Supraoptimal Doses Research Articles

Antigen binding to receptors on immunocompetent cells: II. Thermodynamic and biological implications of the receptor cross-linking requirement for B-cell activation

If one assumes that receptor cross-linking is a necessary, but not sufficient condition for cellular activation, a number of predictions can be made bearing on the physical chemical properties of the cells selected. In this paper we show that the following response characteristics can be consequences of a cross-linking requirement. (1) Small sparsely haptenated antigens such as DNP 10BSA are expected to elicit a response that matures, and such maturation can occur even with antigenic determinant density in excess over the concentration of cellular receptors. (2) There is an optimal concentration for activation of cells with a given affinity, and therefore an optimally immunogenic dose. (3) The optimal dose is relatively insensitive to antigen valence. (4) Increasing valence increases the breadth of the affinity distribution. (5) For supra optimal doses of antigen, unresponsiveness will be preferentially induced in high affinity cells. (6) Small densely haptenated antigens (e.g. DNP 40BSA) are not expected to elicit responses that mature as quickly as those that are lightly coupled. (7) Large polymeric antigens are not expected to induce responses that mature. (8) Antigens with low determinant density may induce tolerance in vivo but not in vitro. The predictions are briefly discussed in the context of relevant experimental data.

Regulation of in vitro lymphocyte responses : I. Adjuvant effect of lipopolysaccharide (LPS) on low-zone unresponsiveness to concanavalin A

Simultaneous addition of concanavalin A (Con A) and lipopolysaccharide (LPS) to cultures of rat spleen lymphocytes resulted in a synergistic effect on DNA synthesis as measured by increased [ 3H]thymidine uptake after 3 days. This effect was maximal when 10 μg of LPS was added to understimulating doses of Con A (synergistic index = 14) and diminished with increasing doses of the mitogen. In contrast to increasing concentrations of serum factors, LPS was not able to unblock the nonresponse of lymphocytes stimulated with supraoptimal doses of Con A. LPS did not exert its adjuvant effect by stimulating lymphocytes with the help of soluble factors released by Con A-activated cells. Both Con A and LPS seem rather to act together on a distinct population of T-cells which can be separated on nylon columns and respond twice as much as nonseparated cells to their synergistic combination. Rat B-cells were unresponsive to stimulation with Con A and LPS added alone or simultaneously. These results help to better understand some of the mechanisms involved in the immunological enhancement observed with LPS.

Stimulation of the immune response against SRBC by reduction of corticosterone plasma levels: Mediation by mononuclear phagocytes

The effects of reduction in corticosterone plasma levels on the immune response of the mouse have been studied. Reduction of corticosterone levels was achieved by (1) repeated corticosterone injections (feedback), (2) adrenalectomy, and (3) continuous injections of aminoglutethimide phosphate (AGP). With the three methods a reduction in the corticosterone production paralleled by an augmentation in monocyte numbers and DH reactivity was obtained. The humoral response was only affected after immunization with supraoptimal doses of antigen. These results indicate that fall of corticosterone plasma level results in a rise of the blood monocyte number. These monocytes are available (effective) in both cellular and humoral reactions.

The role of macrophages in the amplified in vitro response to sheep red blood cells by spleen cells from Corynebacterium parvum treated mice

Antibody production against sheep red blood cells (SRBC) was studied in Marbrook cultures of spleen cells from C. parvum (CP) treated and normal CBA-T6T6 mice. CP given 7 days prior to spleen cell harvest exerted an adjuvant effect on the direct plaque-forming cell (PFC) response, which was most marked with sub- and supraoptimal doses of SRBC. Under the same conditions no IgG production against SRBC could be detected in the CP treated cultures. When spleen cells were harvested 2 days or 2–4 wk after CP injection IgM production against SRBC was diminished as compared to controls. CP treatment resulted in the emergence and persistence of adherent peritoneal cells (PC) which were more efficient than untreated adherent PC in promoting a direct PFC response to SRBC by normal macrophage depleted spleen cells. The results suggest that the CP adjuvant action is mediated via stimulated macrophages on whose surface antigen presentation to antibody-forming cells is intensified.

A Lectin Model for Unresponsiveness

Abstract A mitogen model for unresponsiveness has been studied. In 72-hr cultures the lowest supraoptimal dose of Concanavalin (Con A) showing no stimulation of rat thymocytes above background was 160 µg/ml. This dose was also found to inhibit substantially (50%) the high spontaneous DNA synthesis by thymocytes during the first 24 hr of culture. This effect was most significant 12 to 24 hr after addition of high-dose Con A. It was not necessary for the cells to remain continuously with high-dose Con A, since washing out unbound Con A after 1 hr still led to reduced 3H-thymidine incorporation at 24 or 72 hr. Furthermore, high-dose Con A treatment suppressed DNA synthesis when added at 48 hr to cultures optimally stimulated with Con A. Interaction with high-dose Con A appears to inhibit the ability of cells to initiate new rounds of DNA synthesis without blocking ongoing DNA replication. Removal of Con A with α-methyl-D-mannopyranoside (mannose) led to only partial reversal of these effects after 1 hr although “unresponsiveness” was reversible at 15 min. Suppression was not due to cytotoxic or agglutinating effects of the high-dose Con A. Two × 106 thymocytes exposed to 160 µg of Con A for 1 hr bound 1.7 µg, 86% of which was elutable with mannose. There was no reduction in the ability of high-dose treated cells to bind a subsequent optimal dose of Con A. Since Con A binds to essentially all thymocytes, this system is useful in following early events in the induction of lectin “unresponsiveness” as a model for immunologic tolerance.

The Splenic Suppressor Cell

Abstract The present study confirms and extends the finding that DA rat spleen cells include a subpopulation of adherent cells which act at high doses of phytohemagglutinin (PHA) or concanavalin H (ConA) to suppress DNA synthesis by other cells. Suppressor activity is lacking in spleens of doubly thymus-deprived rats. Mitomycin C treatment does not reduce the suppressor activity of normal whole spleen. It is inferred that the suppressor cell is a T lymphocyte or depends for its action on the presence of T lymphocytes and that its action does not require cell proliferation. DNA synthesis is much more affected by suppression at high mitogen levels than RNA or protein synthesis. A parallel is drawn between the separate regulation of DNA synthesis and other macromolecular synthesis in lymphocytes and so-called “pleiotypic” effects in other types of cells. Spleen cells of young rats give a relatively low response (3H-thymidine uptake) to optimal or supraoptimal doses of PHA, which is markedly increased by removal of adherent cells on glass wool and is not affected by addition of purified macrophages. In contrast spleen cells of older animals give a high response, which falls when adherent cells are removed but is restored to the original level by addition of macrophages. It is concluded that older animals' spleens differ from those of younger rats by a variable loss of suppressor cells and an increasing macrophage dependence of PHA-responsive cells. The former may account for the reported increase in autoantibody formation with age. Reactivity of cells of a given age was not affected by culturing them in young or old rat serum pools, respectively. Stress or injection of hydrocortisone acetate result in loss of suppressor cell adhesiveness to glass wool and increased macrophage dependence of PHA-responsive cells at 5 days. At 2 to 3 weeks there is a return to normal levels of adhesiveness and macrophage dependence, accompanied by a marked increase of suppressor activity. The 5-day changes are thought to reflect direct effects of steroid on the properties of T lymphocyte membranes, and the later changes altered levels of thymus hormone or possibly a surge of “post-thymic cells” into the peripheral lymphoid organs.

The immunological properties of haptens coupled to thymus-independent carrier molecules. I. The characteristics of the immune response to dinitrophenyl-lysine-substituted pneumococcal polysaccharide (SIII) and levan.

AbstractThe antibody response to the hapten 2,4dinitrophenyl (DNP)lysine coupled to two thymus‐independent carrier molecules (pneumococcal polysaccharide SIII and levan) has been studied in mice. The characteristics of the anti‐hapten response are essentially similar to the response to the carrier itself: both conjugates elicit a strongly dose‐dependent IgM response (with little or no IgG antibody), and supraoptimal doses paralyze both anti‐hapten and anti‐carrier antibody responses. The anti‐hapten response to DNPlyslevan is thymus‐independent; it is also carrier‐independent, since levan‐paralyzed mice give a normal antihapten response to the conjugate and, conversely, paralysis of the antihapten reactive lymphocyte population fails to affect the response to the carrier. The results do not support the concept of B cell‐B cell cooperation that has been proposed to explain the immunogenicity of haptenpolysaccharide conjugates. Instead, they indicate that these conjugates immunize separate and noninteracting B cell populations, the one hapten‐specific and the other carrier‐reactive. Both the numbers and the size of anti‐DNP hemolytic plaques were related to the immunizing dose of DNPlyslevan. Preliminary experiments suggest that the reduction in plaque size in partially tolerant mice may be caused by persisting antigen on the surface of immunized lymphoid cells.

Control of mitogen-induced lymphocyte activation: I. The effect of cyclic AMP on DNA and RNA synthesis in PHA-stimulated cells

The role of cyclic AMP in immune responses remains controversial. Although cyclic AMP and compounds which elevate cyclic AMP levels have been shown to enhance immune responses, several recent reports have shown that the addition of cyclic AMP will depress PHA-induced blastogenesis. The data reported here involve the use of much wider dose ranges of PHA and cyclic AMP than reported heretofore. When supraoptimal doses of PHA are used, the addition of cyclic AMP, or drugs which raise intracellular cyclic AMP levels, results in an enhancement of DNA and RNA synthesis; this effect we have termed cyclic AMP-mediated high dose recovery. Measurements of intracellular cyclic AMP levels following exposure to PHA-MR69 and/or cyclic AMP reveal that elevated cyclic AMP levels occur throughout the cell culture period. This demonstrates that elevated cyclic AMP levels may alter macromolecular synthetic patterns at points in time well after the initial activating event.

Stimulation of guinea pig lymphocytes by Lens culinaris lectin-A : Stimulation by insolubilized LcL-A and effect of erythrocytes and peritoneal macrophages on stimulation by soluble LcL-A. Presence of a lymphokine in culture fluids of LcL-A-stimulated cells

Guinea pig lymphocytes in culture are mitogenically stimulated by either soluble LcL-A or by LcL-A covalently attached to Sepharose beads. The degree of stimulation by LcL-A-Sepharose is directly dependent upon the density of LcL-A molecules per bead ranging from essentially zero with low density to a better stimulation than by soluble LcL-A with high density. Addition of small numbers of either erythrocytes or peritoneal macrophages to lymphocytes cultured with suboptimal doses of soluble LcL-A increases stimulation significantly. With optimal or supraoptimal doses the potentiation effect disappears. These observations support the proposal that a stimulating lectin presented to stimulatable lymphocytes in high local concentrations is more effective than the same concentration of lectin molecules binding more randomly on the lymphocyte surface. During stimulation of human and guinea pig lymphocytes by LcL-A or LcL-A-Sepharose a soluble lymphokine is released which is either itself a lymphocyte stimulant or one which makes lymphocytes more stimulatable by low doses of LcL-A.

Lymphocyte activation: The dualistic effect of cAMP

The effects of exogenously added cyclic nucleotides on DNA synthesis have been investigated in human peripheral blood lymphocytes stimulated with phytohemagglutinin (PHA). At low doses of PHA the addition of exogenous cAMP resulted in an inhibition of DNA synthesis. At optimal or supraoptimal doses of PHA the addition of cAMP, db-cAMP, or 8-Br-cGMP resulted in enhancement of DNA synthesis. Measurement of cell associated cAMP and cGMP levels in lymphocytes exposed to PHA with or without exogenously added cAMP revealed a gradual increase in cAMP levels and a fluctuating decline in cGMP levels.

Tolerance to a protein antigen in a poikilotherm, the marine toad Bufo marinus.

IN spite of all the immunological studies which have been carried out with poikilothermic vertebrates, there have been no quantitative studies of the induction of tolerance to purified anitgens, yet such studies might account for the difficulties often encountered in the immunization of lower vertebrates. Moreover, they should have a direct bearing on the phylogenetic emergence of the mechanisms involved in this crucial aspect of immunity. In this article, we shall show that adult marine toads (Bufo marinus) can be made tolerant to bovine serum albumin (BSA) by pretreatment with supraoptimal doses of the antigen.

The X-Y-Z scheme of immunocyte maturation. IV. The exhaustion of memory cells.

A set of conditions has been described under which primed rabbit lymph nodes produce a secondary antibody response upon in vivo stimulation with a large dose of antigen, but are subsequently "exhausted;" that is, lymph node cultures prepared at intervals following the booster injection cannot be re-stimulated to display tertiary responses. Rabbits given 100-fold less antigen in the booster inoculum were able to give a tertiary response upon in vitro challenge. The system used permits neither induction nor continuation of a primary response to BSA in vitro. Since it could be demonstrated that no memory cells were generated by the booster injection within the intervals between in vivo injection and culture, the tertiary response in nonexhausted nodes must have been due to residual memory cells which remained untriggered by the in vivo booster injection. The unresponsive state was not caused by antibody feedback. These results are interpreted to mean that a population of memory cells can be exhausted by a supraoptimal dose of antigen, rendering the node temporarily incapable of further response. This implies that long-lived memory is not due to asymmetric division of memory cells. The source and fate of memory cells is discussed with regard to this evidence.

Growth and nitrogen distribution in callus and crown-gall tobacco tissue cultures treated with kinetin and (2-Chloroethyl)trimethylammonium chloride (CCC)

1. Kinetin at a supra-optimal dose of 1.0 mg/l reduces the growth inhibitory effect of CCC at 5 × 10−2 M on tobacco callus and increases it on tobacco crown-gall. 2. CCC increases the content of total, acid soluble, protein anti nucleic acid nitrogm in both tissues. Kinetin partially reduces the effects of CCC on nitrogem metabolism in callus tissue, and increases it in crown-gall. 3. It is suggested that (1) CCC affects the synthesis of RNA directing the synthesis of protein, and (2) interaction of kinetin with CCC realizes via the genetic apparatus of the cell.