Human skin-derived mast cells can proliferate while retaining their characteristic functional and protease phenotypes.

Human mast cells in adult tissues have been thought to have limited, if any, proliferative potential. The current study examined mast cells obtained from adult skin and cultured in serum-free medium with recombinant human stem cell factor. During the first 4 weeks of culture, the percentages of mast cells increased from 10 to almost 100. After 8 weeks, a 150-fold increase in the number of mast cells was observed. When freshly dispersed mast cells were individually sorted onto human fibroblast monolayers and cultured for 3 weeks, one or more mast cells were detected in about two thirds of the wells, and in about two thirds of these wells the surviving mast cells showed evidence of proliferation, indicating most mast cells in skin can proliferate. Such mast cells all expressed high surface levels of Kit and Fc epsilon RI, each of which were functional, indicating IgE was not required for Fc epsilon RI expression on mast cells. Such mast cells also retained the MC(TC) protease phenotype of mast cells that normally reside in the dermis. After 4 to 8 weeks of culture these mast cells degranulated in response to substance P and compound 48/80, characteristics of skin-derived mast cells that persist outside of the cutaneous microenvironment. (Blood. 2001;97:2045-2052)

Modulation of high-affinity IgE receptor expression in blood monocytes: opposite effect of IL-4 and glucocorticoids.

BACKGROUND: The expression of high-affinity IgE receptor (Fc epsilon RI) is increased in blood monocytes (BMs) from allergic patients compared with those of nonatopic subjects (NASs). OBJECTIVE: We investigated the in vitro effect of cytokines involved in allergic diseases on the modulation of Fc epsilon RI expression in BMs from allergic asthmatic patients (AAPs) and NASs. The influence of in vitro and in vivo treatments with glucocorticoids (GCs) was also assessed. METHODS: Fc epsilon RI alpha-chain expression on BMs evaluated by flow cytometry analysis was studied ex vivo in AAPs treated or not with GCs and in NASs. IgE receptor expression was also evaluated in vitro with or without stimulation by IL-4, IL-13, GM-CSF, and/or GCs. Messenger (m)RNA expression was also analyzed with RT-PCR. RESULTS: The expression of the Fc epsilon RI alpha chain was significantly increased in BMs from untreated patients, with AAPs compared with NASs (P <.05). In steroid-treated AAPs Fc epsilon RI alpha-chain expression returned to the level found in BMs from NASs. In vitro addition of IL-4 induced a dose-dependent increase in Fc epsilon RI alpha-chain expression on BMs from NASs, and this effect was significantly enhanced with BMs from AAPs. Fc epsilon RI alpha-chain mRNA was significantly upregulated by IL-4, whereas the beta chain was always undetectable. The gamma chain was not modulated by IL-4. Similar findings were obtained with IL-13. In contrast with CD23 expression, GM-CSF alone or in coincubation with IL-4 had no effect on Fc epsilon RI alpha-chain expression in BMs. Lastly, GCs significantly inhibited in vitro the IL-4-induced Fc epsilon RI alpha-chain expression (P <.05). CONCLUSION: Two different pathways by which Fc epsilon RI alpha-chain expression was modulated in BMs were identified: (1) the enhancing effect of IL-4 and IL-13 and (2) the inhibitory effect of GCs. Modulation of Fc epsilon RI alpha-chain expression on BMs may affect their capacity to regulate allergic inflammation.

Recombinant human (rh)IL-4-mediated apoptosis and recombinant human IL-6-mediated protection of recombinant human stem cell factor-dependent human mast cells derived from cord blood mononuclear cell progenitors.

Although stem cell factor (SCF) appears to be the major growth factor for human mast cells, other factors undoubtedly play important roles in the development, survival, and function of these cells. The current study examined the effects of recombinant human (rh) IL-4 and rhIL-6 on rhSCF-dependent development and survival of human mast cells derived in vitro from cord blood progenitor cells. After 4-8 wk of culture with rhSCF and various amounts of rhIL-4, a dramatic decline in mast cell numbers was observed with rhIL-4, the EC50 being about 0.1 ng/ml. Numbers of other cell types remained high. Mast cells derived from cord blood progenitors after 7 wk of culture with rhSCF alone displayed an MCT phenotype and expressed Kit, FcepsilonRI, and IL-4R on their surface. Mast cells examined after purification by immunomagnetic sorting became apoptotic within hours after exposure to rhIL-4, a phenomenon blocked by anti-IL-4 Ab. Because rhIL-4-dependent apoptosis but not the loss of mitochondrial membrane potential was prevented by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(Z-VAD)-fluoromethylketone, mitochondrial perturbation most likely preceded caspase activation. Consistent with this conclusion was the observation that both apoptosis and loss of mitochondrial membrane potential (Deltapsim) were inhibited by cyclosporin A in combination with aristolochic acid. rhIL-6 protected cord blood mast cells from rhIL-4-induced apoptosis. Thus, IL-4 can cause both maturation and apoptosis of human mast cells, the latter effect being abrogated by IL-6.

Splice variants of the OB receptor gene are differentially expressed in brain and peripheral tissues of mice.

A high affinity receptor for OB protein was recently cloned from the choroid plexus of mice. At least six alternatively spliced forms of the OB receptor (OB-R) gene have been described, all of which encode proteins containing the OB-R extracellular domain. One splice variant encodes a receptor with a long intracellular domain, OB-RL, that has been implicated in OB-R signaling. Here, we have used in situ hybridization to examine the localization of OB-R splice variants in brain and peripheral tissues of adult and newborn mice. Using a probe hybridizing with all known splice variants, we confirmed that OB-R mRNA was widely distributed in the adult tissues. In the CNS, choroid plexus was the major site of expression. We now demonstrate that OB-R mRNA is expressed in peripheral tissues; primarily associated with connective tissues. In addition, OB-R mRNA was detected at higher levels in peripheral tissues of newborn mice than in adult mice. With a probe specific for OB-RL, we confirmed that high mRNA expression was detected in hypothalamic nuclei, while low levels were observed in choroid plexus. We now report that in peripheral tissues of adult mice, OB-RL mRNA expression was either very low or undetectable. In newborn mice, the pattern of OB-RL message expression in the CNS was similar to that of adult mice, while bone was the site of highest OB-RL message expression in the peripheral tissue. These data suggest different biological roles for OB-R splice variants encoding the short and long forms of OB-R. The localization of OB-RL to hypothalamic nuclei supports the idea that OB-RL is the brain receptor that mediates OB protein signaling and actions. In addition, the expression of OB-R message in newborn mice also suggests a biological role of OB-R during development in mice.

Leptin receptor long-form splice-variant protein expression in neuron cell bodies of the brain and co-localization with neuropeptide Y mRNA in the arcuate nucleus.

Reduced leptin (Ob protein) signaling is proposed to be a stimulus for the activation of neuropeptide Y (NPY) gene activity and increased expression of mRNA for the long form of the leptin receptor (Ob-Rb) in the hypothalamic arcuate nucleus. To determine if Ob-Rb protein is expressed in arcuate nucleus NPY neurons, we developed an affinity-purified polyclonal antibody against amino acids 956-1102 of human Ob-Rb. This antibody specifically recognizes the cytoplasmic tail of Ob-Rb and does not react with shorter leptin-receptor variants. Western immunoblots of Ob-Rb-transfected COS cells showed a single 150-kD band, and immunofluorescence revealed intense perinuclear staining in the cytoplasm. A 150-kD band was also present in Western immunoblots of hypothalamus. Immunocytochemical staining of brain slices revealed immunoreactive Ob-Rb protein concentrated in many neuronal cell bodies in the same regions of the forebrain that also express Ob-Rb mRNA. In the hypothalamus, Ob-Rb-positive cell bodies were abundant in the arcuate nucleus and ventromedial nucleus, with lesser numbers in the dorsomedial nucleus and paraventricular nucleus. Immunostaining was also detected in cell bodies of pyramidal cell neurons of the pyriform cortex and cerebral cortex, in neurons of the thalamus, and on the surface of ependymal cells lining the third ventricle. The choroid plexus, which expresses the short Ob-Ra form, was negative. Combined immunocytochemistry for Ob-Rb protein and fluorescence in situ hybridization for NPY mRNA identified arcuate nucleus neurons containing both NPY mRNA and Ob-Rb protein. The present finding of Ob-Rb protein in neurons that express NPY mRNA supports the hypothesis that arcuate nucleus NPY neurons are direct targets of leptin and play an important role in regulation of food intake and body weight.

Effect of recombinant human IL-4 on tryptase, chymase, and Fc epsilon receptor type I expression in recombinant human stem cell factor-dependent fetal liver-derived human mast cells.

The effect of recombinant human IL-4 (rhIL-4) on the development of recombinant human stem cell factor-dependent fetal liver-derived mast cells was examined. RhIL-4 attenuates the number of mast cells that develop, preferentially affecting the MC(T) type of mast cell. Cellular levels of tryptase and chymase mRNA normalized to that of glyceraldehyde-3-phosphate dehydrogenase were not appreciably affected. Tryptase mRNA levels peaked at least 2 wk before tryptase protein and before chymase mRNA and protein, indicating that tryptase mRNA expression is an early marker of commitment to a mast cell lineage. In contrast, alpha-tryptase and beta-tryptase mRNA levels increased and decreased in parallel. The most dramatic effect of rhIL-4 was to induce expression of functional surface Fc epsilonRI. Expression was maximal by 21 days with 20 ng/ml of rhIL-4 and reached a plateau by 2 ng/ml of rhIL-4 at 4 wk. Fc epsilonRI+ cells increased modestly when myeloma IgE was added to the developing mast cells, but increased synergistically when both myeloma IgE and rhIL-4 were present together. Delayed addition of rhIL-4 progressively diminished Fc epsilonRI expression, as did withdrawal of rhIL-4 during the first 2 wk of culture. RhIL-4 selectively increased Fc epsilonRI alpha mRNA levels at least 10-fold. Mast cells developed in the presence of rhIL-4 released tryptase when exposed to anti-Fc epsilonRI alpha. In conclusion, induction of functional Fc epislonRI on recombinant human stem cell factor-dependent human fetal liver-derived mast cells by rhIL-4 harmonizes with the well-accepted ability of this cytokine to enhance IgE production by B cells.

Assessment of autoimmunity in patients with chronic urticaria.

BACKGROUND: The etiology of chronic urticaria is unknown, and an exogenous allergen cannot be identified as the cause in the vast majority of subjects. Thus the concept has evolved that it might be autoimmune. OBJECTIVE: We have prospectively assessed sera obtained from 50 consecutive patients with chronic urticaria for the presence of autoantibodies that could be pathogenic. METHODS: We tested sera for their ability to release histamine from human basophils and to activate rat basophil leukemia cells that were transfected with the alpha subunit of the IgE receptor. We also tested selected sera for anti-IgE antibodies and for IgG anti-Fc epsilon RI alpha by Western blot. RESULTS: Sera from 38 of 50 patients with chronic urticaria released beta-hexosaminidase from transfected rat basophil leukemia cells, whereas only one of 20 control sera did so (p < 0.001); in 30 subjects this could be attributed to IgG anti- Fc epsilon RI alpha. When human basophils were used, sera from 20 of 50 patients with chronic urticaria released a significant quantity of histamine compared with one of 20 control subjects (p < 0.01). Six patients with chronic urticaria and one control subject had IgG anti-IgE. In selected sera we could demonstrate IgG anti-Fc epsilon RI alpha by Western blot; however, some sera are positive for histamine release but do not demonstrate such binding. CONCLUSION: A large fraction of patients with chronic urticaria have antibody directed to Fc epsilon RI alpha that is functional (60%). A smaller number have IgG anti-IgE (10%). A third group may also have circulating factors capable of activating basophils or mast cells of which the identity is unknown. Thus chronic urticaria may be autoimmune in origin.

The inositol 5'-phosphatase SHIP binds to immunoreceptor signaling motifs and responds to high affinity IgE receptor aggregation.

Immunoreceptors such as the high affinity IgE receptor, FcepsilonRI, and T-cell receptor-associated proteins share a common motif, the immunoreceptor tyrosine-based activation motif (ITAM). We used the yeast tribrid system to identify downstream effectors of the phosphorylated FcepsilonRI ITAM-containing subunits beta and gamma. One novel cDNA was isolated that encodes a protein that is phosphorylated on tyrosine, contains a Src-homology 2 (SH2) domain, inositolpolyphosphate 5-phosphatase activity, three NXXY motifs, several proline-rich regions, and is called SHIP. Mutation of the conserved tyrosine or leucine residues within the FcepsilonRI beta or gamma ITAMs eliminates SHIP binding and indicates that the SHIP-ITAM interaction is specific. SHIP also binds to ITAMs from the CD3 complex and T cell receptor zeta chain in vitro. SHIP protein possesses both phosphatidylinositol-3,4,5-trisphosphate 5'-phosphatase and inositol-1,3,4,5-tetrakisphosphate 5'-phosphatase activity. Phosphorylation of SHIP by a protein-tyrosine kinase, Lck, results in a reduction in enzyme activity. FcepsilonRI activation induces the association of several tyrosine phosphoproteins with SHIP. SHIP is constitutively tyrosine-phosphorylated and associated with Shc and Grb2. These data suggest that SHIP may serve as a multifunctional linker protein in receptor activation.

Functional contributions of the FcepsilonRIalpha and FepsilonRIgamma subunit domains in FcepsilonRI-mediated signaling in mast cells.

The functional contributions of the alpha and gamma subunit domains of the high affinity receptor for IgE (Fcepsilon-RI) were determined following chimeric receptor aggregation. Chimeric receptors of the extracellular (EC) and cytoplasmic tail (CT) domains of FcepsilonRI and the IL-2R p55 subunit (I) were constructed and stably expressed in RBL-2H3 cells. Signaling (inositol phosphate production, tyrosine phosphorylation, Ca2+ mobilization, and secretion of histamine and arachidonic acid metabolites) via alpha/gamma/gamma or I/gamma/gamma was similar to the native rat receptor, and both were shown to associate with endogenous FcepsilonRIbeta and FcepsilonRIgamma subunits. Therefore, the contributions of the EC domains could not be evaluated. The chimeras alpha/I/gamma and I/I/gamma were found to be single polypeptide chains, as they did not associate with beta and gamma. Signaling via alpha/I/gamma resulted in the appearance of biochemical events common to the native receptor. Cross-linking I/I/gamma elicited histamine release, [14C]arachidonic acid metabolites, tyrosine phosphorylation, Ca2+ mobilization, and only inositol trisphosphate production, which were not of a similar magnitude to the native FcepsilonRI. No biochemical events were elicited by cross-linking alpha/I/I or I/I/I. These results demonstrate that both the FcepsilonRIalpha EC domain and the FcepsilonRIgamma CT domain are essential for the FcepsilonRI signaling process, and that while FcepsilonRIIgamma CT plays a critical role in FepsilonRI signaling, the EC domain of FcepsilonRIalpha has a major contribution in signaling, as well as a role in modulating the magnitude of the biochemical events.

Color Doppler sonography in penetrating injuries of the neck.

PURPOSE: To determine whether color Doppler sonography can be a sensitive alternative to screening arteriography for identifying arterial injury in patients with penetrating traumatic neck injuries. METHODS: Fifty-two patients admitted to our trauma center with penetrating neck injuries (gunshot wounds and lacerations) were examined prospectively with color Doppler sonography, and findings were compared with the results of angiography (n = 44), with findings at surgery (n = 4), and with clinical status (n = 4). RESULTS: Color Doppler sonography correctly detected all serious injuries of the carotid arteries (n = 6; 5 diagnosed at angiography and 1 at surgery) and all injuries of the vertebral arteries (n = 4; all diagnosed at angiography). Sonography missed 1 instance of reversible narrowing of the internal and external carotid arteries and did not show 2 normal vertebral arteries. CONCLUSION: Color Doppler sonography was as accurate as angiography in screening clinically stable patients with zone II or III injuries and no signs of active bleeding. Our initial results suggest that in the future, sonography may be used as a screening examination for arterial lesions in patients with penetrating neck injuries.

The yeast tribrid system--genetic detection of trans-phosphorylated ITAM-SH2-interactions.

Protein-protein interactions are often dependent on the post-translational modification of one component of a complex. To facilitate the study of these interactions in signal transduction, we have developed the yeast tribrid system, a modification of the yeast two-hybrid system. We demonstrate that the interactions are dependent upon the presence of a tyrosine kinase, an SH2 domain and a tyrosine containing substrate. Using the gamma subunit of the high-affinity IgE receptor, Fc epsilon RI, this approach has been used to isolate a novel SH2-containing family member. The mRNA encoding this novel protein is differentially expressed in rat tissues. The yeast tribrid system can be readily adapted for the characterization of novel tyrosine kinases or substrates, as well as the study of protein-protein interactions which involve other post-translational modifications.

Spinal dural arteriovenous fistulas: evaluation with MR angiography.

PURPOSE: To show that postgadolinium three-dimensional time-of-flight MR angiography shows abnormal intradural vessels associated with spinal dural arteriovenous fistula better than routine MR imaging and provides screening information useful for subsequent diagnostic conventional angiography and/or posttreatment evaluation. METHODS: Precontrast and postcontrast MR imaging and MR angiograms, as well as subsequent digital subtraction angiograms, were obtained for eight patients with dural arteriovenous fistulas, diagnosed with digital subtraction angiography and verified with surgery. In four patients, MR studies also were obtained after surgery. RESULTS: All patients had cord hyperintensity of T2-weighted images and postgadolinium enhancement on T1-weighted images. Five had vessellike signal abnormalities in the subarachnoid space on MR. Abnormal intradural vessels were detected in all eight patients with MR angiography. Comparison with digital subtraction angiography revealed these vessels to be primarily enlarged veins of the coronal venous plexus on the cord surface. In six patients, the medullary vein draining the fistula was demonstrated, indicating the level of the fistula, later identified by digital subtraction angiography. After surgical obliteration of the fistula, the draining medullary vein and most or all of the abnormal coronal veins were no longer demonstrated, with decrease or resolution of cord hyperintensity on T2-weighted images. CONCLUSION: Postgadolinium, spinal MR angiography in cases of suspected dural arteriovenous fistula provides information about intradural veins that supplements the diagnostic value of the MR imaging results, facilitates the subsequent digital subtraction angiography study, and, in treated cases, reflects the success of surgery and/or embolization.

Role of glycosylation sites in the IgE Fc molecule.

The Fc region of immunoglobulin E (IgE) comprising the C epsilon 3 and C epsilon 4 domains (residues 329-547) is sufficient for binding to the high-affinity IgE Fc receptor (Fc epsilon RI alpha). Three potential N-linked glycosylation sites are present within the C epsilon 3 domain. To determine the effect of the glycosylation sites on IgE Fc synthesis and on Fc epsilon RI alpha binding, site-directed mutagenesis was performed. Mutant IgE Fc constructs were expressed in COS cells and analyzed for protein synthesis and secretion, and Fc epsilon RI alpha binding activity. We find that only N371 and N394 are glycosylated, and that the residues surrounding the glycosylation site at N394 are required for Fc epsilon RI alpha binding activity.

Functional characterization of the signal transduction events mediated by Fc epsilon RI alpha and gamma chimeric receptors.

Chimeric receptors containing the Fc epsilon RI alpha and gamma subunit domains were constructed, stably transfected into RBL-2H3 cells, and characterized for the biochemical events which are elicited upon receptor aggregation. Chimeric receptors containing the extracellular (EC) domain of the human Fc epsilon RI alpha subunit, or the EC domain of the p55 subunit of the interleukin-2 receptor were fused to the human Fc epsilon RI gamma subunit transmembrane and cytoplasmic (CT) domains or only the CT domain. The chimeras generated included alpha/gamma/gamma, I/gamma/gamma, alpha/I/gamma or I/I/gamma. The results indicate that both the Fc epsilon RI alpha EC domain and the Fc epsilon RI alpha CT domain are essential for signalling.

Characterization of the human IgE Fc-Fc epsilon RI alpha interaction.

A significant amount of progress has been achieved on characterizing the interaction of the IgE Fc molecule with the Fc epsilon RI alpha. However, there is yet no definitive structural information which precisely defines the nature of this interaction. It is clear that this information will only be provided by the resolution of the X-ray crystallographic structures of the IgE Fc molecule, the Fc epsilon RI alpha subunit extracellular domain, and the IgE Fc-Fc epsilon RI alpha complex. It is anticipated that these structures will be determined in the near future, and that they may provide some insight into the development of potential therapeutics effective in the management of IgE-mediated allergic diseases.

Identification of sites on the human Fc epsilon RI alpha subunit which are involved in binding human and rat IgE.

The high affinity IgE Fc receptor (Fc epsilon RI), found on mast cells and basophils, is a tetrameric receptor complex. The extracellular portion of the Fc epsilon RI alpha subunit consists of two immunoglobulin-like domains and binds IgE in the absence of the other subunits. To localize the high affinity IgE binding site within the Fc epsilon RI alpha subunit, we generated a series of chimeric receptor constructs where one of the two immunoglobulin-like domains was either deleted or substituted with those from the human Fc gamma RIIIA alpha or the rat Fc epsilon RI alpha subunit. The chimeric receptors were monitored for their capacity to bind human and rat IgE, and their reactivity with different antireceptor antibodies. Domain I substitutions maintained high affinity human IgE binding. Domain II substitutions resulted in a total loss of both human and rat IgE binding. Single-domain alpha subunits could not bind IgE, suggesting that both extracellular domains are required for proper protein folding or IgE binding. To further localize the IgE binding sites, homolog-scanning mutagenesis was performed. At least three independent regions of domain II encompassing residues 118-129, 136-150, and 148-162 were required for IgE binding. Our results suggest that domain II of the human Fc epsilon RI alpha confers most of the important contributions to the binding of the human IgE Fc molecule, whereas domain I of the rat Fc epsilon RI alpha makes important contributions to the binding of rat IgE.

Autoantibodies against the high-affinity IgE receptor as a cause of histamine release in chronic urticaria.

BACKGROUND: Most urticarias are induced by vasoactive mediators such as histamine released from mast cells. Although mast cells are activated by allergens through cross-linking of cell-surface--bound IgE, this mechanism does not appear to explain most cases of chronic urticaria, which, when allergic, infectious, drug-induced, or physical causes cannot be identified, are classified as idiopathic. METHODS: We recruited 26 patients with chronic idiopathic urticaria, in whom intradermal injection of autologous serum caused a wheal-and-flare response. Serum from four patients that induced marked histamine release from basophils from a donor with very low serum IgE levels was studied with respect to the IgE dependence of the histamine release, the activity of the IgG fractions, and the neutralizing effect of a recombinant preparation of the soluble extracellular domain of the alpha subunit of the high-affinity IgE receptor (sFc epsilon RI alpha). RESULTS: The histamine-releasing activity of the serum was abolished by passive sensitization of basophils with myeloma IgE, enhanced after dissociation of IgE by treatment with lactic acid, and induced by IgG fractions from the serum of all four patients. Preincubation of the serum and isolated IgG with sFc epsilon RI alpha resulted in almost complete neutralization. CONCLUSIONS: Histamine-releasing IgG autoantibodies against the alpha subunit of the high-affinity IgE receptor are present in the circulation of some patients with chronic urticaria. Autoantibody-induced cross-linking of IgE receptors may be an important mechanism in the pathogenesis of chronic urticaria and other diseases mediated by mast cells.