Jan E. Bly

Temperature and teleost immune functions

Low environmental temperatures are immunosuppressive for ectothermic vertebrates, such as teleosts. In particular the available data support the notion that, at least in channel catfish, virgin T cells, rather than memory T cells, B cells or accessory cells are particularly susceptible to the inhibitory influences of lower temperatures on adaptive immune responses. The probable mechanisms involved in such suppression in teleosts are reviewed, and considerations are offered regarding cause and effect relationships between such immunosuppression and the development of infectious diseases in fish.

Phylogeny of lymphocyte heterogeneity: the thymus of the channel catfish

The number of thymocytes (approximately 3 x 10(7)) that were recoverable from fingerling channel catfish remained constant from about 3 to 10 months of age, i.e. from September to April following hatching the previous June. Between 11 and 12 months, i.e. May and June, the thymus dramatically increased in size with 3 x 10(9) thymocytes being recoverable from the tissue of individual fish. The thymus remained enlarged for several months (throughout the summer) but at about 15 months (in September) began to involute such that by 17 months (November) no thymus tissue could be seen macroscopically. This natural involution could be accelerated by subjecting the fish to handling and transport stress. Thymocytes of channel catfish aged 4 to 16 months exhibited reactivity with monoclonal antibodies against peripheral T cells but not B cells. Thymocytes responded to the mitogen Concanavalin A only in the presence of added accessory cells (peripheral blood monocytes) or a monocyte-derived supernatant (presumably containing IL-1) at permissive temperatures (27 degrees C). Thymocytes could also be induced to divide at nonpermissive temperatures (17 degrees C) when incubated in the presence of the following combinations of stimulants, a) the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the calcium ionophore A23187, b) TPA and ConA, or c) A23187 and ConA. In those cases where TPA or A23187 were used, accessory cells or their products were not needed. Collectively, these results support the notion that channel catfish thymocytes functionally mimic those lymphocytes in the peripheral blood previously designated as T cells.

Isolation of an acute-phase phosphorylcholine-reactive pentraxin from channel catfish (Ictalurus punctatus)

1. Channel catfish (Ictalurus punctatus) serum contains a protein that precipitates pneumococcal C-polysaccharide (CPS) in a calcium-dependent fashion. 2. The serum titer of this protein follows an acute-phase pattern in catfish injected with turpentine. 3. A non-glycosylated, phosphorylcholine (PC)-reactive protein (PRP) with molecular mass ca 100 kDa, was isolated from channel catfish acute-phase sera by affinity chromatography on PC-Sepharose 4B. 4. Contaminating proteins with molecular masses ca 700 kDa and ca 20 kDa that co-eluted with PRP from PC-Sepharose appear to be aggregated and native low-molecular weight factors (LMFs), respectively. 5. Purified PRP has gamma mobility but in serum samples PRP has gamma-beta mobility. 6. Electron microscopy confirmed that PRP has planar, pentagonal symmetry. 7. The amino terminus of PRP is blocked, but based on comparison of amino-acid compositions channel catfish PRP is clearly similar to human CRP and is most like CRPs from the dogfish (Mustelus canis) and rainbow trout (Oncorhynchus mykiss).

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.

Identification and characterization of a heat shock protein 70 family member in channel catfish (Ictalurus punctatus).

We have determined the cDNA sequence of a member of the channel catfish heat shock protein 70 (CF Hsp70) family. This protein presumably functions as a molecular chaperone, as is characteristic of this family in other species. Channel catfish peripheral blood leukocytes exhibit a classical heat shock response, in that heat shock (37 degrees C) induces the expression of heat shock genes that are quiescent at normal temperatures (23 degrees C). It was observed that pre-existing synthesis of certain other molecules was suppressed (as evidenced by decreases in actin RNA upon heat shock). Similar trends were observed in mRNA expression for CF Hsp70 in two catfish non-leukocyte cell lines, channel catfish ovary and F59. However, three leukocyte cell lines constitutively expressed high levels of CF Hsp70 mRNA at optimal culture temperature (27 degrees C), whereas heat shock (37 degrees C) elicited only a modest induction of CF Hsp70 expression. Furthermore, continued investigation is warranted to determine whether the apparent upregulation of CF Hsp70 mRNA expression in the catfish long-term leukocyte cell lines is involved in the seemingly immortal phenotype of these 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.

Complement deficiencies in channel catfish (Ictalurus punctatus) associated with temperature and seasonal mortality.

This study examined serum complement levels in channel catfish from two commercial ponds over a period extending from the winter through the summer months. The fish from one pond (6B) experienced significant fungal associated mortality in the month of January. In March, there was also notable mortality of fish in the same pond; moribund fish exhibited external fungal as well as systemic Aeromonas infections. In July, all fish collected were apparently healthy in that there was no observed mortality. In contrast, the fish from the second pond (16A) were apparently healthy at all sample times except March when there was amild incidence of fungal associated mortality. The results of analyses of haemolytic complement activity showed that representative fish from both ponds had severe complement deficiences during January and March. The haemolytic complement activity continued to be monitored from fish from these ponds into the summer months and the CH 50 values were found to increase to within the normal range. To determine if low water temperature alone induced complement deficiencies, labolatory-acclimated catfish were subjected to a rapid down-shift in water temperature from 23°-11° C during a 24-h period, and the serum CH 50 values were monitored over the following 5 weeks. The results showed that low temperature can significantly affect the ability of catfish to maintain normal levels of haemolytic complement activity; however, these results do not appear to solely explain the abnormally low levels of haemolytic complement found in the serum of catfish during January and March. These results suggest that other factors, in addition to low temperature may have contributed to low CH values observed in the catfish during the winter and spring months. These analyses, in conjunction with other studies, suggest that fungal associated winter mortality and perhaps the subsequent bacterial infections associated with channel catfish during the following spring, are likely manifestations of a severe multifaceted immunodeficiency syndrome wherein complement as well as the humoral and cell-mediated immune systems are compromised.

A serum-free culture medium for channel catfish in vitro immune responses

This paper describes a chemically defined serum-free medium which supports channel catfish ( Ictalurus punctatus ) lymphocyte mitogen responses to both LPS and ConA as well as proliferation in mixed lymphocyte reactions. Furthermore, this medium has been successfully used to support catfish antibody production in primary and secondary in vitro responses to both thymus independent and thymus dependent antigens. The medium, designated ‘A-L’, is an isotonic 1:1 mixture of two commercially available media, AIM-V and Leibovitzs L-15. Although the essential nutrients contained in this serum-free medium have not yet been identified, it appears that D(+) galactose is an important ingredient for catfish T cell proliferation. This serum-free culture system will permit investigations into new areas of channel catfish immune regulation.

Temperature-mediated processes in teleost immunity: the effects of temperature on membrane immunoglobulin capping on channel catfish b lymphocytes

1. In order to better understand ligand-induced redistribution of membrane receptors and lymphocyte activation in ectothermic vertebrates, flow cytometry was used to monitor the effects of both in vivo acclimation temperature and in vitro assay temperatures on the kinetics of monoclonal antibody-induced membrane immunoglobulin (mIg) capping on channel catfish lymphocytes. 2. It was observed that the kinetics of mIg capping were dependent on in vitro assay temperatures, in vivo acclimation temperatures, and the length of time of in vivo acclimation. In the latter situation in vivo acclimation of fish to 27, 22 and 17 degrees C was considered complete after 3 weeks, while acclimation to 12 degrees C required a minimum of 5 weeks. 3. The energies of activation required for mIg capping ranged from 33 to 24 kcal/mol; lower energies of activation were observed with lower temperature acclimation. 4. It was also noted that the lower energies of activation were associated with concomitant decreases in cellular phospholipid saturated/unsaturated fatty acid ratios. 5. It appears that channel catfish B cell mIg capping, presumably a requisite for immune function, can be significantly affected by environmental temperatures; most likely such effects are attributable to changes in plasma membrane viscosities.

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.