Guinea Pig Lymph Node Research Articles

The effect of specific cell inactivation on the cellular immune response to ferredoxin peptides.

Previous studies using synthetic immunogenic molecules containing two haptenic peptides (ther NH2-terminal heptapeptide and the COOH-terminal pentapeptide of oxidized ferredoxin (O-Fd) from C. pasteurianum) have shown that both peptides individually are capable of initiating lymphocyte transformation and inhibiting migration in populations of lymphocytes from O-Fd-sensitized guinea pigs. While migration inhibition could readily be stimulated by single haptenic peptides, lymphocyte treasformation appeared to be more easily induced by molecules containing two or more haptenic peptides (these could be identical or different) (Kelly, B., Levy, J.G. and Hull, D., Eur. J. Immunol., 1973. 3:574). If lymphocyte transformation is a T cell-mediated phenomenon, these observations indicate the possibility of T cell-T cell interaction. The two haptenic peptides (designated "N" and "C") were synthesized and conjugated to succinylated bovine serum albumin (S-BSA), forming the conjugates N-S-BSA and C-S-BSA, respectively. These conjugates were labeled to high specific activity with 125iodine and were used in an "antigen suicide" procedure to treat guinea pig lymph node cell preparations previously sensitized to O-Fd and keyhole limpet hemocyanin (KLH). Cell populations exposed to either 125I-labeled C-S-BCA demonstrated decreased lymphocyte transformation in the presence of O-Fd but not in the presence of KLH. These results indicate specific cell inactivation by the radioactive peptide conjugates of those cells responsible for initiating cell transformation. Experiments performed by mixing 125I-labeled N-S-BSA-treated cells with 125I-labeled C-S-BSA-treated cells were successful in partially restoring the response to O-Fd and suggest possible synergy between N and C determinant binding cells in the cellular immune response to O-Fd. Evidence from B cell depletion studies suggests that this is a T cell-T cell interaction.

Lymphokines

Incubation of guinea pig lymph node lymphocytes with insoluble concanavalin A (Con A) in serum-free medium yields sizable amounts of migration inhibition (MIF) and mitogenic factors (MF). Various controls confirmed that the activities detected in supernatants from such cultures are not due to free soluble Con A released during culture. Whereas guinea pig MIF does not act upon human and horse monocytes, guinea pig MF stimulated human but not horse lymphocytes. Upon stimulation by insoluble Con A, guinea pig MIF and MF appear to be produced essentially by T lymphocytes. The technique proposed for producing lymphokines appears convenient for further purification and biochemical characterization of culture supernatants.

The Molecular Dimensions of Mitogenic Factor from Guinea Pig Lymph Node Cells

The molecular dimensions of a mitogenic factor from guinea pig lymph node cells were investigated by means of gel filtration, sucrose density gradient ultracentrifugation, and CsCi isopycnic ultracentrifugation. The mitogenic factor was produced by stimulation of immune lymph node cells from inbred guinea pigs, strain 2 or 13, with the specific antigen, ovalbumin; and mitogenic activity was assayed by measuring incorporation of tritiated thymidine by target lymph node cells from syngeneic animals not immune to ovalbumin. The D-20,w of the mitogenic factor was estimated to be (9.9 plus and minus 0.2) times 10-7 cm2/sec by gel filtration on Sephadex G-100, and the apparent S20,w was estimated to be 2.4 plus and minus 0.2 S by sucrose density gradient ultracentrifugation. The buoyant density was determined to be 1.324 plus and minus 0.006 g/ml by CsCi isopycnic ultracentrifugation, and from this value the partial specific volume of mitogenic factor was estimated to be 0.71. From these data the molecular weight of the mitogenic factor was calculated to be 20,000, and the frictional ratio was calculated to be 1.2.

Macrophage-lymphocyte interaction. II. Antigen-mediated physical interactions between immune guinea pig lymph node lymphocytes and syngeneic macrophages.

The effect of specific antigen on the development of physical interactions between lymph node lymphocytes (LNL) obtained from animals which had been immunized to that antigen and macrophages was examined. We found that the presence of antigen, either limited to the macrophage () or free in the medium, profoundly increased the degree of ) or free in the medium, profoundly increased the degree of Mphi-LNL interaction observed. This enhanced interaction was dependent on the coincidence in the cultures of Mphi bearing antigen and LNL from animals specifically immunized to that antigen. Although antigen-independent interactions developed equally well between syngeneic and allogeneic combinations of lymphocytes and macrophages, antigen mediated interactions required that macrophages and lymphocytes be syngeneic. Prolongation of antigen-mediated Mphi-LNL interactions resulted in the induction of LNL DNA synthesis, initially involving those lymphocytes physically associated with antigen-bearing Mphi. These studies are interpreted to indicate that physical interaction between immune lymphocytes and antigen-bearing Mphi represents a morphological correlate of the functional activation of immune lymphocytes. Further, it is suggested that the physical events involved in lymphocyte proliferation may proceed sequentially from antigen-independent reversible binding of lymphocytes by macrophages to prolonged antigen-stabilized interaction eventuating in the triggering of specifically immune lymphocytes.

Characterization of Guinea Pig Mitogenic Factor

Guinea pig mitogenic factor (M.F.) was identified in antigen-stimulated guinea pig lymph node lymphocyte cultures by the ability of supernatants to stimulate DNA synthesis in non-immune lymphocytes. Its activity is not augmented by the presence of antigen and it will stimulate lymphocytes made unresponsive to the eliciting antigen. M.F. production by lymphocytes is detectable within 6 hr after antigen stimulation and reaches a maximum at 24 hr. DNA synthesis produced by M.F. is maximal at 48 hr. M.F. is heat stable at 60 degrees C. Its molecular weight is in the range of 20,000 to 30,000 daltons. M.F. activity appears to be relatively resistant to the action of proteolytic enzymes.

Inhibition of DNA synthesis by methylglyoxal bis(guanylhydrazone) during lymphocyte transformation.

The phytohemagglutinin induced DNA synthesis in guinea pig lymph node cells was inhibited remarkably by methylglyoxal bis(guanylhydrazone). This inhibitory effect was dependent on the time of its addition to the lymph node cell culture after stimulation with phytohemagglutinin. If methylglyoxal bis(guanylhydrazone) was added 48 hr after the stimulation, no inhibition of DNA synthesis was observed. Exogenous spermidine added at an early time of cell culture reversed the inhibitory effect of methylglyoxal bis(guanylhydrazone). However, no reversion occurred when spermidine was added at a late time of the cell culture.

Immunotherapy of Cancer with Nonliving BCG and Fractions Derived from Mycobacteria: Role of Cord Factor (Trehalose-6, 6′-Dimycolate) in Tumor Regression

Delipidated and deproteinized cell walls from Mycobacterium tuberculosis H37Ra suspended in 1.25% mineral oil emulsion cured established tumors in the skin and metastases in draining lymph nodes of guinea pigs (strain 2) after intratumoral administration in 33% of the cases examined. This was increased to 83% when a mixture of cord factor and the delipidated cell walls was used. A similarly high percentage of cures was obtained after administration of lyophilized, killed BCG alone or with addition of cord factor in 1% mineral oil emulsion. Lyophilized, killed BCG dispersed in a solution of tocopherol acetate in peanut oil or in peanut oil alone showed a limited tumor-regressive activity (36%). However, a mixture of BCG and cord factor suspended in the above media cured 80 and 69% of the treated animals, respectively.

Cell‐Mediated Immunity in Guinea Pig and Man to a Salmonella typhi Glycoprotein Complex Assessed by Migration Inhibition Methods

A Salmonella typhi glycoprotein preparation in concentrations of 5 to 10 μg/ml strongly inhibited the migration of peritoneal exudate cells (PEC), obtained from guinea pigs immunized with S. typhi vaccine in Freund's complete adjuvant (FCA) PEC from animals injected with saline in FCA were also inhibited but to a lesser extent, whereas cells from guinea pigs that had received only S. typhi vaccine or normal guinea pigs showed variable and weaker inhibition. PEC from all guinea pigs receiving FCA were strongly inhibited by 5 to 15 μg PPD/ml. These PPD concentrations had no inhibitory effect on exudate cells from guinea pigs receiving S. typhi vaccine only. The S.typhi glycoprotein concentration (5 and 10 μg/ml) primarily used in the migration inhibition studies had negligible inhibitory effect on the phytohemagglutinin (PHA)‐induced DNA, RNA, and protein syntheses in guinea pig lymph node cells. In the human system only blood leukocytes of donors who had received their third or fourth booster of heat‐killed S. typhi vaccine within the latest 2 years were inhibited in their in vitro migration by the S. typhi glycoprotein complex.

Experimental listeria monocytogenes-lymphadenitis. Pathohistological observations.

The auricular lymph nodes of SPF guinea pigs were examined light microscopically at various stages (30 min to 14 days) after subcutaneous injection of Listeria monocytogenes. The bacteria entered the lymph node parenchyma through the marginal sinuses and were phagocytosed by polymorphonuclear leukocytes and monocytes at early stages of the infection (30–60 min). Whereas retothelial cells of the sinuses did not phagocytose bacteria, submarginal reticulum cells showed a high bacterium-phagocytosing activity. Starting at 12 hours after inoculation lymphocyte activation occurred in the interfollicular diffuse lymphatic tissue of the cortex and in the paracortex. The activated lymphocytes were released into the sinuses. There was also paracortical distension, which reached its peak on the 6th day of infection. The submarginal area at the junction of the afferent lymphatic with the cortex and the perivascular regions of the lymph node paracortex were the preferred sites of granuloma formation. On the 8th day with a dose of 106 bacteria the macrophages of the granulomas revealed only unidentifiable debris in the cytoplasm, whereas in the earlier lesions a large number of mononuclear phagocytes, especially in the submarginal granulomas, contained many intracytoplasmic listeria. The granulomas gradually became smaller and smaller. With larger infectual doses the process did not subside within the experimental period; the granulomas developed the appearance of “reticulocytare abszedierende Lymphadenitis”.

Origin and fate of the multivesicular bodies in PHA stimulated lymphocytes.

Stimulation with PHA induces an important development of the vacuolar system in guinea pig lymph node lymphocytes. The early phases of this transformation are characterized by the occurrence of two types of multivesicular bodies (MVB): a pale, large-sized type and a dense regular type. The possible origins and fate of both kinds of MVB are discussed in connection with the patterns of intracellular distribution of endocytic markers as well as of some enzymes characteristic of Golgi structures (both the genuine and the derived ones) and of the vacuolar system, and of 3H-galactose which is also electively incorporated at the Golgi level.

DNA replication in mixed lymphocyte culture.

THE DNA replication of lymphocytes undergoing blast transformation may be used to study the specific triggering of cells during proliferation responses. In recent experiments1–3, employing CsCl gradient analysis of bromouracil (BU) substituted DNA, in vitro stimulation by the homologous antigen selectively modified the pattern of DNA replication in lymph node cells of guinea-pigs previously primed with a soluble antigen. When primed cells were triggered by homologous antigen, DNA fragments banded in two density peaks (a bimodal profile). In contrast, when such primed cells were stimulated by heterologous antigen, DNA fragments were always distributed according to a unimodal density profile. After the stimulation of guinea-pig lymph node cells by phytomitogens, the sedimentation profile was also unimodal. This bimodal distribution of BU-labelled DNA would result from an immunologically specific response. It has been suggested that such a pattern of replication could be involved in the differentiation process related to the immune response1,4. It is well known, however, that the in vitro transformation of lymphocytes to large blast cells and consequent cell division also take place in mixed lymphocyte culture (MLC). Here we describe experiments on DNA replication carried out in MLC between allogeneic mice.

Antigen-Induced Proliferation of Guinea Pig Lymphocytes in Vitro: Obligatory Role of Macrophages in the Recognition of Antigen by Immune T-Lymphocytes

Abstract Highly purified lymphocytes were obtained from the lymph nodes of CFA-immune guinea pigs. Cultures of these lymphocytes did not exhibit proliferative responses upon stimulation with purified protein derivative (PPD), although they were quite responsive to stimulation with phytohemagglutinin. Addition of macrophages to these cultures resulted in marked enhancement of the PPD-induced lymphoproliferative response, but did not affect lymphocyte responsiveness to phytohemagglutinin. The ability of the purified immune lymphocytes to interact directly with soluble PPD was examined by incubating the cells for 1 hr at 37°C with various concentrations of PPD. The lymphocytes were then washed to remove unbound antigen and placed in culture. Addition of macrophages to cultures of antigen-pulsed lymphocytes did not result in the significant DNA synthesis, suggesting that the specifically immune lymphocytes were unable to bind and carry into culture sufficient antigen to allow for their subsequent activation. These results were not due to the induction of tolerance in the lymphocytes, since cultures of antigen-pulsed and untreated lymphocytes were equally responsive to continuous PPD when macrophages were added to the cultures, even when the antigen-pulsed lymphocytes had been exposed to high concentrations of PPD for 1 or 24 hr. In contrast, macrophages which were incubated for 1 hr at 37°C with a given concentration of PPD were as effective as the same concentration of PPD present continuously in macrophage-lymphocyte cultures in the induction of a lymphoproliferative response. Macrophages appear to play an obligatory role in the presentation of antigen to the immunospecific thymus-derived (T)-lymphocyte.

Regulation of immunoglobulin synthesis by dextran.

Dextran, of the variety commonly used as plasma expander, markedly altered antibody synthesis to an unrelated antigenic stimulus, SRBC, in two animal species, guinea pig and mouse. The time at which dextran was administered relative to antigen was found to be most critical for increasing or decreasing the number of IgM and IgG PFC. Furthermore, these times differed for the two species studied. Typically, when given to guinea pigs 6 h before SRBC, dextran caused a 20-fold rise in IgM-producing cells but had little effect upon IgG synthesis. However, if dextran preceded antigen by 24 h the same magnitude of increase was seen in IgG-forming cells while a decrease in IgM-producing cells occurred. In mice, a short 2 h interval between dextran and antigen favored cells synthesizing IgG and not those producing IgM. A longer 6 to 24 h lapse between dextran and antigen resulted again in an inverted pattern, i.e., an increase in IgM and a decrease in IgG-producing cells. In both species, if dextran was given 48 h before antigen, synthesis of IgG markedly decreased. At the cellular level dextran activated those B cells already in the vascular compartment. In stimulating the IgM response to SRBC, dextran appears either to substitute for T cells or to amplify the effects of that small number of T cells still present in bone marrow preparations. Dextran-altered mouse B cells synthesized SRBC-specific IgG in the presence of normal T cells at an earlier time than did normal B and T cells. However, dextran was unable to cause blastogenesis in vitro of guinea pig lymph node cells or mouse B, T, and spleen cells. The data suggest at least two effects that T cells exert upon B cells. One is to stimulate more B cells to produce IgM, a function accomplished by endotoxins, PWM, PPD, and the simple polysaccharide dextran. The other is to trigger shifts in synthesis of immunoglobulins M and G. Our observations are compatible with the view that a single cell is capable of synthesizing both of these immunoglobulins and that the stimulating factor for one may cause cessation of the other.

Demonstration of Proliferation by Guinea Pig and Rabbit Bone Marrow-Derived Lymphocytes in Vitro in Response to Stimulation by Anti-Immunoglobulin Antisera

Abstract We have employed simultaneous complement receptor lymphocyte rosette and radioautographic techniques to investigate the proliferative response of guinea pig lymph node and rabbit peripheral blood bone marrow-drived (B) lymphocytes to stimulation with anti-immunoglobulin antisera. We have shown directly that all of the proliferative response due to anti-immunoglobulin antisera occurred in the complement receptor lymphocyte (B cell) subpopulation.

The production of vesicular stomatitis virus by antigen- or mitogen-stimulated lymphocytes and continuous lymphoblastoid lines.

A variety of lymphoid cell populations were examined in terms of their ability to replicate vesicular stomatitis virus (VSV), a lytic, RNA-containing virus maturing at the cell surface. The number of cells capable of producing VSV was estimated in terms of infectious centers by the virus plaque assay (VPA), and morphologically by electron microscopy (EM). The lymphoid cells examined in this study included: (a) lymph node cells from delayed hypersensitive guinea pigs stimulated by specific antigen, (b) mouse spleen cells activated by selective bone marrow-derived (B) cell and thymus derived (T) cell mitogens, and (c) cells of human and murine continuous lymphoblastoid or lymphoma lines. In unstimulated cultures of guinea pig lymph node cells there is a background of approximately 1 in 1,000 cells which produces VSV; in purified protein derivative (PPD)-stimulated cultures the number of cells producing virus was 1.6% in the VPA and 1.9% by EM. These cells were large lymphocytes with some morphological features of transformed lymphocytes but were not typical blast cells. A few macrophages were associated with virus in both stimulated and control cultures. These observations indicate that (a) cells responsive to antigens, as detected by a marker virus, were lymphocytes; (b) cells other than lymphocytes (macrophages) were capable of replicating VSV even without antigenic stimulation; and (c) the correlation of results obtained by VPA and morphologic examination was usually quite good. Of the total number of mouse spleen cells stimulated with concanavalin (Con A), a T cell mitogen, 4.5 (EM)-5.7% (VPA) were associated with VSV. These were characteristic transformed lymphocytes, similar to phytohemagglutinin (PHA)-stimulated human lymphocytes. In contrast Escherichia coli lipopolysaccharide (LPS)-treated mouse spleen cultures contained lower numbers of virus plaque-forming cells. The majority of such cells associated with virus displayed extensive rough endoplasmic reticulum. Two cultured murine lymphomas containing lymphocytes with the theta surface marker (L5178Y and EL-4) showed a 15-100-fold higher incidence of virus-producing cells than leukemias (L1210 and C57Bl/6) which did not carry this marker. Similarly, the L2C guinea pig leukemia, a known B cell leukemia, yielded a low percent of virus plaque-forming cells (<2%). However, MOPC-104, a plasma cell tumor presumed to be of B cell origin, was found to be an efficient virus producer. There was a wide variation in the efficiency of VSV replication among human lymphoblastoid lines. One line, Wil-2, produced 80% infectious centers after 24 h of exposure to VSV, and all cells were associated with virus at the EM level. The relationship between the virus-producing cells and different lymphocyte subpopulations as well as the efficiency of the two assays for studying virus-producing lymphocytes is discussed.

Guinea pig lymphotoxin and comparison of various assay methods for cytotoxicity.

SUMMARY The conditions for the production of lymphotoxin from guinea pig lymph node cells stimulated with Phaseolus vulgaris phytohemagglutinin (PHA) are described. A comparison of various assay methods of lymphotoxin, including the incorporation of isotopically labeled compounds, standard cell counting, and the release of isotope from 51Cr-labeled cells, was made. The method utilizing 14C-amino acid or 3H-inositol uptake was the most satisfactory in both sensitivity and accuracy. The advantages and disadvantages of other assay methods are also described.

Proliferation by Bone Marrow-Derived Lymphocytes in Response to Antigenic Stimulation in Vitro

Summary The extent of tritiated thymidine (3H-THY) incorporation into bone marrow derived cells (B cells) in cultures of sensitized guinea pig lymph node cells was determined by the simultaneous identification by radioautography of grained cells and cells bearing the receptor for antigen antibody complement complexes found on B cells. By this criterion 14.2 to 27.3% of the cells incorporating 3H-THY in these cultures are B cells whether or not antigen was present. Furthermore, in the cultures to which antigen was added there is an increase in the per cent of proliferating B cells.

Studies in Experimental Eosinophilia

Abstract Puromycin blocked the eosinophil response which is elicited in guinea pig lymph nodes by a single injection of hemocyanin into footpads. The drug was most effective in interfering with the 24-hr response when it was given just before or no later than 4 hr after the antigen. A partial block was noted when the drug preceded the antigen by 2 to 8 hr. Interference with the 15-min vascular response required a higher dose. The secondary type of eosinophil response, which is seen in an immunized animal, was not inhibited by puromycin. The drug was effective when administered into the footpads, but a higher dose was required when it was given by the intraperitoneal route. The results support the notion that the eosinophilia which occurs during the first few minutes of a primary immune response reflects the de novo synthesis of antibody, rather than the presence of preformed antibody.

The Early Stimulation of Protein Synthesis in Sensitized Guinea Pig Lymph Node Cells by Antigen

Abstract Lymph node cells from immunized guinea pigs were studied in short-term cell culture to determine the effect of specific antigen on protein and RNA synthesis. Both RNA and protein synthesis were stimulated early after the addition of antigen to these cultures. Pulse-labeling techniques disclosed a stimulatory effect on protein synthesis by antigen as early as 4 hr after its addition. This stimulatory effect was associated with the specific release of a soluble non-dialyzable mediator which recruited nonsensitized cells to increase their rate of protein synthesis. The stimulatory effect of antigen on protein synthesis was abolished by the prior addition of actinomycin D.

A kinetic approach to cellular interaction in delayed-type hypersensitivity

The virus plaque assay for enumerating antigen-sensitive cells in delayed-type hypersensitivity has been employed in a kinetic analysis of cell interaction in vitro. When cultures are initiated at varying cell densities of tuberculin-sensitive guinea pig, or human lymphocytes, and logarithmic plots of the cell dose versus response in virus-plaque-forming cells are made, the slopes of the resulting lines indicate theoretically the number of cellular interactions required to produce an observable virus plaque-forming cell. In control cultures, i.e., sensitized lymphocytes cultured in the absence of antigen, or non-sensitized lymphocytes cultured in the presence or absence of tuberculin, the number of cells able to produce virus plaques varied directly proportionately with the number of cells cultured ( m = 1), and constitute the background. In contrast, in the case of sensitized human peripheral blood lymphocytes or guinea pig lymph node cells stimulated by tuberculin, the average slope was found to be two ( m = 2), indicating that interaction of two cells was required to produce one virus plaque-forming cell. When increasing concentrations of sensitized lymphocytes were diluted with syngeneic unsensitized cells, the results indicated that only one of the interacting cells was specific for antigen. Removal of a glass-adherent population diminished the number of plaque-forming cells, and reconstitution of purified glass-adherent cells was partially able to restore the activity. These results are consistent with the view that activation of lymphocytes by specific antigens requires two cells, one of which has specificity, the other of which acts non-specifically and is glass-adherent.