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Exposure to allergens is pivotal in determining sensitization and allergic symptoms in individuals. Pollen grain counts in ambient air have traditionally been assessed to estimate airborne allergen exposure. However, the exact allergen content of ambient air is unknown. We therefore monitored atmospheric concentrations of birch pollen grains and the matched major birch pollen allergen Bet v 1 simultaneously across Europe within the EU-funded project HIALINE (Health Impacts of Airborne Allergen Information Network). Pollen count was assessed with Hirst type pollen traps at 10 I min(-1) at sites in France, United Kingdom, Germany, Italy and Finland. Allergen concentrations in ambient air were sampled at 800 I min(-1) with a Chemvol (R) high-volume cascade impactor equipped with stages PM > 10 mu m, 10 mu m > PM > 2.5 mu m, and in Germany also 2.5 mu m > PM > 0.12 mu m. The major birch pollen allergen Bet v 1 was determined with an allergen specific ELISA. Bet v 1 isoform patterns were analyzed by 2D-SDS-PAGE blots and mass spectrometric identification. Basophil activation was tested in an FC epsilon R1-humanized rat basophil cell line passively sensitized with serum of a birch pollen symptomatic patient. Compared to 10 previous years, 2009 was a representative birch pollen season for all stations. About 90% of the allergen was found in the PM > 10 mu m fraction at all stations. Bet v 1 isoforms pattern did not vary substantially neither during ripening of pollen nor between different geographical locations. The average European allergen release from birch pollen was 3.2 pg Bet v 1/pollen and did not vary much between the European countries. However, in all countries a >10-fold difference in daily allergen release per pollen was measured which could be explained by long-range transport of pollen with a deviating allergen release. Basophil activation by ambient air extracts correlated better with airborne allergen than with pollen concentration. Although Bet v 1 is a mixture of different isoforms, its fingerprint is constant across Europe. Bet v 1 was also exclusively linked to pollen. Pollen from different days varied >10-fold in allergen release. Thus exposure to allergen is inaccurately monitored by only monitoring birch pollen grains. Indeed, a humanized basophil activation test correlated much better with allergen concentrations in ambient air than with pollen count. Monitoring the allergens themselves together with pollen in ambient air might be an improvement in allergen exposure assessment. European Commission Christine Kühne - Center for Allergy Research and Education

Although the human basophil is predominantly a circulating cell, it is known to participate in a variety of inflammatory responses. For example, basophils have clearly been identified in skin at sites of contact hypersensitivity, erythema multiforme, bullous pemphigoid, and cutaneous basophil hypersensitivity reactions and in the airways of subjects with allergic rhinitis and asthma?' 2 In addition, an influx of basophils accompanies the allergic late phase response that occurs after antigen challenge of the skin and airways. 3-s Thus basophils, like other granulocytes, can emigrate from the intravascular compartment into specific tissues. The observation that increased numbers of basophils accumulate at certain inflammatory sites suggests that they are activated and recruited to these locations. During this process it is necessary for these cells to interact with endothelial cells, parenchymal cells, and extracellular matrix proteins as they undergo margination, transendothelial migration, and enter the tissue space. Essentially all of the steps in this process involve the interaction of basophil cell surface adhesion molecules with specific adhesion molecule counterreceptors. Several extensive reviews have recently been written on cell adhesion molecule biology 6s and the potential role of these molecules in allergic diseases. 91x This article will focus on the expression and function of adhesion molecules on human basophils.

In previous studies, we demonstrated several growth factors, including epidermal growth factor (EGF), transforming growth factorβ (TGFβ), insulin-like growth factor-I (IGF-I, somatomedin C), and TGFe in extracts of the human pituitary. Using immunohistochemistry, reactivity for TGFα, EGF, and TGFβ was localized in sections of 12 of 12 autopsy-derived human pituitary glands. IGF-I reactivity was demonstrated in 5 of 5 pituitary glands. Sections staining for TGFα and IGF-I were double-stained for the full spectrum of anterior pituitary hormones (i.e., GH, PRL, ACTH, LH, FSH, TSH, and a-subunit). Intracellular EGF immunostaining was demonstrated within the posterior pituitary and in some squamous cell nests of the pars tuberalis. Occasional extracellular EGF reactivity was also noted within connective tissue of the anterior pituitary. Polyclonal antibody to TGFβ showed extracellular reactivity within connective tissue surrounding the gland as well as that separating cords of secretory cells. TGFβ staining was also noted in the mediae of small vessels. Monoclonal anti-TGFα antibody labeled scattered secretory cells throughout the anterior pituitary, including ones engaged in follicle formation, and some cells lining Rathke's cleft remnants. TGFα reactivity in secretory cells was expressed in cells also staining for PRL, α-subunit, LH, FSH, and TSH. No TGFα reactivity was noticed in cells staining for either GH or ACTH. IGF-I reactivity was observed in occasional secretory cells within the anterior pituitary and in neuronal processes of the posterior pituitary. Anterior pituitary cells reactive for hormones were nonreactive for IGF-I. The presence of EGF and TGFβ reactivity within extracellular matrix suggests that it represents a possible storage site of these growth factors within the anterior pituitary. The presence of EGF and IGF-I within the axons and terminations of posterior pituitary implies either local or possibly hypothalamic production of EGF. Finally, our results indicate that TGFα and IGF-I, as well as perhaps EGF, are manufactured by specific pituitary cells and that they may have a role in the endocrine function of the anterior and posterior pituitary, respectively.

Single alanine substitutions were introduced into the CDR1 region of the beta chain of a Kd-restricted TCR. Mutants and wild-type TCR were attached to the zeta chain of the CD3 complex and expressed at the surface of a rat basophil cell line. Transfectants were tested for the binding of purified soluble Kd-peptide complexes. With this experimental system, accessory molecules are unlikely to play a major role and the contribution of each residue to the interaction can be addressed. Results show that all positions in the CDR1 region are involved in the binding to the Kd-peptide complex but at varying degrees. These effects are discussed in relation to a molecular model of the TCR. Comparison of these results with previous data obtained in a T cell hybridoma system suggests the existence of a threshold in the TCR affinity necessary for mature T cell activation.

Background Quantitative measurement of basophil cell surface markers, CD63 and CD203c, by flow cytometry has been developed as useful tool for the diagnosis of IgE mediated allergies. Available methods require large volume of blood and include extra steps such as prior cell purification, priming with IL-3, stopping cell activation and fixing. We have developed a new basophil based test which can be performed with small amount of whole blood directly from skin prick or from small amount of the whole blood where multiple allergens can be tested at the same time for high-throughput testing.

Study of the T-cell repertoire in humans has been hampered by the lack of monoclonal antibodies (mAbs) to the T-cell receptor (TCR) variable region (V) gene products. We describe a method for producing mAbs to the human TCR beta-chain V (V beta) gene products in which mice were immunized with a rat basophil cell line (RBL-2H3) transfected with the extracellular domain of the TCR heterodimer fused to the lambda chain of CD3. These cells acted as excellent immunogens for raising anti-TCR mAb and also formed the basis of a rapid screening assay. We generated mAbs against V beta protein of the TCR, showed that these mAbs stained approximately 1% of peripheral blood T cells, and further showed that the mAbs could stimulate proliferation of these T cells. We then characterized the mAbs by amplifying TCR cDNA derived from mAb-stimulated cells and sequencing the beta chain. All clones sequenced used the V beta 7.1 chain, proving conclusively that the mAbs generated were specific for V beta 7.1 subfamily. This method generates mAbs to human TCR V beta proteins efficiently and might allow production of a complete panel of mAbs directed against human TCR V beta proteins.

Corticotroph (basophil) invasion or the migration of corticotroph cells into the pars nervosa of the human pituitary gland was found in 35 of 767 (4.4%) consecutive pituitaries obtained at autopsy. The degree of invasion increased with patient age and extensive invasion was more common in men than in women. Immunoreactive ACTH, β-MSH, α-MSH, and galanin were detected both in the anterior lobe and invading corticotroph cells in approximately equal frequency. Fewer cells stained positively for α-MSH than for the three other peptides in both the anterior lobe and invading corticotrophs. Twelve corticotropic pituitary adenomas obtained surgically from patients with Cushing's disease were also examined and expressed varying degrees of immunoreactivity for ACTH, α MSH, β-MSH and galanin. Staining for all major pituitary hormones revealed only ACTH in the invading basophil cells. Peptidylglycine α-amidating monooxygenase (PAM) was present in the anterior pituitary, in invading corticotroph cells, and in some cells lining the cysts of the pars intermedia zone. PAM immunoreactivity was also detected in 4/12 corticotroph adenomas. These results indicate that corticotroph cells invading the pars nervosa are immunohistochemically similar to anterior lobe corticotrophs and have the ability to amidate various peptides such as proopiomelanocortin cleavage products and galanin with PAM.

The aim of this study was to evaluate the function of basophil granulocytes in 37 patients with progressive systemic sclerosis (PSS). The in vitro proteinase release test using Chromozyme TH as a chromogenic substrate was performed to measure the basophil cell releasability. Anti-IgE, calcium-ionophore (A 23187) and N-formyl-methyonil-leucyl-phenylalanine were used as activatory agents. Our results showed that the reactivity pattern to those agents did not differ in PSS compared to controls. Patients whose basophil cells release proteinases to all of the 3 activators tended to have more severe organ symptoms compared to the rest of the patients, while teleangiectasia was significantly less frequently (p < 0.02) found in cases with proteinase release. These findings suggest that there may be a certain relationship between basophil function and severity of the PSS.

It has recently been shown that monoclonal antibody (MoAb) 97A6 detects a surface antigen expressed on basophils and their CD34+ precursor cells, as well as the basophil cell line KU-812. In this report the partial amino acid sequence of affinity chromatography– and sodium dodecyl sulfate–polyacrylamide gel electrophoresis–separated 97A6 antigen(s) from KU-812 lysates was determined. Sequence alignment of high-performance liquid chromatography–selected tryptic peptides from the resulting 130- and 150-kd bands revealed a 100% identity with amino acids 393 to 405 of ectonucleotide pyrophosphatase/phosphodiesterase-3 (E-NPP3; CD203c) but not of the related ectoenzyme PC-1 (E-NPP1). Moreover, MoAb 97A6 selectively recognized 293 cells transfected with human E-NPP3, but did not react with cells transfected with PC-1 or parental 293 cells. In addition, E-NPP3 messenger RNA expression was detected in basophils but not other peripheral blood cells. Finally, MoAb 97A6 immunoprecipitated phosphodiesterase activity from KU-812 cells and peripheral blood basophils, but not from other cell populations. These data demonstrate that MoAb 97A6 recognizes the functionally active type II transmembrane ectoenzyme E-NPP3.

In this review, we analyze the action of estrogens leading to the remodeling of the anterior pituitary gland, especially during the estrous cycle. Proliferation and death of anterior pituitary cells a

Insulin-like growth factors(IGF) are involved in feedback regulation of growth hormone(GH) secretion from the pituitary. Though receptors for IGF-I and IGF-II have been identified on particulate preparations of rat pituitary, their localization and relationship to GH-secreting acidophils has not been determined. We used quantitative in vitro autoradiography and immunocytochemistry to simultaneously determine the distribution of IGF receptors and GH-secreting cells in human infant pituitary gland. Frozen or fixed post-mortem human infant pituitary glands were sectioned for binding studies, or immunocytochemistry. Binding for IGF-I and IGF-II showed characteristic specificity for respectively Type I and Type II receptors. Binding sites were visualized throughout the pituitary gland, with similar density and distribution for IGF-I and IGF-II receptor sites. Receptor density was two-fold higher in anterior than posterior pituitary, with highest density in the lateral horns of the anterior pituitary. The distribution of GH-containing cells (acidophils) was similar to IGF receptor distribution. Increased density of IGF receptors in regions of GH-secreting cells may point to the mechanism whereby IGF uniquely inhibits synthesis of human GH in contrast to its promotion of synthetic processes in other cells.

Pit-1 is a transcription factor which has been reported to regulate differentiation toward GH, PRL and TSH in the anterior pituitary glands. In the human pituitary adenomas, Pit-1 is highly expressed in GH secreting and TSH secreting adenomas as it can well be anticipated. Interestingly, human non-functioning pituitary adenomas also express Pit-1, especially it was expressed in all alpha SU positive nonfunctioning adenomas. The human anterior pituitary cells are special in comparison with rodents in a finding that alpha SU is frequently colocalized with GH. As alpha SU is the first hormone appearing during fetal development in the rodent pituitary glands, it may be postulated that alpha SU Pit-1 positive cells undergo differentiation in the GH cell lineage. From this background, this paper proposes that "alpha SU positive Pit-1 Positive" cells are the ones in the GH cell lineage, more specifically a dedifferentiated cell lineage toward alpha SU/GH/Pit-1.

Basophil histamine release was studied in leukocyte suspensions from normal individuals and from patients allergic to house dust mite or birch pollen. Mediator release caused by IgE-mediated reactions was examined by stimulating the cells with anti-IgE or specific antigens, and the calcium ionophore A23187 was used for a non-immunological histamine release. In all experiments influenza A virus caused a synergic enhancement of the mediator release and the potentiation was abolished by galactose (10(-7) to 10(-6) M) and by 10(-6) to 10(-5) M of N-acetylglucosamine, alpha-methyl-D-glucoside, alpha-methyl-D-mannoside, N-acetylneuraminic acid and lactose, but not by glucose. Wash-out experiments show that the sugars prevent the aggravation of mediator release by a binding of sugar to the basophil cell membrane, thereby causing a blockade of binding sites responsible for the potentiating effect of virus.

This article reviews the hypothalamic control of anterior pituitary hormone secretion. The anterior pituitary gland plays a pivotal role in the hormonal response to stress, as it is the source of adrenocorticotropin and prolactin. The involvement of the posterior and intermediate pituitary lobes in the stress response is also considered.

A pro-inflammatory cytokine, interleukin 18 (IL-18), induces intracellular expression of IL-1 and the release of IL-6. IL-1 and IL-6 has been detected in anterior pituitary cells, suggesting that IL-18 is produced in anterior pituitary cells and may serve to aid immuno-endocrine regulation. In the present study, we addressed this hypothesis by investigating the intracellular localization of IL-18 and its receptor in bovine anterior pituitary gland. IL-18 mRNA and its protein were detected in the anterior pituitary gland by RT-PCR and Western blotting. In situ hybridization showed that IL-18 mRNA was expressed in the anterior pituitary cells. Immunohistochemistry of IL-18 and specific hormones revealed the presence of IL-18 in somatotrophs. Furthermore, the expression of GH mRNA in IL-18 immunoreactive cells was confirmed by immuno-laser microdissection. These results first demonstrated that somatotrophs produced IL-18. Subsequently, the distribution of the IL-18 receptor alpha (IL-18Ralpha) was investigated in order to understand IL-18 signaling among the anterior pituitary cells. Bovine IL-18Ralpha cDNA was partially sequenced and detected in the anterior pituitary gland by RT-PCR. Immunohistochemistry of IL-18Ralpha, IL-18 and GH showed that IL-18Ralpha was co-localized in IL-18 immunoreactive cells or somatotrophs. These data suggest that IL-18 acts on somatotrophs as an immuno-endocrine mediator through the autocrine pathway.

Background: Mast cells and basophils express the high-affinity IgE receptor FcΕRI. We have analysed the human mast cell line LAD2 and four subclones of the basophil cell line KU812 in order to reveal possible differences concerning the FcΕRI surface regulation, anti-IgE-triggered activation, FcΕRIα protein stability and the mRNA level of FcΕRIα-, β- and the truncated β-chain (βT), and thereby determine the utility of these cell lines in investigations of the FcΕRI biology. Methods: The surface expression of FcΕRI was assessed by flow cytometry, using the monoclonal antibody CRA1. The FcΕRI-induced cellular activation (i.e. cross-linking of FcΕRI) was determined by changes in the intracellular level of Ca2+, which was measured by fluorescence of Fura-2. The level of the FcΕRIα protein was determined by a Western blot technique and by a radioimmunoassay. The mRNA level of FcΕRIα, β- and βT-chain was analysed using real-time PCR. Results: Two KU812 subclones and especially LAD2 had FcΕRI surface expression which was capable of inducing cellular activation. Both the FcΕRI expression and stability of the FcΕRIα protein were increased when IgE was present. All the cell lines expressed mRNA of FcΕRIα-, β- and βT, with LAD2 tending to have the highest expression. However, a determination of the β/βT ratio demonstrated no difference between any of the cell clones. Conclusion: These cell lines are important tools in the investigation of both the FcΕRI molecule and the effects induced by its activation.

Signaling mechanisms in pituitary morphogenesis as well as pituitary cell fate determination during early embryonic development are relatively well characterized. In contrast, the cues that determine the progression of the various anterior pituitary cell types during postnatal periods are poorly defined. Pax8-/- mice, which are born without a thyroid gland, were used to study the influence of thyroid hormones on the expression of pituitary hormones during early postnatal life. Serum pituitary hormones were determined by RIAs, and the pituitaries were analyzed by Northern blotting, in situ hybridization histochemistry, and immunocytochemistry. In 21-d-old Pax8-/- mice, the cellular composition of the anterior pituitary was dramatically distorted. Thyrotropes exhibited hypertrophy and hyperplasia, the number of detectable somatotropes was drastically reduced, and lactotropes were almost undetectable. Expression of LH and FSH was also reduced, but ACTH and proopiomelanocortin expression was not significantly different. Serum pituitary hormone levels were changed correspondingly. T(4) replacement therapy for variable time periods normalized TSH and GH mRNA expression within 3 d but not prolactin expression, not even when T(4) was administered for 6 d in combination with estradiol. These findings reveal the importance of thyroid hormones in developing the appropriate proportions of anterior pituitary cell types, especially with regard to lactotropes.

Recent studies using biotechnological methods have achieved significant advances in our knowledge of molecular mechanisms underlying pituitary gland development and the differentiation of pituitary cytotypes. A large number of neuropeptides have been reported in the adult pituitary gland as well as in the central and peripheral nervous system. The early presence of neuropeptides during pituitary development is reviewed here. Neuromedin U (NmU), galanin and the polypeptide 7B2 have been localised to different endocrine cells of the gland. Their expression seems to be manifold even though it is temporally and spatially regulated. There is now firm immunocytochemical evidence that neuropeptides are present during morphogenesis of the pituitary and can be present simultaneously with all pituitary hormones. © 2003 ISDN. Published by Elsevier Ltd. All rights reserved.

Background: IL-3 enhances basophil histamine release upon stimulation with any known secretagogue. The molecular mechanism behind this regulation is not known, although some observations suggest that IL-3 modulates the calcium part of the signal transduction mechanism. The inhibitory action of glucocorticoids on basophils can be reversed by stimulation with IL-3. Methods: Calcium-binding proteins in the basophil cell line KU812 were identified by two-dimensional gel electrophoresis, Calcium-overlay assay, N-terminal sequence analysis, and mass spectometry. The presence of the same proteins in purified human basophil leukocytes was established by comigration of KU812 and human basophil proteins on the two-dimensional gels. The expression of the calcium-binding proteins in the absence and presence of IL-3 and/or anti-IgE was determined by densitometric measurement of the spots on the two-dimensional gels. Results: Calreticulin was identified on the two-dimensional gel of KU812 proteins. A protein with exactly the same migration pattern was found on the gels of proteins from purified human basophils. Immunoblotting with a specific anti-human calreticulin antibody confirmed that this protein was calreticulin. Subsequent analysis showed that the expression of calreticulin in the basophils is upregulated twofold upon stimulation with rhIL-3, even in doses below those needed for enhancement of histamine release. Conclusions: The expression of calreticulin in human basophil leukocytes is regulated by IL-3. Calreticulin is known to modulate IP3-dependent Ca2+ influx in different cell systems, and calreticulin overexpression inhibits steroid-induced transcriptional activation. Therefore, modulation of calreticulin expression may be one mechanism by which IL-3 exerts its effects on human basophils.

TSH secretion from the anterior pituitary is mainly regulated by TRH and thyroid hormones. We hypothesized that in addition the pituitary itself could modulate TSH production by sensing its own TSH release, enabling fine-tuning of TSH secretion. For such an ultra-short loop control, the pituitary should contain a TSH receptor (TSH-R). To find evidence for this we screened a human pituitary complementary DNA library with a digoxigenin-labeled TSH-R probe and found 2 positive clones of 32,000 plaques. One clone was sequenced and found to be completely identical to the thyroid TSH-R. Further proof was obtained by RT-PCR on a human anterior pituitary obtained at autopsy. In situ hybridization and immunohistochemistry confirmed the presence of TSH-R in the anterior pituitary at the messenger ribonucleic acid level as well as the protein level. Moreover, double labeling experiments revealed that TSH-R messenger ribonucleic acid as well as TSH-R protein colocalize with major histocompatibility complex class II expression of folliculo-stellate cells. We conclude that TSH-R is expressed in a subpopulation of folliculo-stellate cells in the human anterior pituitary. This finding suggests ultra-short loop regulation of TSH secretion. Putative recognition of the pituitary TSH-R by TSH-R antibodies might have clinical relevance in Graves' disease.

Developmentally pathological conditions of the anterior pituitary cells include failed separation of the primary pituitary gland into sellar and pharyngeal ones, ectopic migration into the subarachnoid space, and basophil invasion into the posterior lobe although the last is a physiological phenomenon with pathological potentiality in certain circumstances. Pituitary primordium appears at about 4 weeks of gestation. One of the causes of the pituitary gland agenesis may be a formation of the primary hypothalamic ganglionic hamartoma just at the time of occurrence of the pituitary primordium, as analyzed in cases of Pallister-Hall syndrome. A double pituitary in a single individual is a rare malformation. Its pathogenesis is considered as a result of notochordal anomaly. In the 8th gestational week, the primary pituitary gland separates into sellar and pharyngeal parts. The disturbance of this histogenesis results in a rare pituitary malformation, a "pharyngosellar pituitary." Despite the failed separation in this case, differentiation of the pituitary cells proceeds and the hormone production of this malformed pituitary gland can be displayed immunohistochemically. In this case, the distribution of the different hormone producing cells was atypical, particularly in those of gonadotropic hormones and ACTH. Life-long existence of the pharyngeal pituitary is a normal anatomical state in humans. Cell differentiation (hormone production) in the pharyngeal pituitary occurs about 4-10 weeks later than in the sellar pituitary. In pharyngeal pituitary, all kinds of adenohypophyseal hormones are produced. Extracranial pituitary adenomas (with intact sellar pituitary), exclusively found in the nasopharynx, sphenoid sinus, and clivus, may occur from the pharyngeal pituitary while another tumorigenesis can develop from the residual tissue fragment in the craniopharyngeal canal. The "overshoot" of the adenohypophyseal cell migration in the distal part of the sellar pituitary is frequently observed in the leptomeninges of the peri-infundibular or peri-hypothalamic region as ectopic pituitary cell clusters that are apparently independent of the pars tuberalis. It is suggested that these cells, frequently found in "normal" individuals, may be one of the possible origins of the intracranial ectopic pituitary adenomas. However, the reason why a majority of the reported intracranial ectopic pituitary tumors are ACTH-adenomas remains unexplained, since the ectopic cells, found in "normal" individuals, consist of fairly different hormone-producing cells. A further enigmatic phenomenon is a "basophil invasion." ACTH-positive cells invade from the pars intermedia into the posterior lobe of the pituitary. This invasion increases in intensity and frequency according to increase in age. However, the invasion of ACTH cells is observed as early as in the fetal life. The invasive cells display occasionally cell atypia as well as mitotic activity. The origin of extremely rare pituitary adenomas inside the posterior lobe can be explained by the existence and proliferative activity of basophil invasion.

Studies evaluating estrogen-induced anterior pituitary tumors revealed a strong direct correlation between expression of the peptide galanin and tumor growth. To evaluate further the potential roles of galanin in the hyperplastic pituitary, we used a model of estrogen-independent anterior pituitary tumor formation, the male human GH-releasing hormone (hGHRH) transgenic mouse. Pituitaries of hGHRH transgenic mice are characterized by a hyperplasia of somatotrophs and contain markedly elevated levels of galanin. We examined the population of galanin-producing pituitary cells in 4- to 6-month-old male hGHRH transgenic mice and their nontransgenic siblings. The percentage of galanin-containing pituitary cells was significantly increased within the anterior pituitaries of hGHRH transgenic mice. By using the cell immunoblot assay we found that the basal secretion of galanin and GH from individual pituitary cells of hGHRH transgenic mice was significantly greater than that from pituitary cells of nontransgenic mice. By modifying the cell immunoblot assay, we determined that somatotrophs from both hGHRH transgenic and normal mice that were positive for galanin immunoreactivity secreted significantly greater amounts of GH than those somatotrophs devoid of galanin immunoreactivity. Moreover, immunoneutralization of galanin significantly decreased GH secretion from pituitary cells obtained from hGHRH transgenic mice. Thus, we now show that the increased levels of galanin peptide within the hyperplastic pituitaries of hGHRH transgenic mice are due to an increase in the population of cells containing galanin, and that galanin participates in the augmented secretion of GH from hyperplastic proliferating pituitary cells.

Interleukin-6, mainly produced by monocytes and macrophages is known to influence the secretion of anterior pituitary hormones and is, therefore, considered to play an important role in the interaction between the immune system and the endocrine system. However, IL-6 represents not only a lymphocyte message but is also produced within the anterior pituitary. Folliculostellate (FS) cells have been identified as the source of the intrapituitary IL-6 production in the normal pituitary, whereas in pituitary adenomas IL-6 is produced by the tumor cells themselves. The present review summarizes the knowledge about the regulation of the intrapituitary IL-6 synthesis and release in FS cells. Moreover, the possible roles of the intrinsic IL-6 production for function and growth of normal and adenomatous endocrine pituitary cells are discussed.

The pituitary gland expresses cytokines and their receptors. IL-2 receptor transcripts and protein products are co-localized in ACTH-, PRL-, and GH-producing cells (double immunofluorescence). IL-2 and IL-6 (1-1000 IU/ml) are involved in the autocrine/paracrine regulation of normal and tumor (GH3 mammosomatotroph cell line and adenoma cell cultures) anterior pituitary hormone-producing cell growth (cell number, DNA synthesis, c-fos mRNA expression and autoradiography combined with hormone staining). IL-1 regulates the growth of normal pituitary cells but does not act on GH3 cells. IL-1ra, which blocks this action, is expressed in tumoral pituitary (mainly GH- and ACTH-) cells. In ACTH- cells, IL-1 enhances glucocorticoid feedback, stimulating glucocorticoid response element transcriptional activity. Cytokines, through specific functional receptors, act as inter/auto-cellular factors that regulate not only the function but also the growth of anterior pituitary cells.

In cell cultures of dispersed rat anterior pituitary, the specific identification of each cell type based on their staining properties and the ultrastructural features of secretory granules has proved to be unreliable. The existence of pituitary cell subtypes and the striking remodeling of the cell surface and intracellular organelles, further complicate the specific identification of pituitary cell populations. An immunocytochemical study of dissociated pituitary cells in culture was carried out to identify the cellular hormonal content by applying specific antibodies against prolactin (PRL), and growth (GH), luteinizing (LH beta), adrenocorticotrophic (ACTH) and thyrotrophic (TSH) hormones. Specifically bound IgG was exposed by the electron microscope with protein A-gold complex. Typical lactotrophs, somatotrophs and gonadotrophs are easily recognized because they retain the main features described in the pituitary tissue in situ. Other undefined groups of cells bearing small or medium round secretory granules can be identified by immunocytochemistry as PRL, GH or TSH producing cells. The latter technique was critical for the characterization of the hormonal content of secretory granules, the shape, size, electron density and cytoplasmic distribution of which differ substantially from those described in the intact gland. Cells displaying rare small oval or sharp pointed secretory granules were identified as gonadotrophs with anti-LH beta, while corticotrophs showed granules with irregular profiles not previously reported in the gland. These remarkable morphological changes appear to be related to the interruption of the flow of hypothalamic hormones and the disruption of structural and paracrine interrelationships. This investigation reveals that immunocytochemistry is essential for the specific recognition of the various pituitary cell types, and particularly of atypical cells exhibiting morphological features not found in the pituitary gland in situ.

Interleukin-6 (IL-6) is a multifunctional cytokine produced by a variety of cell types in tissues of both the immune and endocrine systems. Among the major functions described for IL-6 are its role in the maturation of B cells to high-output antibody-producing cells and its contribution to the acute physiological responses to infection and inflammation, notably production of hepatic acute phase proteins and activation of the hypothalamic-pituitary-adrenal axis. In addition to these better known functions, IL-6 recently has been found within the pituitary of laboratory rats and also in the human pituitary. In rats, pituitary IL-6 mRNA is upregulated by peripheral exposure to bacterial endotoxin. However, the role of anterior pituitary IL-6 in host responses to infection and inflammation remains uncertain, although it may regulate pituitary hormone secretion. The following brief review summarizes the information available concerning cytokine production within the anterior pituitary of species of domestic livestock. To our knowledge, experiments conducted in our laboratory evaluating regulation of IL-6 mRNA expression and secretion from the porcine anterior pituitary provide most of the data in domestic species confirming the presence of IL-6 in the pituitary. Our data indicate that IL-6 mRNA is present in cultured porcine anterior pituitary cells and that the pituitary directly responds to stimulation with bacterial endotoxin by increasing secretion of IL-6. Furthermore, endotoxin-induced upregulation of IL-6 mRNA expression and secretion appears to be dependent upon production of cyclooxygenase products of arachidonic acid metabolism.

The anterior pituitary-specific transcription factor Pit-1 (also known as GHF-1) was initially identified and cloned as a transactivator of the GH and PRL genes, and later as a regulator of the TSH beta gene. Analysis of Pit-1 expression during mouse embryogenesis revealed that initiation of its expression correlates both temporally and spatially with activation of its target genes. Immunocytochemical studies revealed a high expression of Pit-1 protein in the nuclei of only three cell types: somatotropes, lactotropes and thyrotropes. The importance of Pit-1 as a regulator of the anterior pituitary development has been further demonstrated by the absence of somatotropes, lactotropes and thyrotropes in the pituitary glands of Pit-1-defective mice and humans. Since Pit-1 is required for both cell phenotype and proliferation, one may ask if this transcription factor might be associated with development of pituitary tumors. Several investigators have reported Pit-1 gene expression in human pituitary adenomas. These studies, while not in total agreement, show that pituitary tumorigenesis does not seem to be associated with a gross alteration of Pit-1 gene expression in humans. Pit-1 transcripts, identical in size and sequence to those observed in normal pituitary, were described in human GH-, PRL- and TSH-secreting pituitary adenomas and in most cases the presence of Pit-1 transcripts correlated with the localization of Pit-1 protein. The biological relevance of Pit-1 expression reported in some nonfunctioning adenomas remains to be clarified. As expression of the PRL and GH genes is ultimately confined to distinct lactotropic and somatotropic populations despite the presence of Pit-1 protein in both cell types, there must be additional mechanisms that control the cell-specific activation of the PRL and GH promoters. The Pit-1 beta isoform, raised through alternative splicing of exon 2 of the Pit-1 gene, is a more potent inducer of GH transcription than the major Pit-1 form. This form could, at least in part, account for the cell-specific activation of the PRL and GH genes. Pit-1 beta was invariably found present in all the tumors expressing the Pit-1 major form, no significant difference in the Pit-1 beta/Pit-1 expression ratio being observed between tumors identified as pure GH- or PRL-producing tumors. This lack of selectivity together with its low level of expression is therefore not in favor of a key role for the beta-isoform in the cell type-specific expression of the GH and PRL genes in humans. The failure of somatotropes, lactotropes and thyrotropes to proliferate in Pit-1-defective mice and humans indicates that Pit-1 might be competent to activate genes required for cell proliferation or survival of the three cell types. Recent data indeed suggest that Pit-1 may directly or indirectly regulate somatotropes and lactotropes through activation of the receptors for GRF and SRIF on the one hand, and dopamine on the other hand. Such regulatory mechanisms could contribute to the differentiation of the somatomammotropic lineage in fully differentiated somatotropic and lactotropic cells.

Interleukin-1 alpha (IL-1 alpha) and IL-1 beta bind to either the p80 type I IL-1 receptor (IL-1RI) or the p68 type II IL-1R (IL-1RII) on both T and B lymphocytes. We and others have previously shown that the anterior pituitary gland also has specific high affinity binding sites for IL-1 alpha (Kd = approximately 1 nM) and expresses transcripts for both isoforms of the IL-1R. However, the identity of cells in the anterior pituitary gland that express the IL-1R and whether different populations of adenohypophyseal cells express different isoforms of the IL-1R remain unknown. Here we have used a combination of immunohistochemistry and histochemistry to localize IL-1RI and IL-1RII to specific target cells in the mouse anterior pituitary gland. Perfusion-fixed, paraffin-embedded sections of anterior pituitary gland were immunolabeled with well characterized monoclonal antibodies to either IL-1RI or IL-1RII and counterstained using a modified Gomori's method to document acidophils and basophils. Immunolabeling demonstrated that both IL-1RI and IL-1RII were abundantly expressed on a single population of anterior pituitary gland cells and that these cells could be classified on the basis of histochemical staining as a subpopulation of acidophils. The distribution, morphology, and proportion of cells immunolabeled for IL-1RI and IL-1RII were consistent with GH-synthesizing cells. To confirm this hypothesis, a modified indirect avidin-biotin complex, sequential peroxidase/alkaline phosphatase technique was used to label anterior pituitary gland cells with antibodies to IL-1RI followed by antibodies to IL-1RII, GH, PRL, or ACTH. The IL-1RI-positive cells predominately coexpressed IL-1RII and GH, but very little, if any, PRL or ACTH. These data establish that the predominant cell population in the murine anterior pituitary gland that constitutively expresses IL-1R stain as acidophils histochemically, is round to oval with dense granular cytoplasm and eccentric nuclei, synthesizes GH, and simultaneously expresses IL-1RI and IL-1RII isoforms.

The ability of rat anterior pituitary cells to communicate through gap junctions (GJ) was studied using a fluorescent molecule, Lucifer Yellow (LY), which freely passes through GJ channels. The probe was introduced into the cell cytoplasm by using either the cut-end loading method on intact tissue, or cell microinjection on cultured cells. The identification of communicating cells was performed by immunofluorescence labeling of specific hormones in endocrine cells and of S100 protein in folliculostellate (FS) cells. Rat anterior pituitary cells in their physiological organization, i.e. in the intact tissue, exhibited a high level of coupling through GJ. LY-labeled cells were found up to 300-microns apart from its site of introduction. The communicating cells were primarily PRL cells, GH cells, and FS cells. Only a few LH, TSH, and ACTH cells were labeled with LY. Anterior pituitary cells, isolated from the rat tissue by mild protease treatment and cultured for 3 days, reestablished functional GJ as demonstrated by microinjection of LY into individual cells. By immunolabeling of specific hormones and/or S100 protein, we found a GJ coupling between FS cells, and between FS cells and endocrine cells, including PRL cells. The communication between FS cells was by far the most frequent. In conclusion, we demonstrate the presence of functional GJ between anterior pituitary cells of the same type and between anterior pituitary cells having distinct differentiated functions.

Immunoreactive activin A (ir-activin A) release from cultured rat anterior pituitary cells was examined by measuring ir-activin A in culture medium by a specific radioimmunoassay. Ir-activin A release into the medium increased over 1-18 days, and reached a maximal level at 12-15 days. The basal levels of ir-activin A in the culture media were 0.70 +/- 0.10 (mean +/- SD), 1.30 +/- 0.36 and 1.83 +/- 0.44 ng/10(6) cells, when cultured for 6 days with 0, 2 and 10% fetal calf serum, respectively. LHRH induced an approximate 1.4-fold increase in ir-activin A release in contrast to a 40-60% inhibition with FSH, but LH did not affect the activin A release. In the presence of 12-o-tetradecanoylphorbol acetate (TPA), iractivin A release was enhanced, but no significant effect was induced by forskolin. Activin A was distinctly immunostained in cultured rat anterior pituitary cells. These results suggested that activin A release from the pituitary is modified by FSH and LHRH, and that the activation of protein kinase C may be involved in the action of LHRH.