Temperature mediated processes in teleost immunity: differential abilities of channel catfish T and B lymphocytes to cap membrane antigen

1. The effect of both in vivo acclimation temperature and in vitro assay temperatures on channel catfish T and B lymphocyte membrane antigen (mAg) capping were investigated to determine if capping might be the temperature sensitive step involved in the low temperature immunosuppression of channel catfish T cell responses. 2. Flow cytometry was used to monitor the kinetics of capping induced by a mouse monoclonal antibody (mAb 11G3) specific for a common antigenic determinant present on channel catfish T and B cells. Results indicated that the kinetics of mAg capping were dependent on in vitro assay and in vivo acclimation temperatures and the length of time of in vivo acclimation. 3. T cells from fish appropriately acclimated to 27 degrees C cap mAg more efficiently at low assay temperatures than do B cells. 4. Activation energies were 32 and 47 kcal/mol for B and T cells, respectively, from fish acclimated to 17 degrees C for 3 weeks, but were significantly lower (14 and 22 kcal/mol, respectively) after acclimation for 5 weeks. 5. In summary, it appears that after appropriate in vivo acclimation, channel catfish T cells are better able to cap mAg at low assay temperatures than are B cells. These results suggest that mAg capping is not the low temperature sensitive step involved in T cell immunosuppression in channel catfish.     

Kinetics of membrane immunoglobulin capping on murine B lymphocytes. Effects of phospholipid fatty acid replacement

Detailed analyses regarding the effects of temperature and phospholipid fatty acid replacement on the capping of membrane immunoglobulin (mIg) have been performed using a recently described flow cytometric procedure (Cuchens, M. A., and Buttke, T. M. (1984) Cytometry 5, 601-609). Purified murine B cells were incubated for 12-20 h in the presence of bovine serum albumin-complexed 80 microM stearic (18:0), oleic (cis-18:1), or linoleic (cis, cis-18:2) free fatty acids. Unmodified and free fatty acid-treated cells were stained with fluorescein-conjugated rabbit anti-mouse Ig and subjected to pulse-shape (width) analyses to follow the kinetics of mIg capping. In both unmodified and free fatty acid-treated cells, capping of mIg occurred at all temperatures between 17 and 37 degrees C, but the rate of cap formation was temperature dependent. Arrhenius plots of mIg capping were linear, with activation energies ranging from 14 to 23 kcal/mol depending on the saturated/unsaturated fatty acid ratio of B cell phospholipids. Ligand-induced redistribution of mIg thus appears to be sensitive to changes in membrane acyl chain composition.     

Temperature-mediated processes in teleost immunity: homeoviscous adaptation by channel catfish peripheral blood cells

1. Channel catfish peripheral blood erythrocyte, thrombocyte, T cell and B cell membranes were assayed by fluorescence depolarization using the fluorescent probe 1,6-diphenyl-1,3,5 hexatriene (DPH) to determine the effects of in vivo temperature acclimation on membrane viscosity and the kinetics of homeoviscous adaptation. 2. Erythrocyte membranes did not undergo homeoviscous adaptation during the 8 week time period studied and were more rigid compared with those of the other cell types. 3. The kinetics of homeoviscous adaptation exhibited by membranes from T cells, B cells and thrombocytes differed: B cells required 1-3 weeks while T cells and thrombocytes each required 3-5 weeks. Membranes from T cells, B cells and thrombocytes from fish acclimated for relatively short times (less than or equal to 3 weeks) exhibited similar membrane fluidities. 4. T cells from channel catfish appeared not only to be sensitive to temperature but also to a factor(s) independent of temperature but correlated to long term in vivo acclimation, i.e. T cell membranes underwent additional decreases in membrane viscosity between 3 and 5 weeks. 5. In conclusion, it appears that low temperature-mediated immunosuppression of T cell functions in channel catfish is probably not due to an inherent non-adaptability or rigid nature of the T cell membranes.     

Effect of temperature on the immune system of channel catfish (Ictalurus punctatus)--I. Leucocyte distribution and phagocyte function in the anterior kidney at 10 degrees C.

1. Temperatures of 18 degrees C for acclimation or assay had minimal or no effect on channel catfish phagocyte function. Significant suppression was observed at 10 degrees C acclimation and assay temperature. 2. According to the results of a multiple acclimation/assay temperature combination study, the primary impact of temperature on phagocyte function was due to the assay temperature. 3. The only functional change caused by acclimation temperature was the possible adaptation of the respiratory burst. However, 10 degrees C acclimation did cause a decline in the number of lymphocytes in the anterior kidney but not the number of neutrophils. In a temperature-kinetic study, the suppressive effect of 10 degrees C assay temperature was confirmed. 4. Results of our study indicated that phagocytosis in channel catfish is temperature-mediated. However, phagocytes appeared to be more resistant to low temperature than lymphocytes, which implies the importance of phagocytosis in the defense mechanisms of channel catfish at low temperatures.     

Rhodamine 123 permeability through the catfish intestinal wall: Relationship to thermal acclimation and acute temperature change

Temperature is known to influence xenobiotic retention in fish. The effect of acute and acclimatory temperature change upon Rhodamine 123 (Rho123) permeability through an in vitro catfish multi-segment (3) everted sac intestinal wall model was examined in a 9 cell matrix of acclimation and assay temperatures (10, 20 and 30 degrees C). Changes in Rho123 permeability were examined in context with membrane fluidity, xenobiotic solubility and intestinal morphology. When assayed at the acclimation temperature greater Rho123 permeability was noted at warmer acclimation temperatures for the proximal and middle intestinal segments, while the distal segment exhibited little change and apparent compensation across temperatures. Rho123 permeability was increased as assay temperatures were elevated above the acclimation temperature for most comparisons. Cold acclimation significantly increased total intestinal length (43.2%) and proximal intestine weights while total body weights did not differ. Brush border membranes (BBM) increased fluidity with increased assay temperatures, however, composite anisotropy lines were not significantly different between acclimation treatments. In an additive manner, the membrane probe DPH exhibited increased solubility in BBM with increases in acclimation and assay temperatures. Compositely, these results suggest that acclimation and acute temperature change may differentially influence xenobiotic permeability among intestinal segments with interacting mechanisms.     

Temperature-mediated processes in teleost immunity: differential effects of temperature on catfish in vitro antibody responses to thymus-dependent and thymus-independent antigens

An in vitro culture system was employed to ascertain the effects of different temperatures on the anti-hapten antibody-secreting cell responses of channel catfish leucocytes to murine thymus-dependent (TD) and thymus-independent (TI) antigens. The magnitudes of primary responses to a TI antigen (TNP-LPS) and secondary responses to a TD antigen (DNP-KLH) were relatively independent of in vitro culture temperature. The kinetics of each of these responses as a function of temperature was characterized by a Q10 of 2. In contrast, the magnitudes of primary responses to TD antigens (DNP-KLH and DNP-HoSA) were suppressed at lower in vitro temperatures. Furthermore, it was observed that some of the low temperature suppression of primary responses to TD antigens could be abrogated by appropriate low temperature in vivo acclimation. These findings are interpreted as supporting the hypothesis that low environmental temperatures immunosuppress fish by virtue of differential inhibitory effects on the generation of carrier-specific helper cells.     

The effects of in vivo acclimation temperature on the fatty acid composition of channel catfish (Ictalurus punctatus) peripheral blood cells

Channel catfish were acclimated in vivo to 12, 17, 22 or 27 degrees C and their peripheral blood erythrocytes, thrombocytes, T lymphocytes and B lymphocytes assayed for cellular fatty acid composition. Excepting cells from 12 degrees C acclimated fish, all cells responded to acclimation to lower temperatures by exhibiting increased levels of phospholipid unsaturated fatty acids. Although temperature independent differences were observed between erythrocytes, thrombocytes and lymphocytes, no differences between T lymphocytes and B lymphocytes were seen.     

The effect of temperature acclimation on myocardial beta-adrenoceptor density and ligand binding affinity in African catfish (Claris gariepinus)

This study assessed the effects of temperature acclimation on myocardial beta-adrenoceptor density (B(max)) and binding affinity (K(d)) in African catfish (Claris gariepinus) acclimated to 15, 22 and 32 degrees C. B(max) values were not significantly different (P > 0.05) among the three acclimation groups. Conversely, the K(d) value of the 32 degrees C acclimation group (K(d) = 0.88) was significantly higher (P = 0.002) than both the 15 degrees C (K(d) = 0.48) and 22 degrees C (K(d) = 0.46) acclimation groups. In addition, K(d) of rainbow trout (Oncorhynchus mykiss) was significantly lower (P < 0.001) and B(max) significantly higher (P < 0.05) than that of African catfish at all three acclimation temperatures. These results contrast with those reported previously for temperate species, in which B(max) is inversely related to acclimation temperature, and counter a previous suggestion that B(max) is higher in tropical versus temperate species.     

Differential effects of temperature and exogenous fatty acids on mitogen-induced proliferation in channel catfish T and B lymphocytes

1. Exogenously supplied, BSA complexed saturated and unsaturated fatty acids were compared for their effects on mitogen-induced DNA synthesis in channel catfish T and B lymphocytes. 2. At "permissive" in vitro temperatures (27 degrees C), high concentrations (greater than or equal to 240 microM) of all the fatty acids used were inhibitory. However, at lower concentrations (80-160 microM), differences were noted in the ability of some fatty acids to modulate mitogen responses. While palmitic acid (16:0) and linoleic acid (18:2) had little effect on LPS-induced B cell- or Con A-induced T cell proliferation, stearic acid (18:0) suppressed while oleic acid (18:1) enhanced T cell responses only. 3. Adding equimolar amounts of 18:0 and 18:1 obviated the effects of singularly added fatty acids on T cell mitogenesis. 4. 18:1 was used to successfully "rescue" approximately 60% of the Con A-induced T cell proliferation normally inhibited at "nonpermissive" in vitro temperatures (17 degrees C). 5. While B cells readily appear to desaturate 18:0 and synthesize unsaturated fatty acids, T cells accumulate comparatively large amounts of 18:0 in membrane associated phospholipids. 6. It is proposed that 18:1 enhances T cell responses at permissive high temperatures and rescues suppressed T cell responses at nonpermissive low temperatures by increasing membrane fluidity.     

Critical thermal minima and maxima of three freshwater game-fish species acclimated to constant temperatures

A total of 120 critical thermal maxima (CT maxima) and 120 critical thermal minima (CT minima) were determined for channel catfish, largemouth bass and rainbow trout acclimated to three constant temperatures: 20, 25 and 30 °C in catfish and bass, and 10, 15 and 20 °C in trout. Highest mean CT maximum and lowest mean CT minimum measured over these acclimation temperatures were 40.3 and 2.7 °C (catfish), 38.5 and 3.2 °C (bass) and 29.8 and ∼ 0.0 °C (trout). Temperature tolerance data were precise with standard deviations generally less than 0.5 °C. Channel catfish had the largest thermal tolerance scope of the three species while rainbow trout had the lowest tolerance of high temperatures and the highest tolerance of low temperatures. In all species CT minima and CT maxima were highly significantly linearly related to acclimation temperature. Within each species, slopes relating CT maxima to acclimation temperature were approximately half as large as those relating CT minima to acclimation temperature, suggesting that acclimation temperature has a greater influence on tolerance to low rather than high temperatures. Slopes relating both CT minima and CT maxima to acclimation temperature for the two warm-water species were similar and approximately twice those for the rainbow trout. This revised version was published online in July 2006 with corrections to the Cover Date.     

Temperature dependence and Arrhenius activation energy of F-actin velocity generated in vitro by skeletal myosin.

The effect of temperature on the velocity of rhodamine phalloidin-labelled F-actin moving in vitro on rabbit skeletal myosin has been studied. Translating actin filaments were visualized by epi-fluorescence in an inverted microscope, equipped with temperature control (+/- 0.2 K) of the stage and objective. Images were recorded in real time at magnifications of 400x or 160x by an intensified CCD camera on video tape. Motion of individual filaments was tracked by hand and velocities determined using frame times recorded simultaneously on the video tape. Velocity changed from 12 microns per second at 42 degrees C to 11 nm per second at 3 degrees C. The Arrhenius plot is non-linear, with the data following a cubic regression curve with no evident breaks or jumps. Data taken over the temperature range from single preparations followed the same curve for both heating and cooling; this indicates reversibility and absence of hysteresis. A hyperbolic model that smoothly translates with temperature between two asymptotic activation energies fits the data above 7 degrees C: these energies are 50(+/- 5) kJ per mole (Q10 = 1.9) at high temperatures and 289(+/- 29) kJ per mole (Q10 = 76.5) at low temperature with a transition temperature of 15.4(+/- 0.6) degrees C. These values are compared with other measurements made in vitro, in solution studies and on muscle fibres. An Arrhenius activation energy of 50 kJ per mole and a transition temperature of 15 degrees C are consistent with previous determinations but 289 kJ per mole is significantly greater than has been seen at low temperatures in other systems. This may indicate a different rate-limiting step in the kinetics of skeletal myosin driving actin filaments in vitro below 15 degrees C. Current determinations of the myosin "step-size" assume that the actin velocity is determined by the rate of ATP hydrolysis; the data confirm similar activation energies above 20 degrees C but they show that the temperature dependencies and activation energies are different at lower temperatures, implying uncoupling of the two processes.     

Effects of temperature on glucose release and glycogen metabolism in isolated hepatocytes from rainbow trout (Salmo gairdneri)

Endogenous glucose release and glycogen metabolism were investigated in isolated hepatocytes from rainbow trout acclimated to 10 and 20 degrees C. Thermal acclimation did not significantly affect hepatocyte glycogen contents and the rates of glucose release during substrate-free incubations. In both acclimation groups glucose production and glycogen metabolism exhibited clearly different dependencies on assay temperature. It was concluded, that there are different sources of glucose release in the lower and upper temperature range--gluconeogenesis from endogenous precursors at low temperatures and glycogenolysis at high temperatures. This conclusion was supported by experiments with 3-mercaptopicolinic acid, which stimulated glycogen breakdown especially in the low temperature range.     

A kinetic study of membrane immunoglobulin capping by flow cytometry

A flow cytometric procedure has been developed for performing kinetic studies on the capping of membrane immunoglobulin (mIg) on B lymphocytes. Mouse B cells were stained with fluorescein-conjugated antimouse-Ig antisera and subjected to pulse-shape (width, peak, and area) analyses prior to, during, and after ligand-induced redistribution of mIg. It was found that ring-stained, patched, and capped cells could be discriminated based on the width of the electronic signal curve generated as the cells passed through the laser beam. Additionally, endocytosis and or shedding of the cap could be correlated with a change in the area under the curve. Using these two parameters (width and area), the effects of temperature, cross-linking, and several pharmacological agents on the capping process were examined. Through the use of flow cytometry, the inhibitory effects of various perturbants could be localized to discrete stages of the capping process.     

Virus receptor interaction in the adenovirus system. II. Capping and cooperative binding of virions on HeLa cells.

Adenovirus type 2 attachment to HeLa cells was analyzed under controlled conditions. The temperature-dependent attachment kinetics revealed an inflection point at around 20 degrees C, and above this temperature the increase of the rate was doubled. In multiplicity dependence experiments, the attachment exhibited positive cooperative binding at 37 degrees C. This binding pattern was inhibited by low temperatures and prefixation of cells with 0.015% glutaraldehyde. Attachment of rhodamine-labeled virions revealed capping of the particles on 15% of the cells at 37 degrees C. Capping was inhibited by low temperatures, glutaraldehyde fixation of cells, and treatment with cytochalasin B, azide, and 2-deoxyglucose. Consequently, we propose that the adenovirus type 2 attachment to cells leads to rearrangements in the plasma membrane, resulting in cooperative binding and capping of the virus particles.     

Mitochondrial function in seasonal acclimatization versus latitudinal adaptation to cold in the lugworm Arenicola marina (L.)

Previous studies in marine ectotherms from a latitudinal cline have led to the hypothesis that eurythermal adaptation to low mean annual temperatures is energetically costly. To obtain more information on the trade-offs and with that the constraints of thermal adaptation, mitochondrial functions were studied in subpolar lugworms (Arenicola marina L.) adapted to summer cold at the White Sea and were compared with those in boreal specimens from the North Sea, either acclimatized to summer temperatures or to winter cold. During summer, a comparison of mitochondria from subpolar and boreal worms revealed higher succinate oxidation rates and reduced Arrhenius activation energies (Ea) in state 3 respiration at low temperatures, as well as higher proton leakage rates in subpolar lugworms. These differences reflect a higher aerobic capacity in subpolar worms, which is required to maintain motor activity at low but variable environmental temperatures--however, at the expense of an elevated metabolic rate. The lower activity of citrate synthase (CS) found in subpolar worms may indicate a shift in metabolic control within mitochondria. In contrast, acclimatization of boreal lugworms to winter conditions elicited elevated mitochondrial CS activities in parallel with enhanced mitochondrial respiration rates. With falling acclimation temperatures, the significant Arrhenius break temperature in state 3 respiration (11 degrees C) became insignificant (5 degrees C) or even disappeared (0 degrees C) at lower levels of Arrhenius activation energies in the cold, similar to a phenomenon known from hibernating vertebrates. The efficiency of aerobic energy production in winter mitochondria rose as proton leakage in relation to state 3 decreased with cold acclimation, indicated by higher respiratory control ratio values and increased adenosine diphosphate/oxygen (ADP/O) ratios. These transitions indicate reduced metabolic flexibility, possibly paralleled by a loss in aerobic scope and metabolic depression during winter cold. Accordingly, these patterns contrast those found in summer-active, cold-adapted eurytherms at high latitudes.     

Effects of high temperature on body temperature and hormonal adjustments in piglets

Effects of a high (33 degrees C) or thermoneutral (23 degrees C) temperature on body temperature and endocrine parameters were studied in weaned piglets. Rectal and skin temperatures were measured in four ad libitum fed animals per temperature during three weeks. After this acclimation period, 11 blood samples were withdrawn on a 24-h period. Over the acclimation period, rectal and skin temperatures were 0.6 and 2.9 degrees C higher, respectively, at 33 degrees C than at 23 degrees C (P < 0.01), this change occurring from the 1st day at 23 or 33 degrees C. A tendency of serum leptin concentrations to be lower after meals at 33 degrees C than at 23 degrees C was also displayed (P = 0.09). Plasmatic concentrations in Insulin-like growth factor I and thyroxine were decreased at 33 degrees C relative to 23 degrees C (P < 0.01 and P < 0.06, respectively), and triiodothyronine concentrations tended to be lower at 33 degrees C than at 23 degrees C (P = 0.1), which could account for the lower heat production and growth observed in pigs exposed to high temperatures.     

Influence of menhaden oil on mitochondrial respiration in BHE rats

The effects of corn or menhaden oil and thyroxine treatment on hepatic mitochondrial respiration was studied. BHE rats were fed a 64% sucrose, 6% corn, or menhaden oil diet until they were 60-70 days of age. Succinate-supported mitochondrial respiration was studied at 3 degrees C intervals from 4 to 40 degrees C. Upper and lower activation energies and transition temperatures were determined through the calculation of Arrhenius plot. Menhaden oil plus daily thyroxine injection resulted in higher and lower activation energies than the other treatments. This combined treatment also resulted in lower state 3 and higher state 4 respiration rates and tighter coupling of respiration to ATP synthesis. These effects were thought to be due to the effect this treatment combination had on membrane fluidity.     

The effect of acclimation temperature on the activation energies of state III respiration and on the unsaturation of membrane lipids of goldfish mitochondria.

The influence of the acclimation temperature on the thermotropic behaviour of mitochondrial respiration and on the degree of unsaturation of mitochondrial membrane lipids has been studied. The mitochondria were isolated from red muscle, white muscle and liver of goldfish acclimated to 5, 20 and 30 degrees C. ADP-activated succinate oxidation was measured at different temperatures and resulted in non-linear Arrhenius-plots with breaks between 10 and 23 degrees C. As for the break-temperatures, there was found a shift downwards in preparations of decreased acclimation temperatures. This could be caused by a changed composition of membrane lipids and a simultaneous shift of the membrane phase transition temperature. Therefore, the fatty acid composition of all membrane preparations was analyzed. However, no consistent change of the degree of unsaturation due to a changed acclimation temperature could be found.     

The effect of thermal acclimation on the activity of arylhydrocarbon hydroxylase in rainbow trout (Oncorhynchus mykiss)

1. The possibility that temperature acclimation (to 10 or 18 degrees C for 28 days) would alter the cytochromes P-450 of rainbow trout was addressed. 2. The specific content of LM4b (P-450 IA1), the trout isozyme responsible for activation of polynuclear aromatic hydrocarbons, was lower in 18 degrees C fish than it was in 10 degrees C fish. 3. Kinetic analysis of aryl hydrocarbon hydroxylase indicated that, while thermal acclimation caused no change in Vmax, it lowered the apparent Km of this enzyme for benzo[a]pyrene when assayed at acutely shifted temperatures. 4. Thermal acclimation of fish may have significance when feral populations are subjected to acute temperature shifts.