Evaluation of intrauterine immune suppression during pregnancy in a species with epitheliochorial placentation

Cells capable of suppressing the immune response of PBL to the mitogen PHA are associated with the epitheliochorial placenta during normal first pregnancy in the pig. Because placentation in the pig is noninvasive, these suppressor cells are not associated with decidua. Cells with similar activity are also found between the implantation sites and in the uterus of pseudopregnant pigs, suggesting that fetal trophoblast is not essential for recruitment of intrauterine suppressor cells. Cell separation studies demonstrate that two independent populations of suppressor cells are present in the pig uterus on day 28 of gestation, as well as a population of PHA-responsive cells. The inability of unseparated porcine uterine cells to respond to PHA, plus the reconstitution of suppression in remixing experiments, demonstrate directly that a functional, putative T effector cell population is blocked within the uterus during normal mammalian pregnancy.     

Evolution of Placentation in Primates: Implications of Mammalian Phylogeny

Primates are quite unique among placental mammals in that the two extreme types of placentation are present within a single order. Strepsirrhines (lemurs and lorisiforms) have non-invasive epitheliochorial placentation, whereas haplorhines (tarsiers and higher primates) have highly invasive haemochorial placentation. Resemblance in placenta type in fact provided the first evidence that tarsiers are linked to higher primates and distinct from lemurs and lorisiforms. Tree-shrews differ from both primate subgroups in having moderately invasive endotheliochorial placentation, while colugos have invasive haemochorial placentation like haplorhines. All three kinds of placentation have been identified as primitive for placentals by different authors, but until recently the prevailing interpretation has been that non-invasive epitheliochorial placentation is primitive and “less efficient”. Opposing this interpretation, Martin (Primate origins and evolution: a phylogenetic reconstruction, 1990) proposed that moderately invasive endotheliochorial placentation is primitive. Epitheliochorial placentation is unlikely to be primitive because it is predominantly associated with large body size, relatively long gestation periods and precocial offspring. Furthermore, some strepsirrhines and other placental mammals with epitheliochorial placentation retain indications of former invasiveness of the placenta. The recent availability of comprehensive molecular phylogenies for placental mammals has provided an independent framework to determine the most parsimonious interpretation of the evolution of placenta types and other reproductive features. It has consistently emerged that epitheliochorial placentation is best explained as a derived condition, although opinions differ as to whether the ancestral condition for placental mammals (and hence for primates) was endotheliochorial or haemochorial. It is argued that on balance the most likely ancestral condition is endotheliochorial. Comparative evidence across placentals clearly indicates that epitheliochorial placentation is not less efficient than more invasive forms of placentation, at least with respect to growth in overall fetal body mass. The ratio of neonate mass to gestation period (a simple indicator of average daily maternal investment in fetal growth) shows no difference according to placenta type. Differential evolution of placentation is hence presumably linked to immunological factors, parent/offspring conflict and/or genomic imprinting.     

Effects of the chromatographic fractions of the pig placental trophoblast on graft-versus-host reaction

The trophoblast has a significant role in regulation of immune reactions at the materno-fetal interface by producing biologically active substances. In our previous studies five fractions with immunomodulatory activities were isolated by gel chromatography from trophoblast of pig placentas. To confirm the immunomodulatory effect of these trophoblast fractions on allogeneic in vivo systems and to obtain more evidence for the relevance of their activity on the maternofetal interface, their effect was studied on graft-versus-host reaction (GVHR). To assess the GVHR, the primary and secondary popliteal lymph nodes assay was used in mice. In the primary GVHR, 100 microg protein of Fraction 2-5, mixed with 5 x 10(6) allogeneic spleen cells (C57BL/6), were injected into one of the foot pads of recipient (BALB/c) mice. The secondary GVHR was induced in F1 (BALB/c x C57BL/6) mice by injection of spleen cells of BALB/c mice intraperitoneally preimmunized with allogeneic cells. The GVHR was measured by the weight of lymph nodes and by the lymphocyte proliferation. Flow cytometric analyses of the cells in the nodes with GVHR and under the influence of Fraction 4 or 5 were performed using monoclonal antibodies. In the primary GVHR, Fraction 4 or 5, injected simultaneously with allogeneic spleen cells, significantly suppressed the lymph nodes reactivity. Fractions 4 and 5 inhibited the ability of the spleen cells of mice intraperitoneally preimmunized with allogeneic cells to induce secondary GVHR in F1 mice. The Fraction 2 and 3 had no effect on GVHR. The results revealed that a group of proteins with Mr 37-7 kDa, isolated from trophoblast of pig placenta, strongly suppressed popliteal lymph node reactivity in the primary and secondary GVHR. The data provide convincing evidence for these fractions in vivo activity, for their effect across the species barrier and suggest the relevance of the same reactions on the materno-fetal interface.     

Comparative aspects of trophoblast development and placentation

Based on the number of tissues separating maternal from fetal blood, placentas are classified as epitheliochorial, endotheliochorial or hemochorial. We review the occurrence of these placental types in the various orders of eutherian mammals within the framework of the four superorders identified by the techniques of molecular phylogenetics. The superorder Afrotheria diversified in ancient Africa and its living representatives include elephants, sea cows, hyraxes, aardvark, elephant shrews and tenrecs. Xenarthra, comprising armadillos, anteaters and sloths, diversified in South America. All placentas examined from members of these two oldest superorders are either endotheliochorial or hemochorial. The superorder Euarchontoglires includes two sister groups, Glires and Euarchonta. The former comprises rodents and lagomorphs, which typically have hemochorial placentas. The most primitive members of Euarchonta, the tree shrews, have endotheliochorial placentation. Flying lemurs and all higher primates have hemochorial placentas. However, the lemurs and lorises are exceptional among primates in having epitheliochorial placentation. Laurasiatheria, the last superorder to arise, includes several orders with epitheliochorial placentation. These comprise whales, camels, pigs, ruminants, horses and pangolins. In contrast, nearly all carnivores have endotheliochorial placentation, whilst bats have endotheliochorial or hemochorial placentas. Also included in Laurasiatheria are a number of insectivores that have many conserved morphological characters; none of these has epitheliochorial placentation. Consideration of placental type in relation to the findings of molecular phylogenetics suggests that the likely path of evolution in Afrotheria was from endotheliochorial to hemochorial placentation. This is also a likely scenario for Xenarthra and the bats. We argue that a definitive epitheliochorial placenta is a secondary specialization and that it evolved twice, once in the Laurasiatheria and once in the lemurs and lorises.     

Placentation

Placental development differs greatly among members of different taxa. Not only does blastocyst attachment take place at different times, the penetration of trophoblast varies considerably. From an epitheliochorial relationship between fetus and mother to the hemochorial placentation of taxa, such as the higher primates, the trophoblast becomes increasingly exposed to maternal immune recognition which may be one cause of rejection in interspecific embryo transfers or hybridization. Shape of uterus and endometrium and genetic factors govern the morphologic form of the placenta. The paper reviews ungulate placentation, successes and failures of interspecific embryo transfers, and the scant knowledge of genetic determinants in primate placentas.     

Cell proliferation patterns during development of the equine placenta

Placentation involves considerable growth and reorganization of both maternal and fetal tissues. In this investigation, immunohistochemical localization of the proliferation marker Ki-67 antigen was used to monitor cell division during placentation in mares. Endometrial biopsies were obtained from eight mares between day 14 and day 26 of pregnancy and from eight anoestrous mares that had been treated with various combinations of progesterone and oestrogen. Samples of endometrium and fetal membranes were obtained from 19 mares carrying normal horse conceptuses between day 30 and day 250 of gestation and from three failing extraspecific donkey-in-horse pregnancies. Proliferation in the superficial strata of the endometrium was increased by day 18 of gestation and this effect could be mimicked by supplementing with oestradiol benzoate during the last 6 days of a prolonged period (18-36 days) of progesterone administration. Fetal chorionic girdle cells were proliferating vigorously at days 30-32 of gestation, but stopped dividing after they invaded the endometrium, while the trophoblast cells of the allantochorion showed an increase in mitotic activity after day 38. The luminal epithelium of the endometrium started to proliferate only after the primary villi of the true epitheliochorial placenta had been formed, and during days 58-70 this effect was seen only in the pregnant horn in which placentation was further advanced. During the second half of gestation, most of the mitotic activity was confined to the periphery of the microcotyledons which were still growing. In the donkey-in-horse pregnancies, proliferation rates of the maternal and fetal epithelial at day 70 of gestation were markedly reduced in areas of heavy endometrial lymphocyte infiltration and poor placentation. These results provide a basis for further studies on factors that influence invasive and non-invasive placentation.     

Variation in macrophage-migration-inhibitory-factor immunoreactivity during porcine gestation

The localization and activity of macrophage migration inhibitory factor (MIF) was investigated in the interhemal region of the noninvasive, diffuse, folded epitheliochorial placenta and in the nonpregnant uterus of the pig. MIF, a proinflammatory cytokine with many actions on macrophages and monocytes, may play an important role in materno-fetal immuno-tolerance during placental establishment, modulation, and growth. Immunohistochemical staining with anti-human MIF polyclonal antibodies was carried out on placental sections from 11 stages of gestation (16-95 days postcoitus) and on nonpregnant uterus at 13 days postestrus. Western blot analysis confirmed the specificity of the anti-human MIF polyclonal antibodies on pig tissues. MIF staining was intense in both the trophoblast and maternal epithelium in the early stages; in the later stages, it decreased dramatically in the maternal epithelium but remained high in the trophoblast. The uterine glands showed immunoreactivity at all stages, and the maternal and fetal epithelial linings of the areolar cavity showed high reactivity at Day 25. The vasculature also showed staining for MIF, and an intense to moderate staining was shown in the nonpregnant uterus, mostly in the surface and glandular epithelium. The high activity of MIF in the maternal and fetal tissues throughout placentation and its expression in the nonpregnant uterus indicate a regulatory role for MIF during embryo receptivity and epitheliochorial placentation.     

Immune response to different fractions of Taenia solium cyst fluid antigens in patients with neurocysticercosis

The immunopathogenesis of neurocysticercosis (NCC) largely remains unknown. We analyzed the immune response to different fractions of Taenia solium cyst fluid antigens in patients with NCC. Lymphocytes were separated from 48 patients with NCC-related active epilepsy and 30 healthy controls. T. solium (isolated from pig muscles) antigens (crude lysate, CL; cyst wall, CW and cyst fluid, CF) at 20 μg/well concentrations were used to stimulate the cells in a lymphocyte transformation test (LTT). Only CF antigen stimulated cell proliferation significantly greater than control (p < 0.001), hence cyst fluid antigens were further studied. The CF antigens were electro-blotted on nitrocellulose membrane (NC), cut at 0.5 cm distance and particulate antigens were prepared. A total of 12 fractions, designated F1 to F12 according to molecular weight were tested in-vitro for LTT. After 72 h of stimulation by the different fractions, Th1 (IL-1β, TNF-α, IL-2) and Th2 (IL-4, IL-10) cytokine responses were determined in culture supernatants by ELISA. Low molecular weight fractions F1 through F4 (Mol. wt. < 25 kDa) were found to be potent inducers of cytokines. Fractions F1, F3 and F4 induced the production of Th1 (IL-1β, TNF-α, IL-2), whereas F2 induced the production of Th2 (IL-4 and IL-10) cytokine. The study shows that the low molecular weight fractions of CF antigens are immuno-dominant. Most of these fractions (F1, F3, F4) induce strong Th1 immune response except F2 which induces Th2 response. Further studies are needed to identify the different antigens present in these fractions to determine the molecules responsible for the immune response.     

Placental invasiveness mediates the evolution of hybrid inviability in mammals

A central question in evolutionary biology is why animal lineages differ strikingly in rates and patterns of the evolution of reproductive isolation. Here, we show that the maximum genetic distance at which interspecific mammalian pregnancies yield viable neonates is significantly greater in clades with invasive (hemochorial) placentation than in clades with noninvasive (epitheliochorial or endotheliochorial) placentation. Moreover, sister species with invasive placentation exhibit higher allopatry in their geographic ranges, suggesting that formerly separated populations in mammals with this placental type fuse more readily on recontact. These differences are apparently driven by the stronger downregulation of maternal immune responses under invasive placentation, where fetal antigens directly contact the maternal bloodstream. Our results suggest that placental invasiveness mediates a major component of reproductive isolation in mammals.     

Placentation in dolphins from the Amazon River Basin: the Boto, Inia geoffrensis, and the Tucuxi, Sotalia fluviatilis

A recent reassessment of the phylogenetic affinities of cetaceans makes it timely to compare their placentation with that of the artiodactyls. We studied the placentae of two sympatric species of dolphin from the Amazon River Basin, representing two distinct families. The umbilical cord branched to supply a bilobed allantoic sac. Small blood vessels and smooth muscle bundles were found within the stroma of the cord. Foci of squamous metaplasia occurred in the allanto-amnion and allantochorion. The interhemal membrane of the placenta was of the epitheliochorial type. Two different types of trophoblastic epithelium were seen. Most was of the simple columnar type and indented by fetal capillaries. However, there were also areolar regions with tall columnar trophoblast and these were more sparsely supplied with capillaries. The endometrium was well vascularised and richly supplied with actively secreting glands. These findings are consistent with the current view that Cetacea are nested within Artiodactyla as sister group to the hippopotamids.     

Role of uteroferrin in transplacental iron transport in the pig

The pig possesses a noninvasive, diffuse type of epitheliochorial placentation in which the blood supply of the mother is well separated from the absorptive surface of the chorion, a feature that must complicate the movement of nutrient molecules across the placenta. Evidence is presented that a protein synthesized and secreted by the glandular epithelial cells of the maternal uterus of the pig is involved in iron transport to the fetus. This protein, uteroferrin, is induced by progesterone; is purple, which results from an unusual iron center; and possesses acid phosphatase activity. Secreted uteroferrin is taken up by specialized chorionic epithelial cells located in domed structures, called areolae, overlying the mouth of each uterine gland. Uteroferrin then enters the placental venous drainage and its iron is efficiently incorporated into fetal hemoglobin. It is taken up by the fetal liver or cleared by the kidney. The liver is the main site of erythropoiesis in the fetus. From the kidney uteroferrin enters the allantoic sac where it exchanges its iron with fetal transferrin. The rate of uteroferrin biosynthesis in the uterus and its rate of metabolism in the fetus can theoretically provide sufficient iron for the needs of pregnancy, at least until around day 70 of the 115-day gestation. Uteroferrin and transferrin, the iron transport protein of plasma, appear to be unrelated proteins.     

Overexpression of Calreticulin in Pre-eclamptic Placentas: Effect on Apoptosis, Cell Invasion and Severity of Pre-eclampsia

Endoplasmic reticulum (ER) stress has recently been identified as an important process involved in the pathology of pre-eclampsia (PE). Calreticulin (CRT) is an important ER resident protein which participates in the regulation of intracellular Ca(2+) homeostasis, cell adhesion, and cell apoptosis. In order to clarify the role of this protein in normal human pregnancy and in PE, this study has examined the expression of CRT in pre-eclamptic placenta compared with control placenta. The expression of CRT mRNA and protein was elevated in the pre-eclamptic placentas in comparison with control placentas. Furthermore, the expression level was related to the severity of symptoms experienced by PE patients. Therefore, this study aimed to identify the biological characteristics of the CRT gene in trophoblast cells. A CRT-expressing vector was transfected into the JEG-3 human choriocarcinoma cell line. Investigations showed that both proliferation and invasion were inhibited and apoptosis was promoted by CRT expression in JEG-3 cells. These data suggest that augmentation of CRT in the placenta may induce cell apoptosis and impair the invasion of extravillous trophoblast cells, thus leading to shallow placentation in PE.     

Localization of chorionic pregnancy-associated glycoprotein family in the pig

The objective of this study was to localize the immuno-positive porcine PAG (pPAG) proteins within chorionic cells throughout the intensive placenta development as pregnancy advances (16-61 days post coitum - dpc). Placental sections were used for double fluorescent histochemistry with selected primary rabbit anti-pPAG sera. The polyclonals were created against recombinant pPAG2 antigen or various secretory porcine native chorionic antigens produced in vitro. Among placental cells stained with fluorescent propidium iodine, the positive pPAG immuno-complexes were visualized by Alexa 488 fluorochrom - conjugated to secondary anti-rabbit goat immunoglobulins. This is the first report concerning cellular localization of the pPAG protein family within diffuse epitheliochorial placenta development throughout the first half of pregnancy in the pig. Fluorescent immuno-positive pPAG signals have been restricted to chorionic cell layers (branched mushroom-like and finger-like structures) that generate a epitheliochorial feto-maternal surface augmented by maternal endometrium interdigitations with the gestation progress in the pig. These results suggest that the pPAG proteins robustly expressed in chorionic cells are involved in the regulation of intensive development of diffuse porcine placenta during the first half of pregnancy.     

Biologically active substances from the skin that influence the proliferation and differentiation of cultured human keratinocytes]

Three fractions of biologically active substances of human skin varying in molecular weight (F1 > 15 kD, F2 1.4-15 kD, F3 < 1.4 kD) were tested in primary and regenerating cultures of human epidermal keratinocytes. F1 and F3 caused stimulation of keratinocyte proliferation, F2 inhibited proliferation and enhanced terminal differentiation of epidermal cells.     

Evolution of the placenta during the early radiation of placental mammals

The chorioallantoic placenta is an organ of gaseous exchange that exhibits a high degree of structural diversity. One factor determining oxygen transfer across the placenta, the diffusion distance, is in part dependent on the number of cell layers separating maternal from fetal blood. This interhaemal barrier occurs in three principal variants. The focus of this review is on determining how the barrier evolved in placental mammals. The analysis was based on current knowledge of placental structure, as far as possible using ultrastructural data, and on current views about the evolution of placental mammals, derived from molecular phylogenetics. We show that epitheliochorial placentation, the least invasive type, is a derived state and discuss factors that may have determined its evolution with reference to conflict theory, as applied to the allocation of resources between mother and fetus. It is not yet possible to determine which of the two more invasive types of placentation occurred in the last common ancestor of crown placentals. Depending on tree topology and taxon sampling, the result achieved is either endotheliochorial, haemochorial or unresolved. Finally we discuss other factors important to placental gas exchange and point to physiological variables that might become amenable to phylogenetic analysis.     

Growth and development of the placenta in the capybara (Hydrochaeris hydrochaeris)

Background  

The guinea pig is an attractive model for human pregnancy and placentation, mainly because of its haemomonochorial placental type, but is rather small in size. Therefore, to better understand the impact of body mass, we studied placental development in the capybara which has a body mass around 50 kg and a gestation period of around 150 days. We paid attention to the development of the lobulated arrangement of the placenta, the growth of the labyrinth in the course of gestation, the differentiation of the subplacenta, and the pattern of invasion by extraplacental trophoblast.     

Cyto-epitheliochorial placenta of the viviparous lizard Pseudemoia entrecasteauxii: a new placental morphotype

The structural features of the uterine epithelium of the chorioallantoic placenta and omphalloplacenta in the viviparous Australian skink, Pseudemoia entrecasteauxii, were investigated using SEM and TEM techniques. In particular, the structural characteristics that would allow interpretation of function were analyzed, particularly those of gas exchange in the chorioallantoic placenta and histotrophy in the omphaloplacenta. Pseudemoia entrecasteauxii has a complex placenta consisting of a placentome, paraplacentome, and omphaloplacenta. The paraplacentome has a well-vascularized lamina propria in which projecting uterine capillaries displace the overlying uterine epithelial cells, reducing them to attenuated cytoplasmic extensions. Associated cell nuclei and organelles are lost from this region, to provide a capillary lumen to uterine lumen barrier of 0.5-1.0 microm. Hence, the paraplacentome is likely a prominent site for gaseous exchange via simple diffusion. The omphaloplacenta has a similar cytology to that of the placentome, but the uterine epithelial cells are hypertrophied and the apical plasma membrane actively secretes vesicles into the uterine lumen. The omphaloplacenta shows features that are associated with histotrophic transport of nutrients via vesicle secretion, very similar to that of lipid apocrine secretion. The placentome consists of cuboidal cells in the uterine epithelium, with large centrally located nuclei overlying the well-vascularized lamina propria. Although the placentome has a similar cytological structure to that of the omphaloplacenta, granules or active vesicle secretion were not observed. Thus, the placentome may be associated with histotrophy, but not via apocrine secretion. Squamate placentation is epitheliochorial; however, we propose a new term be used to describe the type of placentation in P. entrecasteauxii: "cyto-epitheliochorial," because of the extreme attenuation of uterine epithelial cells of the paraplacentome.     

Evolutionary implications of fetal and maternal microvillous surfaces in epitheliochorial placentae

According to the “parent-offspring conflict hypothesis” the rapid evolution and diversification of the mammalian placenta is driven by divergent optima of resource allocation between fetus and mother. The fetus has an interest to maximize its resource intake, while the mother has an interest to restrict the transfer of resources, and thus retain resources for subsequent pregnancies. In the epitheliochorial placenta, the contacting fetal and maternal surfaces at the feto-maternal interface are covered with microvilli, which leads to an increase of membrane surfaces available for transport processes. Because membranes are the site of active transport, the conflict hypothesis predicts that the fetal surfaces at the feto-maternal interfaces are larger than the maternal ones. We use transmission electron microscopy and a stereological method to estimate the factors by which the apical fetal and maternal membranes are enlarged by the microvilli. Ten species with an epitheliochorial placenta were studied. Focused ion beam—scanning electron microscopy (FIB-SEM) was used to create three-dimensional models of the interdigitating microvilli of the bovine and porcine placenta. In all species, the fetal surface was larger than the maternal. This was due to a higher number of fetal microvilli and to the presence of membrane folds at the base of the fetal, but not of maternal microvilli. Our results suggest that the ultrastructural morphology of the feto-maternal interface in the epitheliochorial placenta is shaped by conflicting interests between fetus and mother and thus represent a so far neglected arena of the parent-offspring conflict.     

Analysis of osteopontin at the maternal-placental interface in pigs

Noninvasive, epitheliochorial placentation in the pig follows a prolonged preimplantation period characterized by migration, spacing and elongation of conceptuses, and secretion of estrogen for maternal recognition of pregnancy. Osteopontin (OPN) is an extracellular matrix protein that binds integrins to promote cell-cell attachment and communication. OPN appears to play a key role in conceptus implantation and maintenance of pregnancy in sheep; however, a role for OPN in the porcine uterus has not been established. Therefore, this study examined OPN expression and function in the porcine uterus and conceptus (embryo/fetus and associated extraembryonic membranes). Northern and slot blot hybridization detected an increase in endometrial OPN expression between Days 25 and 30, and levels remained elevated through Day 85 of pregnancy. In situ hybridization localized OPN mRNA to discrete regions of the uterine luminal epithelium (LE) on Day 15 of pregnancy and to the entire LE thereafter. Glandular epithelial (GE) expression of OPN mRNA was first detected on Day 35 of pregnancy and increased through Day 85. Both 70- and 45-kDa forms of OPN protein were detected in cyclic and pregnant endometrium by Western blotting. OPN protein was localized to the LE and GE by immunofluorescence; however, only the 70-kDa OPN was detected in uterine flushings. OPN protein was present along the entire uterine-placental interface after Day 30 of pregnancy. In addition, OPN mRNA and protein were localized to immune-like cells within the stratum compactum of the endometrium in both Day 9 cyclic and pregnant gilts. Incubation of OPN-coated microbeads with porcine trophectoderm and uterine luminal epithelial cells induced Arg-Gly-Asp (RGD)-dependent integrin activation and transmembrane accumulation of cytoskeletal molecules at the apical cell surface as assessed by immunofluorescence detection of talin or alpha-actinin as markers for focal adhesions. These results suggest that OPN, expressed by uterine epithelium and immune cells, may interact with receptors (i.e., integrins) on conceptus and uterus to promote conceptus development and signaling between these tissues as key contributors to attachment and placentation in the pig.     

Biochemical and functional characterisation of macrophage stimulating factors secreted by mitogen-induced goldfish kidney leucocytes

Mitogen-stimulated goldfish kidney leucocytes secrete a number of different macrophage activation factors (MAF) that induce profound physiological changes in macrophages. MAF produced by goldfish kidney leucocytes was characterised using fast performance liquid chromatography (FPLC) and bioassays that measured MAF-induced respiratory burst (RB) and nitric oxide (NO) responses of activated macrophages. Mitogen-induced fish kidney leucocyte supernatants were fractionated using gel permeation FPLC (GP-FPLC) and the ability of different fractions to induce NO or RB measured. A MAF of M(r) 50 kD, that induced a potent nitric oxide response in both a long-term goldfish macrophage cell line (GMCL) and in in vitro-derived fish kidney macrophages (IVDKM) was identified. The GP-FPLC partially purified 50 kD MAF activity occasionally induced significantly higher nitric oxide production than that of the crude MAF preparations. This increase in the NO-inducing activity was due to segregation of the 50 kD MAF from a novel macrophage deactivating molecule of M(r) 10-12 kD present in crude MAF preparations. This 10-12 kD molecule was shown to inhibit nitric oxide production in cytokine-activated goldfish macrophages. Mitogen-induced fish kidney leucocyte supernatants contained two distinct MAFs that induced the respiratory burst in GMCL and IVDKM: the 50 kD and 30 kD proteins. The partially purified 30 kD MAF primed goldfish macrophage for increased RB activity after only 6 h of treatment, and continued to augment the RB activity after 24 h of stimulation. In contrast, the GP-FPLC partially purified 50 kD molecule also primed the RB after only 6 h of stimulation, but subsequently deprimed the RB after 24 h of stimulation, an effect similar to that observed for crude MAF preparations. The 50 kD MAF activity was further purified using chromatofocusing FPLC (C-FPLC) using basic pH gradients and was shown to consist of two distinct NO-inducing molecules (> pI 9.3). Mitogen-stimulated fish kidney leucocytes secrete several factors that profoundly affect the anti-microbial responses of teleost macrophages and which undoubtedly are responsible for regulating teleost macrophage function in vivo.