ) CY given before antigenic stimulation has a long-lasting effect on antibody formation, but no apparent effect on the precursors of activated T cells. ( b ) After antigenic stimulation, T cells also become susceptible to CY. ( c ) The production of a nonspecific participant (monocyte) in the DTH reaction is also suppressed by CY, though the supply of circulating monocytes is not immediately affected by the drug. The differential effect of CY on T and B lymphocytes depends on the differing physiological states of the majority of cells that make up these two populations. The former are resting cells that are insensitive to CY until exposed to specific antigen, while the latter are drawn from a rapidly replicating precursor pool and are susceptible to CY at all times.

The involvement of lactoferrin in the hyposideremia of acute inflammation.

Abstract: The hyposideremia of inflammation was found to be based on a three-step mechanism involving lactoferrin, the iron-binding protein from the specific granules of neutrophilic leukocytes. ( a ) Lactoferrin is Released from Neutrophils in an Iron-Free Form . When phagocytosis was induced in neutrophils by zymosan or bacteria, lactoferrin was recovered in the incubation medium together with other constituents of the specific granules, such as alkaline phosphatase and lysozyme. Lactoferrin extracted from leukocytes was able to bind the amount of iron corresponding to its theoretical iron-binding capacity. After injection of endotoxin into rats, lactoferrin was detected in various tissues where it was normally absent, or in the plasma when the reticuloendothelial system (RES) had previously been blocked by injections of India ink or aggregated albumin. ( b ) Lactoferrin is Able to Remove the Iron from Transferrin . Significant exchange of iron from transferrin to lactoferrin was observed in vitro only at a pH below 7.0 or in the presence of a high concentration of citrate. However, the fast elimination of lactoferrin in vivo, when saturated with iron, might account for the observed transfer of iron to endogenous or administered apolactoferrin. Intravenous injection of human apolactoferrin into rats caused a marked decrease of the plasma iron level. The kinetics of this process, as well as controls with other proteins, ruled out the possibility of a secondary inflammatory effect due to phlogogenic contaminants. ( c ) Fe-Lactoferrin is Taken-up by the RES . By immunofluorescence, lactoferrin was shown to be bound and ingested by monocytes. The rate of elimination of human Fe-lactoferrin injected into rats was particularly fast when compared to that of human apolactoferrin, succinylated Fe-lactoferrin, or other human proteins. Blockade of the RES slowed down the rate of clearance of Fe-lactoferrin and was also found to retard the elimination of endogenous rat lactoferrin released by endotoxin. These experiments suggest the existence of specific receptors for Fe-lactoferrin on the membrane of macrophages.

Selective triggering of human t and b lymphocytes in vitro by polyclonal mitogens

Abstract: Human lymphocytes from spleen and tonsils have been cultured with a variety of polyclonal mitogens. Cultures consisted of either unseparated T and B cells or alternatively purified T or B lymphocytes. The purity of the starting cell populations and the origin of activated lymphoblasts was analyzed with a panel of seven markers which discriminate between T and B cells. The selectivity of the lymphocyte responses was influenced by cell populations in a given culture, the mitogen used, and to a limited extent on culture conditions. Purified T lymphocytes from tonsil and spleen responded to phytohemagglutinin (PHA), pokeweed mitogen (PWM), and staphylococcal enterotoxin B (SEB). Purified B cells from spleen responded well to PWM, weakly to SEB and lipopolysaccharide, but not at all to PHA. Tonsil B cells responded weakly to PWM and SEB but not to PHA. Some B lymphocytes do respond to PHA in the presence of activated T cells. These results are discussed in relation to previously reported selective responses of human cells and parallel studies in animal species.

The viral envelope glycoprotein of murine leukemia virus and the pathogenesis of immune complex glomerulonephritis of new zealand mice

Abstract: The use of monospecific antisera for the analysis by radioimmunoassay and immunofluorescence study of two major viral proteins, gp69/71 and p30 of murine leukemia virus, that could be of significance in the pathogenesis of immune complex glomerulonephritis of mice, particularly NZB and B/WF1 hybrid mice, yielded the following conclusions. A remarkably high concentration of viral envelope glycoprotein, gp69/71, was detected in the spleen and serum of New Zealand mice (NZB, NZW, B/WF1, and W/BF1); the concentration in the spleen was 10-fold greater than that found in AKR mice and 30-fold greater than that present in C57BL/6 mice. The gp69/71 was deposited along with bound immunoglobulins, apparently as an immune complex, in the diseased kidneys of mice, and the glomerular site and extent of deposition of gp69/71 was related to the severity of the glomerulonephritis. This study suggests that the pathogenesis of immune complex glomerulonephritis (and vasculitis) in mice is related to the expression of this specific viral envelope glycoprotein and to the host immune response to this protein.

Direct evidence for Hageman factor (factor XII) activation by bacterial lipopolysaccharides (endotoxins).

Abstract: Purified precursor Hageman factor has been demonstrated to bind to soluble bacterial lipopolysaccharide (LPS, endotoxin) isolated from Escherichia coli 0111:B4, and this complex has been shown to have the capacity to convert prekallikrein to its active form. In addition, LPS-activated Hageman factor substantially reduces clotting times in XII-deficient plasma. The capacity to activate Hageman factor has been demonstrated to reside in the lipid A region of the LPS molecule. Activation of Hageman factor by LPS contrasts with fluid-phase activation (e.g., by kallikrein or trypsin) in that no cleavage to lower molecular weight fragments occurs. High concentrations of LPS inhibit the activity of Hageman factor, probably by a direct LPS-Hageman factor interaction.

Interactions of c-reactive protein with the complement system : i. protamine-induced consumption of complement in acute phase sera

Abstract: Protamine sulfate was found to consume large amounts of C selectively during preincubation with sera of individuals in the "acute phase". Marked depletion of C1, C4, and C2 with minimal, if any, depletion of C3-9, was observed. The consumption was time and temperature dependent, occurring most rapidly and extensively at 37°C, 0.10 M relative salt concentration and pH 7.5–8.0; it required calcium ions. It was mediated by a heat-stable nondialyzable factor which separated with C-reactive protein (CRP) during fractionation and purification, correlated with serum CRP levels, and, like other known reactivities of CRP, was inhibited by phosphoryl choline. Preparations of CRP purified either from serum or ascites resulted in consumption of large amounts of C1, C4, and C2 when preincubated with normal serum and protamine. We conclude that CRP is a potent activator of the C system at the level of C1, and that polycations such as protamine sulfate are substrates of CRP which can bring about this activation. It seems not unlikely that one role of CRP in health and disease involves its ability to interact with the C system.

Evidence for linkage between HL-A histocompatibility genes and those involved in the synthesis of the second component of complement.

Abstract: HL-A analysis of a family with C2 deficiency revealed evidence for close linkage between the C2 defect and the histocompatibility HL-A loci. The propositus was homozygous both for C2 deficiency and the HL-A haplotype 10,W18. Among seven children of three double backcross matings, no recombinants were found. The possible significance of such linkage is discussed.

THE INTERACTION OF IgE WITH RAT BASOPHILIC LEUKEMIA CELLS I. EVIDENCE FOR SPECIFIC BINDING OF IgE

Abstract: A rat basophilic leukemia cell line originally described by Eccleston et al. (3) has been successfully transplanted into eight Wistar strains and adapted to suspension cell culture without noticeable morphological changes. The cells deplete the reaginic activity of mouse and rat immune sera to an extent roughly equivalent to that reported for normal rat mast cells. Studies utilizing radioiodinated antilight-chain antibodies and radioiodinated partially purified rat IgE indicate that the cells bind IgE to their surface membrane with high specificity. Heating or mildly reducing the rat IgE destroyed its binding activity. The binding is unaffected by the presence or absence of Ca(++) and Mg(++), and is markedly inhibited by reaginic but not by normal rat or mouse serums. The affinity of these cells for human IgE, if present at all, is substantially lower than for rat IgE.

GENETICS OF A NEW IgVH (T15 IDIOTYPE) MARKER IN THE MOUSE REGULATING NATURAL ANTIBODY TO PHOSPHORYLCHOLINE

Abstract: The idiotype present on the Fab of a phosphorylcholine-binding IgA myeloma protein TEPC 15 (T15) of BALB/c origin was found in normal serum of BALB/c mice. Molecules carrying the T15 idiotype in normal serum could be adsorbed with Sepharose phosphorylcholine beads and R36A pneumococci. The T15 idiotype is absent in germ-free BALB/c but appears when the mice are conventionalized. A survey of normal sera of inbred strains for the T15 idiotype showed it to be present in BALB/c, 129, C57L, C58, and ST and absent or in low levels in CBA, C3H, C57BL/6, C57BL/Ka, C57BL/10, SJL, B10.D2, DBA/2, RIII, A, AL, AKR, NZB, and NH inbred strains of mice. The T15 idiotype is associated with some but not all strains carrying the IgCH allotypes found in BALB/c. Linkage of genes controlling the T15 idiotype in normal serum to the IgCH locus of BALB/c was demonstrated in F2 progeny of a BALB/c and C57BL cross, Bailey's recombinant inbred strains, C x BD, C x BE, C x BG, C x BH, C x BI, C x BJ, C x BK, and CB20 congenic strains. Among these strains, only those possessing the IgCH locus of BALB/c including the F2 progeny consisting of BALB/c homozygotes and BALB/c/C57BL heterozygotes and C x BG and C x BJ recombinants showed the T15 idiotype.

Mechanism of thymus-independent immunocyte triggering : mitogenic activation of b cells results in specific immune responses

Abstract: The present experiments were performed in order to analyze the mechanism by which thymus-independent antigens (nonspecific B-cell mitogens) can induce specific immune responses to antigenic determinants present on the same molecule. The hapten NNP was coupled to the B-cell mitogen, lipopolysaccharide (LPS). The conjugate retained full mitogenic activity and bound specifically to NNP-reactive cells. NNP-LPS activated polyclonal as well as specific anti-NNP antibody synthesis, but the optimal concentrations for induction of specific anti-NNP cells were several orders of magnitude lower than the concentrations required for polyclonal activation. These low concentrations failed to activate nonspecific cells, but they induced specific thymus-independent responses of high-avidity NNP-specific cells with the typical kinetics of antigenic responses in vitro. Furthermore, hapten-specific cells were paralyzed by NNP-LPS concentrations that were optimal for induction of polyclonal activation. Specific activation and paralysis could be abolished by free hapten indicating that selective binding of NNP-LPS to hapten-specific cells was responsible for the specificity of the response. However, the triggering signal lacked specificity, since high-avidity specific anti-NNP cells could still be activated by stimulating concentrations of NNP-LPS in the presence of free hapten, even though the Ig receptor combining sites were presumably occupied by NNP. The findings show that B cells with specific Ig receptors for the antigenic determinants on mitogen molecules preferentially bind these molecules and become activated at concentrations still unsufficient to trigger other B cells that lack specific receptors. It is suggested that activation for primary IgM responses in B cells is the result of "one nonspecific signal." This nonspecific signal is provided by the mitogenic properties of some antigens (highly thymus independent or, alternatively, by nonspecific T-cell factors (for highly T cell-dependent antigens), or both, and the surface structures responsible for triggering are not the Ig receptors. The specific Ig receptors only act as passive focusing devices for nonspecific stimuli, entitling the cell to be selectively activated, even though both the signal and the receptors for the triggering are nonspecific.

Activation of T and B lymphocytes in vitro. II. Biological and biochemical properties of an allogeneic effect factor (AEF) active in triggering specific B lymphocytes.

Abstract: The studies presented herein have focused on the biological and biochemical properties of a nonspecific mediator produced by populations of activated T lymphocytes during short-term in vitro reactions with foreign alloantigens. We have analyzed the activity of the unseparated and of chromatographically separated fractions of the supernatants from such cultures on the in vitro responses of mouse lymphocytes to soluble and macrophage-bound DNP-carrier conjugates and also to particulate heterologous erythrocytes. The results demonstrate that a highly active protein moiety, termed allogeneic effect factors (AEF), in the mol wt range of 30,000-40,000, is capable of acting directly on B lymphocytes, in the presence of antigen, probably to effect triggering and subsequent differentiation and proliferation to antibody-forming cells in vitro. The active molecule, although not manifesting specificity for antigen, does exhibit some strain-specific properties suggesting a possible relationship to cell surface antigens or other gene products coded in the major histocompatibility gene complex.

Evidence for the expression of Ia (H-2-associated) antigens on thymus-derived lymphocytes

Abstract: We have demonstrated in an anti-Ia serum the presence of specific antibodies reacting with T cells, as well as with B cells, using a highly sensitive dye exclusion test. This antiserum reacts with both spleen and lymph node in a characteristic biphasic titration curve killing up to 70% of these cells. It also reacts with cortisone-resistant thymocytes. The A.TH-α-A.TL serum can be absorbed with spleen, lymph node, cortisone-resistant thymus, or normal thymus cells. Further in vivo absorptions in BALB/c nude cannot remove all of the cytotoxic activity for normal BALB lymph node lymphocytes, while completely removing the activity for nude cells. A Thy-1 positive cell line derived from a C57Br leukemia is reactive with this anti-Ia serum.

Studies on mediator production by highly purified human T and B lymphocytes.

Abstract: Highly purified populations of T and B lymphocytes obtained by affinity column separation were stimulated by antigen and their ability to produce two mediators, migration inhibitory factor (MIF) and lymphocyte mitogenic factor (LMF) was assessed. Both T- and B-cell populations made MIF; the production of MIF was antigen-specific using purified protein derivative of tuberculin, streptokinase-streptodornase, and Candida antigens. The MIF activity from both populations could not be attributed to antigen-antibody complexes as the inhibitory activity eluted from Sephadex G-100 columns in the same region corresponding to mol wt 23,000 daltons. Further studies indicate that the T cells producing MIF are proliferating cells whereas the B cells producing this mediator are not. In contrast, LMF was made only by T cells and not B cells when these populations were stimulated by antigen. The LMF induced the [3H]thymidine incorporation into both T and B cells obtained from donors lacking sensitivity to the antigens used to elicit the factor. Chromatographic studies indicate that LMF eluted from Sephadex G-100 in a fraction of mol wt 23,000 daltons where MIF is also found; however, since B cells produce MIF but not LMF, these two factors appear to be distinct from one another. Some of the implications of these findings are discussed. The explanation for the production or lack of production of MIF by lymphocytes obtained from patients with immunodeficiency disorders requires reinterpretation.

Studies of the human lymphocyte receptor for heat-aggregated or antigen-complexed immunoglobulin

Abstract: The lymphocyte receptor for complexed immunoglobulin was shown not to bind heat-aggregated human serum albumin, bovine serum albumin, transferrin, F(ab')2, reduced and alkylated Ig, and mildly oxidized Ig, which indicated that the receptor is specific for a site dependent on disulfide bond(s) on the Fc portion of complexed Ig. Inhibition experiments provided evidence that the same receptor binds both heat-aggregated Ig and antigen-antibody complexes. Lymphocytes treated with pronase were no longer able to bind Ig complexes, which suggested that the receptor is a protein or glycoprotein. Additional evidence was obtained that lymphocyte surface Ig and the receptor for complexed Ig are distinct since the former could be capped without affecting the distribution of the latter, and surface Ig was not detectable after trypsinization of lymphocytes, whereas the binding of Ig complexes was unaffected by such treatment. Incubation of lymphocytes which had bound Ig complexes in tissue culture medium at 37°C revealed that the complexes remained on the surface membrane for several hours, and that only a minority of lymphocytes binding complexes showed cap formation. Lymphocytes which had heat-aggregated IgG specifically bound to their receptors for complexed Ig were markedly inhibited in their ability to mediate antibody-dependent cytotoxicity, thus providing strong evidence for the necessity of the receptor in this immune activity. Titration of this inhibition with varying amounts of complexes revealed distinct plateaus in the dose-response curve. This suggested that there may be more than one kind of receptor and/or different populations of lymphocytes which bear the receptor.

Effects of concanavalin A on mouse peritoneal macrophages. I. Stimulation of endocytic activity and inhibition of phago-lysosome formation.

Abstract: Concanavalin A (Con A) binds to saccharide residues on the mouse peritoneal macrophage plasma membrane and stimulates extensive pinocytic interiorization of the membrane. The overall pinocytic rate is increased 3.5–4.5 times by the addition of Con A, and the surface marker enzyme adenosine triphosphatase can be identified histochemically in association with the cytoplasmic vesicles generated after exposure of the cells to Con A. Once formed, these pinocytic vesicles may persist for several days and fail to show morphologic evidence of fusion with primary or preformed secondary lysosomes. There is no apparent effect on the capacity of the macrophage to ingest either latex particles or IgG-coated SRBC administered either simultaneously with or subsequent to the Con A.

Properties of plasminogen activators formed by neoplastic human cell cultures

Abstract: A series of human cell lines has been examined for fibrinolysis in culture. The sera that are activating for fibrinolysis by human cells are mouse, monkey, human, horse, and bovine. Individual human sera show considerable variation in the ability to activate fibrinolysis. In common with other neoplastic or transformed mammalian and avian cell cultures, human cell lines of neoplastic origin produce substantial amounts of plasminogen activator. Several cultures of nonmalignant origin also produce plasminogen activator, whereas cultures obtained from trypsinized human embryos, or from human embryonic skin do not. The human melanoma plasminogen activators are of two kinds: a major component with a mol wt of 50,000, and a minor species with a mol wt of approximately 60,000. Both are DFP sensitive, serine proteases.

Genetic control of immune responses in vitro v. stimulation of suppressor t cells in nonresponder mice by the terpolymer l-glutamic acid60-l-alanine30-l-tyrosine10 (gat)

Abstract: In recent studies we have found that GAT not only fails to elicit a GAT-specific response in nonresponder mice but also specifically decreases the ability of nonresponder mice to develop a GAT-specific PFC response to a subsequent challenge with GAT bound to the immunogenic carrier, MBSA. Studies presented in this paper demonstrate that B cells from nonresponder, DBA/1 mice rendered unresponsive by GAT in vivo can respond in vitro to GAT-MBSA if exogenous, carrier-primed T cells are added to the cultures. The unresponsiveness was shown to be the result of impaired carrier-specific helper T-cell function in the spleen cells of GAT-primed mice. Spleen cells from GAT-primed mice specifically suppressed the GAT-specific PFC response of spleen cells from normal DBA/1 mice incubated with GAT-MBSA. This suppression was prevented by pretreatment of GAT-primed spleen cells with anti-theta serum plus C or X irradiation. Identification of the suppressor cells as T cells was confirmed by the demonstration that suppressor cells were confined to the fraction of the column-purified lymphocytes which contained theta-positive cells and a few non-Ig-bearing cells. The significance of these data to our understanding of Ir-gene regulation of the immune response is discussed.