TNP-keyhole Limpet Hemocyanin Research Articles

Long-Lived Plasma Cells Secrete High-Affinity Antibodies Responding to a T-Dependent Immunization in a Teleost Fish.

The recent discovery of long-lived plasma cells (LLPCs) in mammals, which provide a constant expression of specific high-affinity antibodies that mediate humoral memory, has caused a dramatic paradigm shift in the study of immunity and vaccine development. In teleost fish, there are few studies regarding the association between LLPCs and antibody production, and the affinity of the antibodies secreted by the LLPCs is poorly understood. In the present study, channel catfish (Ictalurus punctatus) were immunized with trinitrophenylated-keyhole limpet hemocyanin (TNP-KLH) to examine TNP-specific antibody titers, affinities, antibody-secreting cell (ASC) dynamic changes, and especially the affinity of secreted antibodies by LLPCs post-immunization. We demonstrated that TNP-specific LLPCs were generated starting at week 4 post-immunization, achieved a maximal number at week 8, and maintained a comparable level throughout the 18-week post-immunization period, which was correlated with the dynamics of serum antibody titers and affinity maturation in the response. The LLPCs appeared to mostly reside within, or migrate to, the anterior kidney (bone marrow-like tissue in mammals), but a small portion was also located in the spleen and peripheral blood. The antibodies produced by the LLPCs possessed high affinities, indicating that the generation and development of LLPCs were driven by affinity selection in teleosts. Collectively, the results of this study provide insights toward the evolutionary understanding of the affinity-dependent mechanism of LLPC generation and development.

Aryl hydrocarbon receptor modulates the development and activity of innate-like B cells (HEM1P.226)

Abstract The Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that recognizes polyaromatic hydrocarbons. While the contributions of AhR ligands to the differentiation and functions of Th17/Treg cells are relatively well understood, little is known about AhR in B cells. Microarray and quantitative PCR analysis of B cell subsets revealed that B1 and marginal zone (MZ) B cells expressed higher levels of AhR compared to follicular (FO) B cells. We found ~ two-fold more B1 B cells, but fewer MZ B cells in AhR-/- mice compared to AhR+/+ mice. Following immunization with Streptococcus pneumonia, AhR-/- mice had significantly higher titers of anti-phosphorylcholine IgM antibodies. In contrast, after immunization with a model T cell-dependent antigen, Trinitrophenyl Keyhole Limpet Hemocyanin (TNP-KLH), AhR-/- mice had anti-TNP titers that were comparable to those in AhR+/+ mice. Further, we found significantly fewer B cells in C57BL/6 mice raised on AhR ligand free diet, and an increase in titers of IgM and IgG3 in mice following a switch to an AhR ligand free diet when compared to littermates raised and maintained on conventional diet. Taken together, these findings suggests that AhR plays a role in the development of B1 and MZ B cells and regulates T cell-independent but not T cell-dependent humoral response. Further studies using mice with a B cell specific deletion of AhR are underway to elucidate the mechanism by which AhR ligands influence humoral immunity.

Wiskott-Aldrich syndrome protein (WASP) and N-WASP are critical for peripheral B-cell development and function

AbstractThe Wiskott-Aldrich syndrome protein (WASP) is a key cytoskeletal regulator of hematopoietic cells. Although WASP-knockout (WKO) mice have aberrant B-cell cytoskeletal responses, B-cell development is relatively normal. We hypothesized that N-WASP, a ubiquitously expressed homolog of WASP, may serve some redundant functions with WASP in B cells. In the present study, we generated mice lacking WASP and N-WASP in B cells (conditional double knockout [cDKO] B cells) and show that cDKO mice had decreased numbers of follicular and marginal zone B cells in the spleen. Receptor-induced activation of cDKO B cells led to normal proliferation but a marked reduction of spreading compared with wild-type and WKO B cells. Whereas WKO B cells showed decreased migration in vitro and homing in vivo compared with wild-type cells, cDKO B cells showed an even more pronounced decrease in the migratory response in vivo. After injection of 2,4,6-trinitrophenol (TNP)–Ficoll, cDKO B cells had reduced antigen uptake in the splenic marginal zone. Despite high basal serum IgM, cDKO mice mounted a reduced immune response to the T cell–independent antigen TNP-Ficoll and to the T cell–dependent antigen TNP–keyhole limpet hemocyanin. Our results reveal that the combined activity of WASP and N-WASP is required for peripheral B-cell development and function.

The differential dynamics of antibody subpopulation expression during affinity maturation in a teleost

A compositional analysis of the antibody response in rainbow trout was conducted using an affinity-based immunopartitioning assay. Trout were immunized with TNP-keyhole limpet hemocyanin (TNP-KLH) and individual serum titers and their affinity distributions analyzed over a period of 27 weeks. The kinetics of antibody affinity subpopulation development revealed certain key features: 1) the lowest affinity subpopulation (log aK, 3.5–3.99) appears early, does not achieve high titer, and was more transient than the higher affinity subpopulations; 2) intermediate affinity subpopulations (log aK, 5.0–5.99) appear later (week 5), achieve relatively high titers and persist longer; and 3) the highest affinity subpopulations (log aK, 6.0–7.49) emerge much later (post week 15), and have comparable titers to the intermediate affinity group. We find that the affinity maturation of the serum antibody response can be resolved into each affinity subpopulation’s contribution both in quantity and timing.

CD86 Regulates IgG1 Production via a CD19-Dependent Mechanism

CD86 signals directly in a B cell to activate PI3K and increase the rate of IgG(1) production, without affecting germline transcription. However, the mechanism by which CD86 activates PI3K in a B cell and the relevance of CD86 stimulation in vivo remains unknown. We show that the addition of CD28/Ig to CD40 ligand/IL-4-activated wild-type, but not CD86- or CD19-deficient, B cells increased the level of phosphorylation for Lyn and CD19, as well as the amount of Lyn, Vav, and PI3K that immunoprecipitated with CD19. Adoptive transfer of CD86-deficient B cells and wild-type CD4(+) T cells into RAG2-deficient mice and immunization with trinitrophenylated keyhole limpet hemocyanin resulted in an IL-4 and germline IgG(1) response equivalent to control mice, but a decrease in serum IgG(1). Thus, our findings suggest that CD86 plays a key role in regulating the level of IgG(1) produced in vitro and in vivo, and that Lyn and CD19 may be the signaling intermediates activated by CD86 proximal to PI3K.

Antibody structural variation in rainbow trout fluids

Rainbow trout ( Oncorhynchus mykiss) were immunized with trinitrophenylated-keyhole limpet hemocyanin (TNP-KLH) and the redox structure of induced anti-TNP antibodies from the serum, mucus, egg and ovarian fluid was examined. In conducting these studies it was determined that all TNP-specific antibody from each source possessed the mAb-specific H chain (1–14) epitopes, which facilitated the direct structural analysis of the induced antibodies. A protocol was developed which ensured complete adsorption of all specific anti-TNP antibody from each fluid. Together these protocols permitted the unbiased compositional analysis of all redox forms of the anti-TNP antibodies from each source. All antibodies, regardless of source, possessed the same molecular mass, characteristic of the trout tetramer (800 kDa). It was found that specific antibody titers were significantly higher in male than female trout, while the degree of disulfide polymerization was relatively invariant in male antibodies, while being highly variable in female antibodies. Within the females, no distinctively different redox ratios were between antibodies isolated from sera, ovarian fluid or eggs: however, mucus antibodies possessed a unique redox structure consisting of halfmeric constituents that were not observed in antibodies from other fluids.

Flow immunoassay of trinitrophenol based on a surface plasmon resonance sensor using a one-pot immunoreaction with a high molecular weight conjugate

A surface plasmon resonance (SPR) immunosensor based on a competitive immunoreaction for the determination of trinitrophenol (TNP) is described. A goat anti-mouse IgG (1st antibody), which recognizes an Fc moiety of an antibody, was immobilized on a gold film of an SPR sensor chip by physical adsorption. A TNP solution containing a fixed concentration of a mouse anti-TNP monoclonal antibody (2nd antibody) and a TNP-keyhole limpet hemocyanin (KLH) conjugate was incubated in one-pot and introduced into the sensor chip. The TNP-KLH conjugate competes with TNP for binding with the 2nd antibody. The resulting complex of the 2nd antibody with the TNP-KLH conjugate was bound to the 1st antibody, which is immobilized on the sensor chip. The SPR sensor signal based on resonance angle shift is dependent on the concentration of TNP in the incubation solution in the range from 25 ppt to 25 ppb, and the coefficient of variation of the SPR signals for the 25 ppb TNP solution was determined to be 13% ( n = 4). The experimental results for the adsorption constant of the 1st antibody on the sensor chip and the binding constant of the 1st antibody complex with the 2nd antibody are discussed, together with theoretical considerations.

Plasmablast and Plasma Cell Production and Distribution in Trout Immune Tissues

These studies describe the in vitro and ex vivo generation of plasmablasts and plasma cells in trout (Oncorhynchus mykiss) peripheral blood and splenic and anterior kidney tissues. Cells were derived either from naive trout and cultured with the polyclonal activator, Escherichia coli LPS, or from trout that had been immunized with trinitrophenyl-keyhole limpet hemocyanin. Hydroxyurea was used to resolve populations of replicating (plasmablast) and nonreplicating (plasma cell) Ab-secreting cells (ASC). Complete inhibition of Ig secretion was only observed within the PBL. Both anterior kidney and splenic lymphocytes possessed a subset of ASCs that were hydroxyurea resistant. Thus, in vitro production of plasma cells appears to be restricted to the latter two tissues, whereas peripheral blood is exclusively restricted to the production of plasmablasts. After immunization with trinitrophenyl-keyhole limpet hemocyanin, specific ASC could be isolated from all immune organs; however, the anterior kidney contained 98% of all ASC. Late in the response (>10 wk), anterior kidney ASC secreted specific Ab for at least 15 days in culture, indicating that they were long-lived plasma cells. Cells from spleen and peripheral blood lost all capacity to secrete specific Ab in the absence of Ag. Late in the Ab response, high serum titer levels are solely the result of Ig secretion from anterior kidney plasma cells.

Selective augmenting effects of nitric oxide on antigen-specific IgE response in mice

Here, we report the enhancing effects of nitric oxide (NO) on an IgE antibody response in mice. Anti-trinitrophenyl (TNP) IgE production induced in vitro in TNP keyhole limpet hemocyanin (KLH)-primed spleen cells was inhibited by approximately 70% when an NO synthase (NOS) inhibitor, l-NG-monomethyl-l-arginine, was added at 10−7–10−6 M to the lymphocyte culture. On the other hand, addition of NO-generating agents to the culture resulted in a marked enhancement of the IgE production. In contrast, anti-TNP IgM and IgG1 responses were affected only marginally when the IgE production was either suppressed or augmented by these agents. NO did not directly augment IgE class switching in normal B cells stimulated with lipopolysaccharide and interleukin (IL)-4. NO-mediated augmentation of the IgE response is considered to be of a physiological significance because administration of aminoguanidine (AG), an inhibitor of inducible NOS, to immunized mice resulted in a preferential suppression of anti-TNP IgE production in vivo. This may be explained by the observation that AG-administration increased interferon-γ expression without changing that of IL-4 in the immunized mice. Taken together, these observations suggest a pathophysiological role of NO in the development of IgE-mediated allergic diseases.

Suppression of Antigen-Specific Th2 Cell-Dependent IgM and IgG1 Production Following Norepinephrine Depletion In Vivo

The mechanism by which the Th2 cell-dependent Ab response is modulated by the sympathetic neurotransmitter norepinephrine (NE) was investigated. Our model system used the severe combined immunodeficient (scid) mouse that was depleted of NE with 6-hydroxydopamine before reconstitution with a clone of beta2-adrenergic receptor (beta2AR)neg KLH-specific Th2 cells and resting trinitrophenyl (TNP)-specific beta2ARpos B cells enriched from the spleens of unimmunized mice. Following challenge with TNP-keyhole limpet hemocyanin (KLH), Ab production in these mice was hapten-, carrier-, and allotype-specific as well as MHC restricted. Depletion of NE resulted in a 50-75% suppression of the primary anti-TNP IgM response compared with that of NE-intact controls, while the secondary IgM response returned to control levels. In contrast, both the primary and secondary anti-TNP IgG1 responses were suppressed by 85 and 40%, respectively. Using NE-intact mice exposed to either a betaAR- or alphaAR-selective antagonist, the effect of NE on the Ab response was shown to be mediated by the betaAR. In addition, administration of a beta2AR-selective agonist to NE-depleted mice partially reversed the suppressed Ab response that resulted from NE depletion. Expression of the beta2AR on TNP-specific B cells was confirmed by radioligand binding, immunofluorescence, and cAMP analysis. Also, while splenic histology was comparable in NE-intact and NE-depleted mice before Ag exposure, follicle expansion and germinal center formation were suppressed in NE-depleted mice after Ag exposure. Taken together, these results suggest that NE stimulation of the beta2AR expressed on B cells is necessary for the maintenance of an optimal primary and secondary Th2 cell-dependent Ab response in vivo.

Dopamine beta-hydroxylase deficiency impairs cellular immunity.

Norepinephrine, released from sympathetic neurons, and epinephrine, released from the adrenal medulla, participate in a number of physiological processes including those that facilitate adaptation to stressful conditions. The thymus, spleen, and lymph nodes are richly innervated by the sympathetic nervous system, and catecholamines are thought to modulate the immune response. However, the importance of this modulatory role in vivo remains uncertain. We addressed this question genetically by using mice that lack dopamine beta-hydroxylase (dbh-/- mice). dbh-/- mice cannot produce norepinephrine or epinephrine, but produce dopamine instead. When housed in specific pathogen-free conditions, dbh-/- mice had normal numbers of blood leukocytes, and normal T and B cell development and in vitro function. However, when challenged in vivo by infection with the intracellular pathogens Listeria monocytogenes or Mycobacterium tuberculosis, dbh-/- mice were more susceptible to infection, exhibited extreme thymic involution, and had impaired T cell function, including Th1 cytokine production. When immunized with trinitrophenyl-keyhole limpet hemocyanin, dbh-/- mice produced less Th1 cytokine-dependent-IgG2a antitrinitrophenyl antibody. These results indicate that physiological catecholamine production is not required for normal development of the immune system, but plays an important role in the modulation of T cell-mediated immunity to infection and immunization.

CTLA4 (CD152) modulates the Th subset response and alters the course of experimental Leishmania major infection.

Since both the nature and the amplitude of an antigen-specific T cell response are dependent on co-stimulatory signals, we have investigated the role of CD28/CD152-mediated T cell co-stimulation in the regulation of experimental cutaneous leishmaniasis. CD28-deficient mice and their wild-type littermates are equally susceptible to Leishmania major infection. Whole anti-CD152 antibody significantly exacerbates the disease while anti-CD152 Fab ameliorates the disease in genetically susceptible BALB/c mice but not in C57BL/6, a resistant strain. The anti-CD152-induced exacerbation of the disease is accompanied by increased IL-4-secreting cell number, diminished parasite-specific delayed-type hypersensitivity (DTH) response and augmented anti-2,4,6-trinitrophenyl (TNP) IgG1 in response to TNP-leishmanial antigen crude soluble antigen (CSA), suggesting an exaggerated Th2 type of response. Anti-CD152 Fab-mediated amelioration of the disease is associated with increased IFN-gamma-secreting cell number, increased parasite-specific DTH response and enhanced IgG2a isotype in response to TNP-CSA suggesting a Th1 type of response. Unlike TNP-CSA, TNP-keyhole limpet hemocyanin does not induce the change in Ig isotype, indicating that the immunomodulatory effect of anti-CD152 is antigen specific. Anti-CD152 antibody-induced early change in Th subsets suggests an important role for CD152 in determining the course of L. major infection, perhaps by alteration of Th subset differentiation.

Chemical Chaperones Enhance Superantigen and Conventional Antigen Presentation by HLA-DM-Deficient as well as HLA-DM-Sufficient Antigen-Presenting Cells and Enhance IgG2a Production In Vivo

Chemical chaperones, first defined in studies of mutant cystic fibrosis transmembrane conductance regulator proteins, are small molecules that act as stabilizers of proteins in their native state and have the ability in some cases to rescue protein-folding mutants within cells. HLA-DM is an MHC II-specific molecular chaperone that facilitates peptide loading onto MHC II proteins and also stabilizes empty MHC II molecules prior to their acquisition of antigenic peptides. APC that lack HLA-DM exhibit quantitative defects in protein Ag as well as superantigen presentation. Here we show that both the superantigen and protein presentation defect in MHC II-transfected, HLA-DM-deficient T2 cells can be partially overcome by treating the APC with the chemical chaperones glycerol, DMSO, or trimethylamine oxide. These chemical chaperones also enhance superantigen and conventional Ag presentation by wild-type APC. In vivo, glycerol was found to act as an adjuvant and resulted in enhanced IgG2a production to trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH). In vitro, the enhancement of Ag presentation by chemical chaperones was found to take place at the level of the APC and took several hours to develop. Subcellular fractionation experiments show that HLA-DM enhances presentation of peptides by dense endosome fractions whereas chemical chaperones enhance presentation by light membrane fractions (early endosome or plasma membrane). The mechanism by which these chemical chaperones augment Ag presentation is not defined, but flow cytometric analysis suggests that the enhancement may be due to a subtle effect on the stability of several different proteins at the cell surface.

The role of NK cells during in vivo antigen-specific antibody responses.

Abstract Investigations into the role of NK cells in regulating Ab responses have yielded variable results, some suggesting that NK cells can down-regulate Ag-specific Ig production and others proposing an enhancing effect. These apparently inconsistent findings may stem partially from the specificity of reagents used in purifying cell populations and/or the nature of the in vitro systems used to study these events. We chose to investigate the ability of either resting or poly(I:C)-activated NK cells to alter an in vivo Ab response in mice given a T-independent (TNP-LPS) or T-dependent (TNP-keyhole limpet hemocyanin (KLH)) Ag. By using a more specific Ab, anti-NK-1.1, to deplete NK cells, we were able to clearly show that resting, endogenous NK cells do not affect either type of response, as measured by serum Ag-specific Ig levels quantitated by isotype-specific ELISA. In contrast, activation of NK cells by poly(I:C) increased Ag-specific IgC2a as well as IgG1 levels. Interestingly, only the effect on IgG2a production is reversible by depletion of NK cells.

Induction of antigen-specific IgE response in murine lymphocytes by IL-10

When murine spleen cells that had been primed with trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) were stimulated in vitro with the same antigen, anti-TNP IgE, as well as anti-TNP IgM and IgG1, was secreted into the culture medium. On the other hand, anti-TNP IgM and IgG1 were produced, but anti-TNP IgE secretion was negligible when the carrier (KLH)-primed spleen cells were cultured with the hapten-carrier antigen (TNP-KLH) under the same conditions. Anti-TNP Ig responses in the latter cultures are thought to reflect the interaction between normal TNP-specific B cells and KLH-primed helper T cells. By using this culture system, we investigated the requirements of exogenous cytokines for inducing anti-TNP IgE response. The addition of interleukin-4 (IL-4), that is known to induce IgE response in LPS-stimulated murine B cells, failed to elicit anti-TNP IgE response. The combination of IL-4 with IL-2 and/or IL-5 was also ineffective. Interestingly, a significant level of anti-TNP IgE was induced when IL-10, another cytokine from type 2 helper T cells, was added to the culture. Although IL-10 enhanced the production of anti-TNP IgM and IgG1, as well as that of anti-TNP IgE, the rate of enhancement was at least 3-fold higher in the IgE response than in the IgM and IgG1 responses. Simultaneous addition of IL-4, IL-5 or IL-13 with IL-10 did not augment but rather reduced the enhancing effects of IL-10. IL-10 did not further stimulate the spontaneous secretion of IgE from antigen-primed B cells. These results suggest that IL-10 plays a positive role in the induction of antigen-specific IgE responses.

Immunological assessment of hybrid striped bass at three culture temperatures

Hybrid striped bass ( Morone saxatilisWalbaum× Morone chrysopsRafinesque) were maintained at three water temperatures (23°C, 26°C and 29°C) within their preferred temperature range. Physiological and immunological parameters were measured in each temperature group to determine if an optimal culture temperature exists for various immune functions. No differences were found in the mean haematocrit, leukocrit, and serum cortisol of the three temperature groups; however, changes in the relative numbers of leukocytes in the highest temperature group were suggestive of thermal stress. Nonspecific immunity as evaluated by granulocyte respiratory burst activity and serum spontaneous haemolytic activity was found to be temperature sensitive with the highest responses occurring in the 26°C group. Following immunization with trinitrophenyl-lipopolysaccharide antigen, the passive haemagglutinating antibody response was very rapid (3 days) at 29°C but was delayed (6 days) in the 23°C group, and lower numbers of plaque-forming cells were evident at 7 days post-immunization in the 29°C group reflecting possible faster kinetics. The secondary in vitroplaque forming cell response to trinitrophenyl-keyhole limpet haemocyanin was also decreased in the highest temperature group indicating fewer memory cells or suppression of the 29°C acclimated cells when stimulated at 23°C in vitro.

Suppression of B-cell mediated immunity in juvenile chinook salmon (Oncorhynchus tshawytscha) after exposure to either a polycyclic aromatic hydrocarbon or to polychlorinated biphenyls.

Juvenile chinook salmon (Oncorhynchus tshawytscha) were injected intraperitoneally with either the polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene (DMBA)1 or with the commercial polychlorinated biphenyl (PCB) mixture, Aroclor 1254, to assess effects on the B-cell mediated immune response. B-cell mediated immunity was assessed by examination of the primary and secondary plaque-forming cell (PFC) responses of anterior kidney and splenic leukocytes to a T-independent antigen, TNP-keyhole limpet hemocyanin (TNP-KLH). Salmon exposed to DMBA at dosages of 20% or 1% of the 96 hr LD50 (12.7 mg and 0.6 mg/kg of salmon, respectively) or to PCBs at a dosage of 20% of the 96 hr LD50 (54.0 mg/kg of salmon) exhibited a suppressed PFC response. The secondary PFC response of anterior kidney and splenic leukocytes to both antigens and the primary splenic PFC response to TNP-LPS were suppressed in salmon exposed to either DMBA or PCBs. However, only the primary PFC response of anterior kidney leukocytes to TNP-LPS was suppressed in salmon exposed to PCBs and no suppression of this response was observed in salmon exposed to DMBA. Neither anterior kidney or splenic leukocytes from salmon exposed to DMBA or PCBs showed an altered primary PFC response to the T-dependent antigen, TNP-KLH. These results suggest that B-cell mediated immunity in salmon is suppressed by known mammalian immunosuppressants and that suppression of the PFC response observed previously in salmon from an urban estuary may be due to contaminant exposure.

Suppression of IgE antibody response in mice by a naphthalene derivative, TEI-6472

In the present report, we investigated suppressive effects of a naphthalene derivative, (7E)- N-(2-carboxyphenyl)-8-(2-naphthyl)-5,6-trans-5, 6-methano-7-octenamide L-lysine salt (TEI-6472), on in vitro and in vivo antigen-specific IgE response. Anti-trinitrophenyl (TNP) IgE response induced in vitro in TNP-keyhole limpet hemocyanin (KLH)-primed murine spleen cells was suppressed by about 60% in the presence of 10 −6 M TEI-6472. On the other hand, anti-TNP IgG1 and IgM response were not significantly suppressed under the same conditions. Proliferative responses of BALB/c spleen cells stimulated by lipopolysaccharide, concanavalin A or allogeneic spleen cells were not inhibited by TEI-6472 at 10 −6-10 −5 M. Interleukin 4 production from helper T-cell clone, D10.G4.1 was suppressed only slightly (less than 20%) at 10 −6 M TEI-6472. This compound was also effective in suppressing secondary anti-TNP IgE response in mice that were immunized twice with TNP-KLH and alum. When 10–20 mg/kg/day TEI-6472 was administered s.c. for 5 consecutive days starting from one day before the first and the second immunization, secondary anti-TNP IgE response was inhibited most strongly (40–45%). Anti-TNP IgG1 response was also inhibited but to a smaller extent (20–24%), while anti-TNP IgM response was suppressed only slightly (0–15%). These results suggest that, under appropriate conditions, TEI-6472 can suppress IgE responses more preferentially both in vitro and in vivo.

In vivo cortisol administration suppresses the in vitro primary immune response of winter flounder lymphocytes

Prolonged (24-day) administration of cortisol to adult winter flounder, Pleuronectes americanus , was found to decrease the in vitro primary immune response of splenic lymphocytes as measured by the passive haemolytic plaque assay. Intraperitoneal implantation of fish with cortisol mixed into a vegetable shortening and cocoa butter vehicle resulted in elevated serum cortisol levels that were within physiological ranges. Existing techniques for piscine lymphocyte culture were modified for use with winter flounder. Longer durations of elevated serum cortisol in the fish produced a more marked suppression of the plaque-forming cell (PFC) response. The suppression was evident as early as 10 days postimplantation when winter flounder cultures were stimulated for 14 days with trinitrophenyl-keyhole limpet haemocyanin (TNP-KLH). Suppression of the PFC response was not apparent until 17 days postimplantation in those cultures stimulated with trinitrophenyl-lipopolysaccharide (TNP-LPS) and was less apparent when the culture period was lengthened from 9 days to 14 days. Our results indicate that the in vitro primary PFC response can be used to evaluate the presence of chronic stress in fish.

In vitro antigen-specific IgE response is refractory to suppression by interferon-gamma.

Although interferon (IFN)-gamma has been shown to be involved in the down-regulation of polyclonal IgE response in murine B cells that were activated by lipopolysaccharide (LPS) and interleukin 4 (IL4), effects of IFN-gamma on antigen-specific IgE responses have not been fully investigated. We have developed the following culture systems for inducing antigen-specific IgE responses in murine lymphocytes, and examined the effects of IFN-gamma on the following responses in vitro. (1) Anti-trinitrophenyl (TNP) IgE response induced by the stimulation with TNP-keyhole limpet hemocyanin (KLH) of BALB/c spleen cells that had been primed in vivo with the same antigen. (2) Anti-TNP IgE response induced by the coculture of unprimed C3H B cells with conalbumin (CA)-specific helper T cell clone, D10.G4.1, in the presence of TNP-CA. The former anti-TNP IgE response was not suppressed, and the latter suppressed only partially (less than 30%) by the addition of 100-200 U/ml IFN-gamma. In contrast, polyclonal IgE response in murine B cells that were stimulated by LPS and IL4 was abolished by 10 U/ml IFN-gamma. These results indicate that IgE production from antigen-stimulated B cells, in contrast to those activated polyclonally, are refractory to direct suppression by IFN-gamma.