Neuroendocrine-immune interactions in carp: a role for cortisol and interleukin-1
Engelsma, M. - \ 2002
Wageningen University. Promotor(en): W.B. van Muiswinkel; B.M.L. Verburg-van Kemenade. - S.l. : S.n. - ISBN 9789058086655 - 158
karper - cyprinus - hydrocortison - interleukine 1 - endocrien systeem - immuunsysteem - leukocyten - interacties - stress - neurofysiologie - carp - cyprinus - hydrocortisone - interleukin 1 - endocrine system - immune system - leukocytes - interactions - stress - neurophysiology
Maintaining a dynamic internal equilibrium, homeostasis, is crucial for survival of an organism. Disturbances in the environment may threaten the homeostasis and this will subsequently evoke an adaptive response in order to restore homeostasis. In vertebrates the adaptive response is mediated via the neuroendocrine system by adrenocortical and adrenergic activation. Glucocorticoids (GC) and catecholamines are the main actors in the response and can affect a whole range of processes, including those in the immune system. In response to pathogenic challenges the immune system is triggered, resulting in activation of components of innate and acquired immunity. Bi-directional communication between the Hypothalamus-Pituitary-Adrenal (HPA)-axis, sympathetic nervous system and the immune system is crucial to ensure homeostasis in mammals. Shared use of ligands and especially receptors forms a key component of this mutual interaction.
The Hypothalamus-Pituitary-Interrenal (HPI)-axis is the teleost equivalent of the HPA-axis. Stress induced immuno-suppression in fish is mostly attributed to actions of cortisol, major GC in fish and end-product of the HPI-axis. Stress in aquaculture is one of the potential factors causing increased susceptibility of fish to pathogens and subsequently considerable losses in production.
As part of a programme investigating adaptive strategies of carp ( Cyprinus carpio L.) after temperature stress, this study focuses on the possible neuroendocrine modulation of immune functioning during acute stress. We studied the effects of in vitro cortisol and in vivo acute temperature stress on carp leucocytes and functioning of these leucocytes. Moreover, the cortisol influence on gene expression of the cytokine interleukin-1b(IL-1b) was studied. IL-1bin mammals is part of the reciprocal signalling between neuroendocrine and immune system, therefore it may be an important candidate for modulating hormone secretion in carp.
Cortisol acts upon lymphocytes differentially; in previous research it was demonstrated that in carp, in particular the B lymphocytes are affected. In vertebrates B lymphocytes play an important role in acquired immunity as precursors of antibody producing cells. Maturation and activation state of B lymphocytes may have consequences for the influence cortisol has on these cells. Therefore, carp B lymphocytes were isolated from different tissues and compared with regard to their proliferation, apoptosis and the effects of cortisol on these processes. Head kidney and spleen B lymphocytes were characterised by high basal proliferation. Peripheral blood B lymphocytes showed a low basal proliferation which could be up-regulated by stimulation with lipopolysaccharide (LPS), a major constituent of the cell wall of gram-negative bacteria. LPS could not alter proliferation of head kidney B lymphocytes. In addition, Ig-crosslinking induced higher intracellular calcium responses in circulating B lymphocytes compared with B lymphocytes from head kidney or spleen origin. With respect to apoptosis, stimulation could enhance cell viability in all organs. However, in combination with cortisol high levels of apoptosis were induced. Especially activated peripheral blood B lymphocytes were sensitive to cortisol-induced apoptosis. Also head kidney and to a lesser extent spleen B lymphocytes, although less sensitive than their equivalent in circulation, underwent cortisol-induced apoptosis irrespective of extra stimulation. Proliferation was suppressed by cortisol in blood and spleen B lymphocytes and to a more limited extent in head kidney, regardless of LPS stimulation. It is suggested that cortisol may be important for immunoregulation in both stress and non-stress conditions, because the relatively modest concentration of cortisol used (compared to plasma values measured during stress conditions) could induce a significant increase in apoptosis in all three populations of B lymphocytes. This implies an impact of stress on B lymphocyte development and activity.
Stress-induced immunological changes that may contribute to a decreased disease resistance in carp were investigated. A 3 h drop in ambient water temperature was used as model for a relative mild and acute stressor for carp. After single or multiple temperature shocks, the relative number of circulating B lymphocytes decreased significantly within 4 h after the onset of the stressor, which was even more pronounced than after challenging the immune system. After a single temperature shock the relative number of B lymphocytes returned to control levels within 24 hours. In head kidney, an increase was measured in the relative number of B lymphocytes. Migration of B lymphocytes resulting in a redistribution of these cells to other body compartments may contribute to the relative drop in B lymphocytes in the circulation. Granulocyte numbers showed opposite reactions, doubling in circulation and decreasing significantly in head kidney. This demonstrates differential modulation of immune cells in vivo by a relative mild stressor. Freshly isolated blood lymphocytes from stressed carp showed a considerable higher number of apoptotic cells than lymphocytes from unstressed animals. Besides B lymphocytes, Ig -lymphocytes contributed significantly to this stress-induced apoptosis. Glucocorticoid receptors could be detected in the vast majority of the B lymphocytes and also part of the Ig -lymphocytes. As distribution of B lymphocytes was substantially affected by temperature stress, the effects of multiple temperature shocks on humoral antibody responses were determined. The kinetics of the antibody response to both, T lymphocyte independent (TI) antigens and T lymphocyte dependent (TD) antigens consistently showed a trend to decreased antibody response in stressed carp. In carp immunised with the TI-antigen TNP-LPS the antibody response was significantly slower in the stressed carp. These observations confirm the effect of temperature stress on the B lymphocyte population.
These results show that even a mild stressor can affect distribution of B lymphocyte and granulocyte cell populations reversibly with differential effects and thus can have implications for a subsequent immune response. However, during acute stress, the role of cortisol is most probably not purely immunosuppressive but more immunomodulatory. A stress-induced enhancement of an innate type of response could facilitate a fast and effective reaction of the immune system.
Cytokines, like IL-1b, play a pivotal role in the regulation of the immune system. Macrophages and a whole range of other cells release IL-1bas a response to infection or tissue damage. IL-1bhas pleiotropic effects as an immune and inflammatory mediator. Furthermore, IL-1bis an important candidate able to affect the HPI-axis by altering the release of corticotropin releasing hormone (CRH) and adrenocorticotropic hormone (ACTH).
In fish, most interleukin molecules await identification but the IL-1bsequences of several teleost fishes were recently elucidated. In the tetraploid carp we describe gene organisation and expression of two IL-1bgenes: IL-1b1 and IL-1b2. The two carp mRNA sequences share about 74% amino acid identity. The existence of two IL-1bcopies in the carp genome probably originates from the tetraploid nature of the species. In contrast to carp IL-1b1, the IL-1b2 locus is represented by multiple sequences with 95-99% identity. Detection of up to 6 distinct IL-1b2 sequences within single homozygous fish suggests the presence of multiple copies of the IL-1b2 gene in the carp genome. Both IL-1b1 and IL-1b2 comprise seven exons with typical IL-1 characteristics as an IL-1 family motif and instability motifs in the 3'-untranslated region. A general discrepancy of teleost IL-1bsequences described thus far with mammalian IL-1b, is the lack of a clear caspase-1 (interleukin- 1b-converting enzyme; ICE) cleavage site. Three IL-1b1 RNA transcripts could be detected in carp: (1) a fully spliced product, (2) exon 1-7 with introns 5 and 6 and (3) exon 1-7 with intron 5 only. Intron-containing products were also detected for IL-1b2. These intron-containing products probably represent partially spliced transcripts.
IL-1bmRNA expression in carp was semi-quantitatively analysed by RT-PCR in multiple organs, including brain and pituitary. In vivo , mRNA of both IL-1bsequences were constitutively expressed in healthy carp, for IL-1b1 this was predominantly in the immune organs head kidney and spleen. Furthermore, a scattered distribution of IL-1b1 producing cells was shown by in situ hybridisations of head kidney tissue. Administration of phorbol-myristate-acetate (PMA) or LPS to phagocytes isolated from the head kidney, resulted in up-regulation of IL-1b1 expression. Also IL-1b2 transcripts could be up-regulated by in vitro LPS stimulation of head kidney phagocytes. Interestingly, by determining the ratio of expression it was demonstrated that IL-1b2 is expressed at a maximum of one tenth of the amount of the IL-1b1 sequence. Together with the high number of amino acid substitutions in the IL-1b2 sequences this suggests either that IL-1b2 is approaching a pseudogene status or IL-1b2 is part of a complex receptor - ligand interaction network. The involvement of nuclear factor (NF)-kB in carp IL-1b1 expression was shown with suppression of the LPS-induced IL-1bexpression by the NF-kB inhibitor, pyrrolidine dithiocarbamate (PDTC). Data suggests also that carp IL-1b2 is regulated via NF-kB and consequently both IL-1bsequences appear to have similar promoter regions.
Cortisol, as endocrine-derived factor potentially mediating carp IL-1bexpression, was able to inhibit constitutive expression of IL-1b1 as well as IL-1b2 transcripts in vitro. However, when cortisol was added in combination with LPS at a physiological dose, cortisol could not inhibit LPS-induced expression. Moreover, it appears that cortisol synergistically enhances LPS-induced IL-1bexpression in carp. Probably LPS overrules the glucocorticoid receptor mediated inhibition via the NF-kB pathway. This might imply that cortisol can not suppress IL-1bactivation during infection. At a tenfold higher cortisol dose, however, the expression is inhibited.
In conclusion, data presented in this thesis show that carp leucocytes are differentially sensitive to cortisol and in vivo stress, with regard to cell type, location and maturation or activation state. This affects cell viability, replication and migration with subsequent consequences for the immune status of carp. Also interaction of the neuroendocrine system with immune regulating factors was demonstrated: cortisol affects carp IL-1bmRNA expression. IL-1bin carp consists of multiple forms and is part of an immune regulating mechanism which probably matches that of mammals in complexity.
Modulation of gap junctional intercellular communication between human smooth muscle cells by leukocyte-derived growth factors and cytokines in relation to atherogenesis
Mensink, A. - \ 1997
Agricultural University. Promotor(en): J.H. Koeman. - S.l. : Mensink - ISBN 9789054857716 - 125
atherosclerose - leukocyten - signaaltransductie - celinteracties - atherosclerosis - leukocytes - signal transduction - cell interactions
In this thesis, the effect of leukocyte-derived growth factors and cytokines on GJIC between SMC was investigated. GJIC is regarded as an important mechanism in the control of cell growth, cell differentiation and tissue homeostasis. Disturbance of SMC growth control is regarded to be a key event in the pathogenesis of atherosclerosis in which growth factors and cytokines are thought to play a central role. In the present study, cultured human SMC were incubated with (human) recombinant growth factors and cytokines. TNFα, IFN-γ, PDGF, bFGF and IL-6 were chosen as representatives of several classes of growth modulating factors. These growth factors and cytokines are known to be products of macrophages and/or T lymphocytes and have been detected in human atherosclerotic lesions. After an incubation period, GJIC between SMC was measured. In addition, human SMC were co-cultured with J774A.1 murine macrophages or human monocyte-macrophages in the Transwell-COL cell culture system, to account for the complexity of macrophage secretion patterns. After removal of the macrophages, GJIC between the co-cultured SMC was determined.
The experiments described in chapter 2 and 3 clearly demonstrate that all factors tested reduced GJIC between SMC with ~20 - 50%, except for bFGF which strongly increased GJIC. Furthermore, these experiments revealed that effects of growth factors and cytokines on GJIC are not univocal and thus cannot be generalized. PDGF, IL-6 and bFGF caused transient effects on GJIC, whereas in experiments with TNFαor IFN-γ, a persistent inhibition of GJlC was obtained.
The most remarkable result of the study described in chapter 4 was that upon combining TNFα and IFN-γ, GJIC between SMC strongly reduced (up to 86%) in an additive or synergistic manner. Upon long term incubation with the combination of TNFα and IFN-γ, some SMC did not communicate with neighbouring cells at all. This may result in an escape from growth control mechanisms, which, in turn, may lead to disturbance of SMC proliferation, a key event in atherosclerosis. In incubations with other combinations of growth factors and cytokines, (antagonistic) interactive effects on GJIC were observed.
The present investigation provided evidence that reactive oxygen species may play a role in cytokine-induced inhibition of GJIC between SMC. Experiments described in chapter 2 revealed that pretreatment of SMC with antioxidants like ascorbic acid, α-tocopherol or GSH prevented the inhibition of GJIC upon exposure of SMC to TNFα. Studies with SOD (chapter 4)demonstrated that the superoxide radical may be involved in GJIC reduction by TNFα, since incubation with SOD, even hours after the addition of TNFα, restored GJIC to control values. Furthermore, SOD partly restored IFN-γeffects on GJIC in the short - but not in the long term. When SMC were incubated with TNFα and IFN-γsimultaneously for 24 h, high levels of SOD could not even partly counteract the strong inhibition of GJIC caused by these cytokines. Thus, other, superoxide-unrelated mechanisms may affect GJIC more predominantly in long term incubations with the combination of TNFα, and IFN-γ, One such mechanism may be represented by the reduced Cx43 staining which was observed in immunofluorescence studies on SMC cultures incubated with these cytokines (chapter 4) ,which may be an indication for the reduced presence of functional gap junction channels.
PDGF-AA, PDGF-BB, IL-6, IFN-γ, TNFαand bFGF all stimulated SMC proliferation in our cell culture system, as individual factors as well as in combinations (chapter 3 and 4). Upon comparing these cell proliferation results with GJIC data, a complex relationship between modulation of GJIC, cell proliferation and the process of atherosclerosis is suggested.
Experiments described in chapter 5 demonstrated that macrophages cultured on pore membrane inserts modulate GJIC between SMC co-cultured in Transwell-COL cell culture chambers. Since these results were obtained in an indirect co-culture system which prevents direct cell-cell contact, it was hypothesized that soluble factors, released by macrophages, may be involved in the modulation of GJIC between SMC. At this moment, one can only speculate about the nature of the factors involved in this macrophage-dependent modulation of GJIC. The results clearly indicate that the source and activation state of macrophages were of importance in these co-culture experiments. Therefore, further research should be aimed at studying the effect of different types of macrophages on GJIC between co-cultured SMC. Heterogeneity in atheroma macrophages exists; the most noticeable difference being the presence of 'normal' macrophages and the presence of macrophage-derived foam cells, which are likely to differ in endocytic and secretory repertoire. Furthermore, macrophages should be exposed to different (patho-)physiological agents with relevance to the process of atherosclerosis, in order to study their effects on GJIC between SMC in even more detail.
The present study provides a good starting point for further research aimed at the understanding of mechanisms by which enviromnental contaminants or drugs might interfere with atherogenesis. It is already known that widespread food chain and cigarette smoke contaminants like for example benzo[a]pyrene, polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo- p -dioxin may affect the pathogenesis of atherosclerosis in several ways, for instance by damaging SMC DNA, disrupting endothelial barrier function, or by modulation of plasma cholesterol and lipoprotein levels). Furthermore, chemicals like components in cigarette smoke condensate may modulate GJIC between SMC. Considering that growth factors and cytokines like TNFα, and IFN-γ, may have marked effects on GJIC, one may assume that enviromnental contaminants and drugs capable of affecting the expression of growth factors and cytokines or their receptors may interfere with GJIC in an indirect manner. In the case of atherogenesis for instance, chemicals may stimulate growth factor- and cytokine production by SMC and/or macrophages which, in turn, may influence homologous GJIC between SMC. In addition, exogenous chemicals may influence heterologous GJIC between macrophages and SMC as well; either directly, or indirectly via the induction of growth factor and cytokine expression by these cells. As a consequence, macrophage-derived reactive substances will have more - or just less- impact on SMC functioning.
Relatively short exposures to enviromnental contaminants or drugs in individuals in which plaques have already passed some critical phases in the atherosclerotic process might enhance the severity of the lesions. Further research along this line may also lead to the identification of nutritional or chemical factors that may have beneficial (protective
Modulation of GJIC by growth factors and cytokines may affect a response-to-injury. On the other hand, modulation of GJIC may also play a role in the monoclonal expansion of cells. Therefore, the response-to-injury hypothesis and the monoclonal theory may be compatible is some respects, as was previously suggested by Zwijsen.
The results of the present study may also be applicable to other pathophysiological phenomena, in which growth factors and cytokines may play a prominent role in the onset or progression of the disease. Proliferative diseases like pulmonary fibrosis, glomerulosclerosis and liver cirrhosis share some pathobiologic mechanisms with atherosclerosis, including leukocyte infiltration, mesenchymal cell proliferation and enhanced matrix synthesis. Leukocyte-derived growth factors and cytokines may modulate GJIC between the mesenchymal cells concerned, which in turn may result in abnormal cell proliferation. It is known that certain chemicals may contribute to the development of these diseases. The mechanisms by which these chemicals act may be linked to the processes studied and discussed in this thesis.
Overall, the information presented in this thesis concerning the possible role of growth factors and cytokines in the pathophysiology of atherosclerosis provides a useful instrument to study possible modulatory effects of chemicals on the process of atherosclerosis via the mechanisms mentioned above.
Characterisation of fish leucocytes : an immunocytochemical and functional study in carp (Cyprinus carpio L.)
Koumans - van Diepen, J.C.E. - \ 1993
Agricultural University. Promotor(en): W.B. van Muiswinkel, co-promotor(en): J.H.W.M. Rombout. - S.l. : Koumans-van Diepen - ISBN 9789054851110 - 167
cyprinidae - karper - bloedserum - fibrine - bloedplaatjes - bloed - erytrocyten - leukocyten - bloedplasma - reticulo-endotheliaal systeem - antilichamen - immunoglobulinen - immunocytochemie - cyprinidae - carp - blood serum - fibrin - platelets - blood - erythrocytes - leukocytes - blood plasma - reticuloendothelial system - antibodies - immunoglobulins - immunocytochemistry
A panel of monoclonal antibodies (MAbs) against carp serum immunoglobulin (Ig), WCIs or carp thymocytes (T), WCTs were used for the characterisation of carp leucocytes. Unfortunately, all WCTs and some WCIs react with common carbohydrate determinants present on all leucocytes and Ig. Most WCIs react specific with protein determinants at the heavy chain of Ig. Consequently, B lymphocyte (sub) populations, plasma cells and Ig-binding cells could be studied. Ig molecules are found in clusters at the cell membrane of B cells and plasma cells, and in contrast to mammalian plasma cells, most carp plasma cells still have Ig at their surface membrane. Mainly the dull surface Ig-positive (sIg +) cells were stimulated by the mammalian B cell mitogen LPS and not by PHA (T cell mitogen) in vitro , whereas the sIg-negative (sIg -) cells were stimulated by PHA and not by LPS. The percentages of B cells and plasma cells showed an increase during ontogeny and reached a plateau at about 3 months and 8 months of age respectively. It is suggested that full development of the carp (humoral) immune system needs at least 8 months (at 21-22 °C). Three different subpopulations of B cells and plasma cells and at least two Ig isotypes can be distinguished based upon their reactivity with WCI 4 arid WCI 12. The distribution of the three B cell subpopulations appeared to be organ and age dependent which indicates functional differences between the Ig isotypes. Fc-like receptors were mainly demonstrated on gut macrophages while pronephros macrophages and neutrophilic granulocytes did not show Ig binding. Consequently, other forms of antigen opsonisation (e.g. complement) may play a role in phagocytosis by these non Ig-binding cells. Several procedures were tested for obtaining MAbs specific for Ig -lymphoid cells. It is concluded that the presence of immunodominant carbohydrate determinants is the major problem for obtaining specific MAbs. Tolerisation of mice against these determinants or the use of isolated membrane lysates from (sIg -) PBL appeared promising but till now only specific thrombocyte markers have been obtained. The use of more purified antigen is recommended in further attempts. The data presented in this thesis can be used for fundamental studies on cell interactions in the immune response, but also for more applied investigations on fish health control.
Genetical and some environmental influences affecting the level of leucocyte counts in the milk of cows
Afifi, Y.A. - \ 1967
Wageningen University. Promotor(en): T. Stegenga. - Wageningen : Veenman - 81
rundvee - rauwe melk - diergeneeskunde - melkklieren - melksecretie - lactatie - dierlijke producten - vervalsing - besmetting - verouderen - gebreken - achteruitgang (deterioration) - bloedserum - fibrine - bloedplaatjes - bloed - erytrocyten - leukocyten - bloedplasma - mastitis - cattle - raw milk - veterinary science - mammary glands - milk secretion - lactation - animal products - adulteration - contamination - aging - defects - deterioration - blood serum - fibrin - platelets - blood - erythrocytes - leukocytes - blood plasma - mastitis
The progeny groups of different sires varied widely in white-cell count in milk, even after exclusion of all cows which had suffered from mastitis. The sire had a demonstrable effect on white-cell count in milk, especially during the second half of lactation. Heritability estimates of white-cell count in milk showed that values for the fourth lactation were higher than those for heifers. But at the end of lactation heritability values for 4th lactation cows and heifers were nearly equal (about 0.40). The daughter groups with high average white-cell counts mostly showed frequent mastitis. There was a high phenotypic and genetic correlation between clinical mastitis and white-cell count. Within seasons for cows which, so far known, had never mastitis, very high and very low producers had higher white-cell counts than other cows. White-cell counts increased remarkably with advancing lactation. A relation between white-cell count and ease of milking could not be demonstrated.Increasing milking vacuum over 40 cm mercury pressure, especially at the end of lactation, or increasing pulsation to over 50 per min. tended to increase white-cells. Milking routine (man/machine ratio) affected white-cell count in the end of lactation. More cows per milker increased the number of white-cells.
Verspreiding van bloedgroepen in het Nederlandse zwartbonte rundvee : een onderzoek naar de frequenties van bloedgroepen en naar enige factoren, die de frequenties beinvloeden
Kraaij, G.J. - \ 1967
Wageningen University. Promotor(en): T. Stegenga. - Wageningen : Veenman - 127
bloedserum - fibrine - bloedplaatjes - bloed - erytrocyten - leukocyten - bloedplasma - rundvee - melkveerassen - nederland - genetica - heritability - genetische variatie - blood serum - fibrin - platelets - blood - erythrocytes - leukocytes - blood plasma - cattle - dairy breeds - netherlands - genetics - heritability - genetic variation
Blood groups are genetically determined components of the red blood cells. In cattle there were 13 loci known to determine blood groups and some of these loci had large series of alleles. There were also 14 other loci known to determine proteins and enzymes in blood and milk of cattle.
The author examined how the distribution of blood groups in the Dutch Friesian population had been influenced by the restrictions of breeders. He found that parent cattle were paired independently of blood group. In offspring there was no selection for a certain phenotype until the end of the first year.
There were clear differences in the frequency of some genes between adult bulls and cows. The gene for blood group A was less frequent in bulls and that for blood group F was less frequent in cows. This occurred in some foreign breeds of cattle.
The distribution of blood groups over the population was not even. There were differences in gene frequency between breeding areas and in the breeding areas there were large differences between artificial insemination stations. Differences within farms and within breeding pedigrees were even greater. These differences could be ascribed largely to the use of one or only a few sires.
Het kopergehalte van lever en bloedserum bij het Fries-Hollandse rund
Grift, J. van der - \ 1955
's-Gravenhage : Staatsdrukkerij (Verslagen van landbouwkundige onderzoekingen 61.10) - 61
melkveerassen - rundvee - bloedserum - fibrine - bloedplaatjes - bloed - erytrocyten - leukocyten - bloedplasma - dairy breeds - cattle - blood serum - fibrin - platelets - blood - erythrocytes - leukocytes - blood plasma