Prostaglandin-induced enhancement of the anamnestic response

The ability of various prostaglandins (PGs) to affect the anamnestic immune response of keyhole limpet hemocyanin (KLH)-primed rabbit popliteal lymph node cells was investigated. Of the four PGs studied (PGA₁, PGE₂ and PGF_2α), PGE₁ was found to have a stimulatory effect, whereas PGA₁, PGE₂ and PGF_2α were ineffective in stimulating or inhibiting the anamnestic response. Under the conditions studied, a 3.5-fold increase in antibody production was obtained in PGE₁-treated, KLH-stimulated cultures. Maximum enhancement was obtained when 0.2 μg of PGE₁ were added at the time of culture initiation and were allowed to remain in contact with the lymph node cells for 24 hours.     

Prostaglandin-induced enhancement of the in vitro anamnestic response

The ability of various prostaglandins (PGs) to affect the $\text{in vitro}$ anamnestic immune response of keyhole limpet hemocyanin (KLH)-primed rabbit popliteal lymph node cells was investigated. Of the four PGs studied (PGA₁, PGE₂ and PGF_2α), PGE₁ was found to have a stimulatory effect, whereas PGA₁, PGE₂ and PGF_2α were ineffective in stimulating or inhibiting the $\text{in vitro}$ anamnestic response. Under the conditions studied, a 3.5-fold increase in antibody production was obtained in PGE₁-treated, KLH-stimulated cultures. Maximum enhancement was obtained when 0.2 μg of PGE₁ were added at the time of culture initiation and were allowed to remain in contact with the lymph node cells for 24 hours.     

Prostaglandin-induced enhancement of the in vitro anamenstic response

The ability of various prostaglandins (PGs) to affect the in vitro anamnestic immune response of keyhole limpet hemocyanin (KLH)-primed rabbit popliteal lymph node cells was investigated. Of the four PGs studied (PGA₁, PGE₂ and PGF_2α), PGE₁ was found to have a stimulatory effect, whereas PGA₁, PGE₂ and PGF_2α were ineffective in stimulating or inhibiting the in vitro anamnestic response. Under the conditions studied, a 3.5-fold increase in antibody production was obtained in PGE₁-treated, KLH-stimulated cultures. Maximum enhancement was obtained when 0.2 μg of PGE₁ were added at the time of culture initiation and were allowed to remain in contact with the lymph node cells for 24 hours.     

Effect of corticosteroids on human in vitro anamnestic response.

When human peripheral blood lymphocytes were cultured in the presence of the antigens streptodornase (SKSD) and allogeneic lymphocytes (MLC), increased rates of DNA synthesis were observed 4 to 6 days laer. If these cell cultures were continued to 12 days, the rates of DNA synthesis returned to control levels. On rechallenging these cells with the same antigens, an accelerated response was observed. The corticosteroid preparations hydrocortisone and methylprednisolone suppressed both of these responses and also impaired the ability of the 12-day cultures to generate cells responsible for the accelerated secondary response. Although cells cultured for 12 days in the presence of SKSD and hydrocortisone responded poorly to SKSD rechallenge, they responded well in MLC. Hence, the suppressive effect was not completely unselective.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. II. Antigen-dependent enhancement by exogenous prostaglandins of the E series.

The spleen cells from CFW/D mice injected with dimethylbenzanthracene-induced leukemia virus exhibited a progressive decline in the in vitro response to heterologous erythrocyte antigens in parallel with tumor growth. Cell transfer experiments revealed that this immunodepressed state may involve a B-cell defect rather than extrinsic factors in the cellular environment since: (i) nonresponsiveness could be transferred to irradiated non-tumor-bearing mice with spleen cells, and (ii) T cells from tumorbearing mice cooperated with normal bone marrow cells, but bone marrow from tumorbearing mice did not cooperate with normal T cells. In addition, T cells from the thymic tumor could cooperate with normal bone marrow cells upon transfer to irradiated recipients. TL 485-2 cells, a T-cell line derived from the tumor, could be specifically activated with SRBC thereby indicating that the virus transformed T cells were immunocompetent. Suppressor cells, which appeared in the spleen concomitant with immunodepression and tumor development, may directly raise B-cell thresholds for T-dependent triggering signals since the antibody response of spleen cells from tumor-bearing mice could be restored by adding agents such as LPS, 2 mercaptoethanol, or T cells exogenously preactivated in normal animals. The suppressor cell could be enriched by adherence to plastic and was removed by treatment with carbonyl iron. In addition, it was unlikely that the suppressor cell was a virus-infected cell since transformed, virus-infected cells from the tumor or TL 485-2 cells were not suppressive when added to spleen cells in vitro but rather resulted in a marked, polyclonal enhancement of the PFC response. The interaction of TL 485-2 cells and normal spleen cells resulted in the release of a stimulatory factor which increased DNA synthesis in resting cells as well as increasing PFC. The role of these enhancing factors and suppressor cells in controlling tumor growth remains to be elucidated.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP: I. Antigen-induced uptake of nucleotides and nucleosides by lymphocytes

Addition of N⁶, O²′-dibutyryl cAMP (DbcAMP) to keyhole limpet hemocyanin (KLH)-primed rabbit lymph node cells for 1 hr, followed by its removal and the addition of KLH, had no effect on the subsequent antibody response, whereas addition of KLH for 1 hr followed by DbcAMP resulted in a 100% enhancement of antibody synthesis. Addition of cholera enterotoxin (CT), which rapidly and irreversibly binds to lymphocytes and activates adenylate cyclase, either before or after the addition of antigen, elevated the antibody response by 100%. These results suggested that some antigen-induced event(s) may be required for DbcAMP to exert its enhancing effects on the antibody response. The effect of KLH on the uptake of DbcAMP by KLH-primed lymph node cells was investigated. One and one hundred micrograms of KLH, which induce optimal and supraoptimal antibody synthesis, respectively, promoted maximal uptake of DbcAMP. This induced uptake was first detectable about 12 hr after addition of KLH, and it peaked during 24–48 hr of culture. DbcAMP uptake induced by a brief exposure of KLH (0–1 hr) was equivalent to that observed with long-term KLH addition (0–24 hr). KLH-induced DbcAMP uptake required KLH-reactive lymphocytes and represented active transport. Antibody to rabbit T lymphocytes inhibited this antigen-induced uptake. The mitogens concanavalin A (Con A) (T cells) and goat anti-rabbit Fab' (anti-Fab') (B cells) also stimulated DbcAMP uptake, as did human serum albumin (HSA) and myoglobulin (Mb) when added to homologously primed cells, indicating the generality of the phenomenon. [³H]DbcAMP entered the cells as di- or monobutyryl cAMP with about 40% metabolized to 5′AMP. This uptake could be competitively inhibited by other adenine or guanine nucleotides and nucleosides.     

Effect of Corynebacterium parvum on the immune response in guinea pigs. I. Mode of enhancement of the anamnestic response and development of delayted hypersensitivity after treatment with Corynebacterium parvum]

The effect of Corynebacterium parvum (C. parvuum) on the immune response of the guinea pig to ovalbumin varies with the protocol of immunization. The marked effect of C. parvum on the anamnestic response in the rabbit has been confirmed in the guinea pig when immunization is carried out intradermally with a mixture of C. parvum and ovalbumin. When C. parvum is given intravenously or subcutaneously or intradermally but separately from the antigen, this effect is not observed. Whatever the route of injection guinea pigs treated with C. parvum show skin reactions of delayed type hypersensitivity at the site of an intradermal booster when the latter is given at least 27 days after primary immunization.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. IV. Evidence for participation of prostaglandins of the E series in the early events.

The addition of KLH to KLH-primed rabbit lymph node cell cultures induced an anamnestic antibody response. The further addition of prostaglandins of the E series, but not PGF1^α, enhanced this antibody response manifold. The addition to these cultures of prostaglandin synthetase inhibitors together with KLH inhibited antibody production. At the concentration (10^?4) required to inhibit antibody synthesis, by a variety of criteria one of these inhibitors, indomethacin, was shown not to exert its effects through cytotoxicity. By contrast, two other inhibitors of prostaglandin synthesis, Ro-20-5720 and Ro-3-1314, inhibited antibody synthesis because of their cytotoxicity. The inhibition of the antibody response by indomethacin did not occur when PGE1 or PGE2 was added concurrently to these cultures, clearly showing that inhibition was due to a deficiency of prostaglandins. These findings strongly suggest that induction and/or regulation of the in vitro anamnestic antibody response of KLH-primed lymph node cells to 1 and 100 μg KLH requires continued prostaglandin synthesis. Potential mechanisms for the regulation of the antibody response by prostaglandins are discussed.     

In vitro anamnestic immune responses and modulating factors

Summary The term anamnestic refers to the specific and enhanced immune responses of antigen-immunized (primed) lymphoid memory cells to secondary challenge with a foreign substance (antigen). These responses include the accelerated and quantitatively greater syntheses of antibody and other macromolecules than upon primary challenge of such cells. Rabbits were primarily immunized with keyhole limpet hemocyanin (KLH). Six days later their memory lymph node cells (LNC) were removed, and upon culture with KLH, responded with the synthesis of antibody, immunoglobulin (Ig), protein, DNA and RNA, as well as with active transport of dibutryl cyclic AMP (DbcAMP). Purified thymus-derived (T) LNC were prepared on anti-rabbit Ig affinity columns. Bursal-equivalent (B) cells were prepared by binding to a complex of sheep erythrocytes (SRBC)-antibody to SRBC-complement and centrifugation of these complexes on suitable gradients. When these T and B KLH-primed LNC were mixed and challenged with KLH the aforementioned macromolecular syntheses and active transport occurred. Indeed, by a variety of criteria, the reconstituted anamnestic immune responses were indistinguishable from these responses of unfractionated LNC. Antigenic stimulation of KLH-primed T cells induced the synthesis of proteins and DNA, but not antibody, but antigenic challenge of KLH-primed B cells did not evoke these syntheses. However, added KLH induced a mixture of T and B antigen-primed LNC to synthesize more protein, Ig, DNA than either population alone and more antibody than T cells per se; B cells required help for all of these responses. The thymus (T) cell-dependent phase of in vitro anamnestic antibody response lasted the first 24–36 hr.The antibody response was regulated by antigen-concentration. One g KLH evoked maximal antibody synthesis, 10 and 100 g KLH much less. Challenge of the separated T and B cell populations with different KLH concentrations, followed by recombination and eventual assay of antibody synthesis revealed different optima. The optimal concentration for T cell help was 0.01–0.1 g KLH; higher amounts induced much less antibody production. The optimum for B cells was 1–10 g KLH; 100 g inhibited antibody formation.The antibody response to KLH and human serum albumin (HSA) was regulated nonspecifically utilizing LNC from rabbits immunized simultaneously with these two antigens. Thus stimulation of LNC from these rabbits with either antigen induced the synthesis of antibodies to both antigens. HSA and KLH did not cross-react either serologically or cellularly. Cross-stimulation of antibody synthesis also was observed when rabbit LNC were primed with KLH and Mb. However, in this instance, cross-reaction between KLH and sperm-whale myoglobulin (Mb) was observed at the cellular, presumably the T cell, level, although not at the antibody (B cell) level. The antibody response could also be modulated by exogenous cholera enterotoxin (CT), dibutyryl cyclic AMP (DbcAMP) and prostaglandins of the E series. The addition of each substance together with 1–100 g KLH to KLH-primed LNC enhanced the antibody response many-fold. CT-induced non-immunized LNC to produce soluble factor(s) (SF) which, when added to KLH-primed LNC together with KLH, enhanced antibody synthesis significantly. The addition of Indomethacin, an inhibitor of PGE synthesis to KLH-immunized cells together with KLH inhibited antibody production, suggesting that PGE was involved in this response. Evidence was adduced that neither cyclic AMP nor PGE was required for the antibody response: Ca²⁺ was not required for induction of this response by KLH, but only its regulation by cAMP.Moreover, when KLH-primed LNC were fractionated on Nylon columns, the effluent cells were induced by KLH to synthesize antibody, but this synthesis was not enhanced by added DbcAMP or PGE; presumably, regulatory cells were removed on the column. Added KLH induced PGE synthsis in these cultures; this synthesis required macrophages. In all of the LNC cultures — including cultures from rabbits immunized with KLH, HSA, and MB months or a year earlier — much antibody synthesis occurred even when antigen was not added to the cultures. This spontaneous antibody was anamnestic, thymus (T cell)-dependent and involved the interaction of residual immunogen on dendritic cells with T and B memory cells. This spontaneous antibody response provides a model for the study of the factors involved in the longterm maintenance of humoral immunity.Mb was employed as a source of more refined antigenic determinants. Rabbits were immunized with Mb in complete Freunds adjuvant. The addition of small synthetic peptides corresponding to the five antigenic sites of Mb to the Mb-primed LNC induced the synthesis of antibody, Ig, protein, DNA, RNA, and macrophage migration inhibitory factor (MIF). The N terminal 1–6 peptide, which is not antigenic, i.e. does not combine with antibody to Mb, also induced all of these syntheses, except MIF. These peptide-induced responses appeared to be thymus-dependent.Abbreviations AP alum-precipitated - AFab goat IgG antibody to rabbit Fab - ATG goat IgG antibody to rabbit thymocytes - BGG bovine gamma globulin - Bsa bovine serum albumin - BAC bromo acetyl cellulose - B bursalequivalent lymphocytes - CT cholera enterotoxin - CRL complement receptor lymphocytes - DFA complete Freund's adjuvant-, - cAMP adenosine 3:5-cyclic monophosphate - cGMP guanosine 3:5-cyclic monophosphate - DbcAMP N⁶,O²-dibutryl cyclic AMP - EAC sheep erythrocytes sensitized with antibody and complement - FITC fluorescein isothiocyanate - HSA human serum albumin - KLH keyhole limpet hemocyanin - LNC lymph node cells - MEM minimum essectial Eagle's medium - medium; MIF m crophage migration inhibitory factor - Mb sperm-whale myoglobin - PHA phytohemagglutinin - PGE prostaglandins of the E series - PGF prostaglandins of the F series - PGSI inhibitors of prostaglandin systhesis - Slg surface immunoglobulin - T thymus-derived lymphocytes     

Differentiation of antibody forming cells in lymph nodes during the anamnestic response

The cellular and subcellular events in the anamnestic response were considered. Rabbits previously immunized with key hole limpet hemocyanin (KLH) were given an anamnestic challenge in the hind footpads. The popliteal lymph nodes were removed at intervals after immunization and the following correlated on a temporal basis: the changes in the number and types of cells in the lymph nodes; the formation and regression of ribosomes, polyribosomes, endoplasmic reticulum and Golgi apparatus in plasma cells; the changes in intracellular immunofluorescence for anti-hemocyanin; and, the incorporation of ¹⁴C labeled amino acids by lymph node cells into anti-KLH during a brief in vitro culture period. Maximum intracellular fluorescence for anti-KLH and the largest incorporation of ¹⁴C labeled amino acids into antibody occurred between the third and fourth day after immunization. During this interval highly differentiated plasma cells were most numerous with respect to the total cellular population. These events took place in a 12 to 24 hour period. This was followed by an abrupt decline in the synthesis of antibody. Coincident with this was a reduction in the number of recognizable plasma cells in the nodes, diminished intracellular fluorescence for anti-KLH and a simplification of the cytoplasm of the plasma cells toward a lymphocytic form.     

In vitro anamnestic antibody response: differential cellular and calcium requirements for induction by antigen and regulation by cyclic AMP.

Lymph node cells from rabbits, immunized 6 clays previously with keyhole limpet hemocyanin (KLH) were fractionated on columns containing nylon fibers. The non-retained population (effluent cells) and the retained population (adherent cells) were subsequently characterized by various criteria. The addition of dibutyryl cAMP (DbcAMP) or cholera enterotoxin (CT) during induction by 1 and 100 μg KLH resulted in a >100% increase in antibody synthesis over the control (KLH only) responses in the unfractionated and adherent cell populations. In the effluent population CT and DbcAMP failed to enhance the 1 μg response, but did increase the 100 μg response. Antibody forming cells, as judged by ongoing antibody synthesis during the first 24 hr of culture, were deficient in the effluent population. Both the effluent and adherent cells responded to the mitogens concanavalin A, phytohemagglutinin, and goat anti-rabbit Fab'. The control, effluent, and adherent populations each contained approximately 45% surface Ig positive cells as judged by direct immunofluorescence. The removal of calcium from the medium during induction (0–24 hr) also demonstrated that induction of the antibody response by KLH was separable from the cAMP mediated enhancement of antibody synthesis.