IL-3-producing basophils are required to exacerbate airway hyperresponsiveness in a murine inflammatory model.

BACKGROUND: Basophils are commonly associated with allergic responses because of their ability to produce large amounts of pro-Th2 cytokines and histamine. However, the mechanisms through which bone marrow-resident basophils (BMRB) become fully competent cytokine and histamine producers in response to IgE crosslinking are poorly understood. Here, we sought to determine the role of IL-3 in promoting pro-Th2 basophils. METHODS: BMRB and basophils exposed to IL-3 in vitro and in vivo were evaluated for their production of Th2 cytokines and histamine in response to FcεRI crosslinking on both protein and gene expression levels. In vivo relevance of our findings was assessed in a model of ovalbumin-induced allergic asthma using IL-3-deficient and wild-type mice in a protocol of adoptive basophil transfer. RESULTS: We show that BMRB and basophils previously exposed to IL-3 differ in their ability to generate cytokines (IL-4, IL-6, IL-13, and GM-CSF) and histamine in response to FcεRI crosslinking, reflecting two stages of maturation. Exposure to IL-3 initiated an autocrine loop of endogenous IL-3 production that enhanced histamine and cytokine production upon FcεRI crosslinking. This increased responsiveness required calcium flux and was dependent on calcineurin and store-operated calcium channels. Our findings are of pathophysiological relevance, as assessed by the failure of IL-3-deficient mice to develop airway hyperreactivity, which could be restored by adoptive transfer of IL-3-derived basophils recovered from wild-type mice. CONCLUSION: IL-3-dependent basophils promote Th2 allergic AHR, which designates the IL-3/basophil axis as a promising therapeutic target for the treatment of basophil-dependent asthma.

Activation of basophils by the double-stranded RNA poly(A:U) exacerbates allergic inflammation.

BACKGROUND: It is commonly acknowledged that asthma is exacerbated by viral infections. On the other hand, basophil infiltration of lung tissues has been evidenced postmortem in cases of fatal disease, raising the question of a possible link between these two observations. OBJECTIVES: Herein, we addressed the relationship between asthma exacerbation by viral infection and basophil activation and expansion by investigating how stimulation with the dsRNA polyadenylic/polyuridylic acid [poly(A:U)] affected basophil activities and recruitment in an allergic airway inflammation model. METHODS: The effect of dsRNA on basophils was assessed by measuring the cytokine levels produced upon stimulation. We used an OVA-induced experimental model of allergic asthma. Airway hyperreactivity, recruitment of infiltrating cells, and cytokine production were determined in the lung of mice having received poly(A:U), as compared with untreated controls. The exacerbating effect of basophils was assessed both by adoptive transfer of poly(A:U)-treated basophils and by their in vivo depletion with Ba103 antibody. RESULTS: We found that in vitro treatment with poly(A:U) increased basophil functions by inducing TH 2-type cytokine and histamine production, whereas in vivo treatment increased peripheral basophil recruitment. Furthermore, we provide the first demonstration for increased infiltration of basophils in the lung of mice suffering from airway inflammation. In this model, disease symptoms were clearly exacerbated upon adoptive transfer of basophils exposed to poly(A:U), relative to their unstimulated counterpart. Conversely, in vivo basophil depletion alleviated disease syndromes, thus validating the transfer data. CONCLUSIONS: Our findings provide the first evidence for airway inflammation exacerbation by basophils following dsRNA stimulation.

Histamine production during the anti-allograft response. Demonstration of a new lymphokine enhancing histamine synthesis.

Histamine production is greatly increased during culture of allograft recipient spleen cells in the presence of immunizing cells (secondary mixed leukocyte cultures [MLC]) as compared to that found in primary MLC (i.e., without previous allograft). This phenomenon appears after 24 h of culture and reaches its maximum at 48 h. Optimal increased histamine production is observed when MLC is performed with spleen cells removed from mice during rejection. This increased production of histamine during secondary MLC results from the action of a lymphokine: the histamine-producing cell stimulating factor (HCSF). This factor is released by T lymphocytes. Its production requires specific stimulation of the recipient lymphocytes because increase in histamine production during secondary MLC can be only observed when recipient cells are cultured with stimulating cells bearing at least one homology at K or D loci with immunizing cells. HCSF acts on a cell which is present in bone marrow, spleen, blood, and peritoneal cells but absent in thymus or lymph node cells. This target cell is found in the less-dense layer of a discontinuous Ficoll-gradient of bone marrow cells. HCSF is heat stable, destroyed by trypsin treatment, and has a molecular weight between 50,000 and 100,000. It acts on its target cells by increasing histidine decarboxylase activity.

Gut mucosal mast cells. Origin, traffic, and differentiation.

Gut mucosal mast cells (MMC), which are nearly absent in normal mice are abundant during nematode infection. In normal mice, study of MMC precursors (MMC-P: cells giving rise to MMC colonies in the presence of IL-3) show that: (a) their frequency, judged by limiting dilution is very high in bone marrow (BM) and gut, and very low in most lymphoid organs and thoracic duct lymph (TDL); (b) gut MMC-P are Thy-1- Lyt-1-2- and are not rapidly replicating; (c) they are the progeny of less differentiated BM MMC-P which are attracted from the blood to the gut mucosa by local factor(s), other than antigen and T cell factors (since normal amounts of gut MMC-P are found in germ-free, nude, and newborn mice). In mice bearing the Wehi 3 tumor (which releases enough IL-3 to produce detectable blood levels) spleen and mesenteric lymph nodes (LN) show increased MMC-P frequency, the greatest increase being in the gut and BM, where numerous differentiated MMC are found. In Nippostrongylus brasiliensis (Nb)-infested mice (known to develop a large, T cell-dependent, gut MMC infiltration), gut MMC-P proliferation is induced by IL-3 released from gut mucosal Thy-1+ Lyt-2- cells, whose in vitro IL-3 release capability is much higher than that of similar cells from normal mice. Both Nb-stimulated T blasts and proliferating MMC-P undergo cyclic traffic, migrating into the TDL and then seeding the whole length of the gut (a process which allows a widespread immune defense after a local antigenic stimulus). Experiments using 2-d interruption of this traffic and fetal gut grafts, suggest that the continuous homing of T blasts back to the gut which leads to permanent Nb-stimulated IL-3 release, is essential for the full maturation of MMC. Transfer experiments in the rat show that TDL circulating MMC-P rapidly mature into MMC when they home back to the Nb-infested gut. It is proposed that gut MMC arise after several stages of progressive differentiation of MMC-P, influenced both by IL-3 and unidentified gut factor(s).

Transforming growth factor beta1, in the presence of granulocyte/macrophage colony-stimulating factor and interleukin 4, induces differentiation of human peripheral blood monocytes into dendritic Langerhans cells.

Langerhans cells (LCs) are dendritic cells (DCs) that are present in the epidermis, bronchi, and mucosae. Although LCs originate in bone marrow, little is known about their lineage of origin. In this study, we demonstrate that in vitro LCs may originate from monocytes. Adult peripheral blood CD14+ monocytes differentiate into LCs (CD1a+, E-cadherin+, cutaneous lymphocyte-associated antigen+, Birbeck granules+, Lag+) in the presence of granulocyte/macrophage colony-stimulating factor, interleukin 4, and transforming growth factor beta1 (TGF-beta1). This process occurs with virtually no cell proliferation and is not impaired by 30 Gy irradiation. Selection of monocyte subpopulations is ruled out since monocyte-derived DCs can further differentiate into LCs. Our data suggest that in vivo LC differentiation may be induced peripherally, from a nonproliferating myeloid precursor, i.e., the monocyte, in response to a TGF-beta1-rich microenvironment, as found in the skin and epithelia. Therefore, the monocyte may represent a circulating precursor critical to the immune response in vivo.

Interleukin 7 induces preferential expansion of V beta 8.2+CD4-8- and V beta 8.2+CD4+8- murine thymocytes positively selected by class I molecules.

We analyzed the phenotype and V beta-T cell receptor (TCR) repertoire, together with interleukin 7 receptor (IL-7R) expression in unfractionated thymocytes stimulated in vitro with IL-7. This culture system results in a specific proliferation of mature thymocytes belonging to the CD3+CD4-, CD4+8-, and CD4-8+ subsets. IL-7 induced a preferential expansion of V beta 8.2+CD4-8- and V beta 8.2+CD4-8- thymocytes. This phenomenon is not observed in beta 2-microglobulin-deficient mice, showing that a fraction of CD4+8- thymocytes, enriched in V beta 8.2+ cells, is selected by class I molecules in normal mice, as are a large proportion of CD4-8- alpha beta TCR+ thymocytes. Our findings also establish that IL-7 plays a major role in the expansion of rare thymocyte subsets, which could exert important functions in inflammatory and immune responses.

Poly(A)-poly(U) induces circulating colony-stimulating activity resulting from interactions between endogeneous interleukin 6 and serum components.

The immunostimulant poly(A)-poly(U) induces a rapid enhancement of circulating colony-stimulating activity (CSA) in normal mice, culminating 2 h after i.v. injection. A dose of 200 micrograms per mouse is sufficient for a maximal effect. The colonies formed in response to sera from poly(A)-poly(U)-injected mice are mainly granulocytic with few macrophages. These sera are devoid of detectable interleukin 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF), but contain large amounts of interleukin 6 (IL-6) that are perfectly correlated with circulating CSA levels. Although, in our hands, IL-6 alone induces no colony formation in the standard methylcellulose colony assay, it is nevertheless requisite for this biological activity because 1) monoclonal antibodies against IL-6 strongly diminish colony formation in response to sera from poly(A)-poly(U)-injected mice, and 2) recombinant (r)IL-6 induces colonies when tested in combination with low amounts of normal murine serum. At the concentrations used (0.3%-2.5%), the latter has no or a very slight effect alone. Low amounts of hematopoietic growth factors, that is, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), GM-CSF, or IL-3 that are almost ineffective in the absence of IL-6 can replace normal serum. Taken together, these data suggest that circulating IL-6, induced by i.v. injection of poly(A)-poly(U), promotes colony formation by interacting with serum components that might be identical with hematopoietic growth factors present in normal serum at subliminal concentrations. Finally, the involvement of lipopolysaccharide (LPS) in this phenomenon has been ruled out by the use of the low responder strain of mice (C3H/HeJ) that leads to similar results.

Interleukin 3 induces histamine synthesis in the human hemopoietic system.

Recombinant human interleukin 3 (rhIL-3) induces an increase in histamine production by human bone marrow, fetal liver, and cord blood cells. This phenomenon, already significant after 3 days of incubation, is strikingly enhanced following either enrichment in immature cell subpopulations or CD8+ cell depletion. It results from an increase in histamine synthesis because of 1) the low level of histamine cell content before any incubation, 2) the parallel increase in both extra- and intracellular histamine levels in response to rhIL-3, and 3) the early IL-3-induced increase in L-histidine decarboxylase (HDC; EC 4.1.1.22) activity. Moreover, rhIL-3 has no similar activity on adult peripheral blood cells, suggesting that it might be specific to the hemopoietic system.

Concomitant histamine, interleukin 4, and interleukin 6 production by hematopoietic progenitor subsets in response to interleukin 3.

Murine interleukin 3 (IL-3) induces a strong, concomitant increase in histamine, interleukin 6 (IL-6), and interleukin 4 (IL-4) synthesis by progenitor-enriched bone marrow cell populations, whereas interleukin 2 (IL-2) or interferon-gamma (IFN-gamma) are undetectable. This phenomenon is observed between 4 and 12 h after exposure to the growth factor and attains maximal cytokine and histamine levels within 24 and 48 h, respectively. None of these mediators is produced by lymphoid populations such as lymph node cells or by granulocytes. Splenocytes secrete only low histamine and IL-6 levels, in accordance with the lower incidence of progenitors in the spleen, whereas total bone marrow cells generate substantial amounts of the three mediators even before enrichment. Histamine, IL-4-, and IL-6-producing cells copurify with immature cells and cannot be separated from each other throughout the sorting procedures used herein. They are concentrated in the low-density layers (buoyant density 1.069-1.086 g/cm3) of a discontinuous Ficoll gradient (less than 4% of the total bone marrow) together with the majority of hematopoietic progenitors (marrow-repopulating ability [MRA] cells, spleen colony-forming units [CFU-S] day-8 and day-12, granulocyte-macrophage colony-forming units [CFU-GM], and mast cell precursors). Their lightscatter characteristics are those of relatively large, granular cells. They do not belong to the most primitive stem cell subset (MRA and part of CFU-S day-12), but to a population with high mitochondrial activity identified by their important rhodamine retention (colony-forming unit cells [CFU-C], blast cells). In addition, we provide evidence that histamine, IL-4, and IL-6 do not depend on each other for their respective expression. Taken together, our data are consistent with the notion that in certain conditions, immature hematopoietic cells are a potent source of histamine and cytokines.

Carbohydrate does not modulate the in vivo effects of injected interleukin-3.

Murine interleukin-3 (IL-3) has extensive N-linked glycosylation. Experiments were performed to determine whether the T cell-derived glycosylated IL-3 differs in its biological activity in vivo when compared with a chemically synthesized form of nonglycosylated IL-3. Groups of mice were treated by intravenous injection with identical units of IL-3 bioactivity as determined in vitro in a cell-proliferation assay. Mice that were treated with seven 5000-unit doses of either form of IL-3, given in 12-hour intervals, showed a small but significant increase in the frequency of mast cell precursor cells in the spleen and of IL-3-responsive colony-forming unit cells (CFU-C). There was no difference in potency of glycosylated and nonglycosylated IL-3. Induction, by IL-3, of histidine decarboxylase in bone marrow and spleen cells was used as a second measure for IL-3 bioactivity. Both IL-3 preparations showed good in vivo histidine decarboxylase inducing activity; however, T cell-derived glycosylated IL-3 was significantly more effective than synthetic IL-3 in inducing the enzyme histidine decarboxylase in bone marrow and in spleen cells. Pharmacokinetic studies showed that chemically synthesized IL-3 was cleared about twice as fast as the T cell-derived IL-3 and that there may be some tissue trapping of glycosylated IL-3. The shorter in vivo half-life of nonglycosylated IL-3 appears to have significant pharmacological consequences on the short-term effect of inducing histidine decarboxylase activity, but not on the effect of the long-term treatment of IL-3 on stimulating the increase of hematopoietic progenitor cells.

Fas cross-linking mimics the inhibitory effect of anti-CD3 on IL-3-induced histamine and cytokine production by murine myeloid spleen cell precursors.

In the present study we investigated the effect of anti-CD3 stimulation on IL-3-induced histamine, IL-6, and IL-4 synthesis by murine hematopoietic precursor cells. These activities were strikingly decreased in splenocytes from mice that had received a single intravenous injection of 10 microg of anti-CD3 monoclonal antibody (mAb) 24 hours previously. A similar inhibition occurred after 24-hour in vitro stimulation of normal spleen cells with 1 microg/mL of anti-CD3 mAb. In both situations the inhibitory effect depended on T cell activation in that treatment with F(ab')2 fragments of anti-CD3 did not diminish secretion of histamine and cytokines. Cross-linking of Fas antigen on spleen cells mimicked the action of anti-CD3, provided that interferon (IFN)-gamma was present during the incubation period. Substantial amounts of this cytokine were detected in spleen cell supernatants, which were able to replace recombinant IFN-gamma during Fas receptor cross-linking. This effect was entirely mediated by IFN-gamma, as assessed by its neutralization in the presence of anti-IFN-gamma mAbs. In contrast to splenocytes, bone marrow cells responded normally to IL-3 after in vivo or in vitro stimulation with anti-CD3. They were also not affected by combined treatment with anti-Fas mAb and IFN-gamma. Together, our data support the notion that the decrease in IL-3-induced histamine and IL-6 production by splenocytes pretreated with anti-CD3 is mediated, at least in part, by Fas/FasL interactions, suggesting that the activity of extramedullary myeloid precursor cells can be modulated by molecules involved in apoptosis.

Murine hematopoietic progenitor cells produce IL-6 in response to IgE.

Similarly to interleukin-3 (IL-3), IgE is capable of inducing IL-6 production by murine bone marrow cells (BMC). IgE responder cells do not belong to the mature bone marrow compartment but coenrich with hematopoietic progenitors in the low-density fraction of a discontinuous Ficoll gradient. A significant enhancement of IL-6 production is observed after a 4-hour stimulation, reaching a maximum between 24 and 48 hours and is preceded by increased mRNA expression. The effect of IgE on IL-6 production is not mediated by IL-3 since it is not modified by anti-IL-3 antibodies. Upon a 4-hour exposure to IgE or IL-3, a similar percentage of progenitor-enriched BMC expresses IL-6 mRNA (3.9 and 5.4%, respectively, as determined by in situ hybridization), which is not further increased by a combination of both stimuli. IgE and IL-3 responder cells also cannot be distinguished on the basis of size, internal structure, and rhodamine (Rh) retention. The BMC sorted in the most fluorescent Rhbright subset (approximately 0.2% of total BMC) produce 30- to 40-fold more IL-6 than unfractionated cells and are similarly enriched for CFU-cells (CFU-C). The most primitive cells concentrated in the Rhdull fraction do not express this biological activity. The sorted Rhbright population does not contain mature mast cells/basophils or monocytes, and IL-6 is not produced in response to Fc epsilon RI cross-linkage after presensitization with IgE.

Modulation of histidine decarboxylase activity and cytokine synthesis in human leukemic cell lines: relationship with basophilic and/or megakaryocytic differentiation.

In the present study, we show that UT7D1 cells, derived from the pluripotent cell line UT7, express high levels of histidine decarboxylase (HDC) mRNA spontaneously. These cells conserve the ability to differentiate into megakaryocytes upon stimulation with PMA, while greatly increasing their HDC activity. We provide evidence that enhanced HDC activity reflects the basophil rather than the megakaryocytic differentiation potential of UT7DI cells. Indeed, in addition to HDC mRNA, they express spontaneously several other mRNA coding for molecules present in basophils (FcepsilonRI, CCR3, IL-4Ralpha, IL-5Ralpha). Furthermore, the basophil antigen Bsp-1 is displayed on the surface of some UT7D1 cells in response to PMA concomitantly with increased histamine synthesis and mRNA expression of typical basophil-derived cytokines (IL-6, IL-4, and IL-13). Nevertheless, PMA cannot sustain the differentiation of this lineage, because mRNAs for basophil markers gradually diminish during long-term culture, whereas molecules associated with the megakaryocytic lineage remain prominent. In support of the notion that HDC activity is not related with megakaryopoiesis, we show that PMA-induced CD41 expression and PDGF transcription occurs in the K562 cells, though neither HDC mRNA nor any known basophil marker are expressed in these conditions. In contrast, all these markers are expressed in the basophilic leukemia cell line KU812F. Interestingly, the megakaryocytic cell line HEL produces also substantial amounts of histamine and expresses FcepsilonRI, thus revealing its basophil differentiation potential. HEL as well as KU812F need not be stimulated with PMA to react with Bsp-1 mAb, suggesting that they are more engaged into the basophil differentiation scheme than UT7D1. Other leukemic cell lines unrelated to the megakaryocyte or basophil lineage, like HL60 and U937 do neither synthesize histamine nor express basophil markers before or after PMA stimulation. To our knowledge, this is the first evidence for a factor-dependent cell line with megakaryocyte/basophil bipotentiality with which early stages of basophil commitment can be analyzed.

Evidence for bidirectional histamine transport by murine hematopoietic progenitor cells.

Murine hematopoietic progenitor cells synthesize substantial amounts of histamine in response to IL-3 or calcium ionophore. They also take up extracellular histamine by an active transport system. In the present study we demonstrate that this system mediates both influx and efflux of histamine. Indeed, MR16155 and thioperamide, the two H3 antagonists which are most effective in inhibiting histamine uptake, likewise diminish the release of preloaded histamine from bone marrow cells. These compounds also inhibit the release of histamine which has been newly synthesized by hematopoietic progenitors in response to IL-3 or calcium ionophore, as assessed by the accumulation of the mediator inside the cells in the presence of the antagonists. The potency of different histamine receptor antagonists as inhibitors of histamine release increases with their capacity to block histamine uptake.

Superoxide-induced deimination of arginine in hematopoietic cells.

Murine bone marrow cells can produce citrulline directly from L-arginine without intermediate ornithine. An L-arginine-dependent biochemical pathway synthesizing L-citrulline and nitrate, coupled to an effector mechanism has also been recently demonstrated in murine cytotoxic activated macrophages. We show herein that L-citrulline synthesis in murine bone marrow cells can be induced by the generation of superoxide. It can take place in an arginine-free medium, suggesting the implication of a superoxide-dependent peptidyl arginine deiminase.

Binding of histamine H3-receptor antagonists to hematopoietic progenitor cells. Evidence for a histamine transporter unrelated to histamine H3 receptors.

Hematopoietic progenitor cells can take up histamine or release IL-3-induced histamine through a bi-directional transport system that is blocked by H3-receptor antagonists. In the present study we demonstrate a correlation between the affinity of various H3-receptor antagonists and their potency as inhibitors of histamine uptake. All compounds that blocked histamine uptake also inhibited IL-3-induced histamine release. Yet, classical H3 receptors are not involved in this biological activity, since highly specific histamine H3-receptor agonists neither alter histamine uptake nor affect the release of endogenous histamine synthesized in response to IL-3. Furthermore, the inhibitory effect of H3-receptor antagonists on histamine uptake was not reversed by the agonists. Unlike H3-receptor antagonists, the agonists did not displace the binding of the labeled antagonist iodoproxyfan.

Macrophage arming factor release by allografted mouse lymphocytes stimulated by phytohermgglutinin.

Spleen cells from a C57BL/6 mouse allografted with DBA/2 skin may release a macrophage arming factor when stimulated with phytohemagglutinin. This in vitro nonspecific release is observed only when the recipient cells are collected during a limited period preceding or coinciding with graft rejection. The phenomenon disappears if the skin allograft has been removed before cell collection. It appears if an i.v. injection of donor cells is given to the recipient after graft removal, on the day preceding cell collection. These data suggest that this in vitro apparently nonspecific macrophage arming factor release by phytohemagglutinin-stimulated recipient cells may in fact disclose a previous specific in vivo immune cell triggering by graft antigens.

Reduction of morbidity and cytokine release in anti-CD3 MoAb-treated mice by corticosteroids.

In keeping with the in vitro mitogenic properties of anti-CD3 MoAbs, the first injections of anti-CD3 are invariably responsible for an in vivo cellular activation. This activation induces a massive cytokine release in the circulation (TNF, IFN gamma, IL-2, IL-6, and IL-3). Paralleling this release, a severe clinical reaction occurs in OKT3-treated patients and in 145 2C11-treated mice. Corticosteroids both in vitro and in vivo inhibit the production of several cytokines involved in the anti-CD3 reaction. A single 1 mg hydrocortisone dose was administered to 145 2C11-treated mice according to different kinetics schedules. When given 1 hr prior to the anti-CD3 MoAb, hydrocortisone exerted a beneficial effect on the mouse physical reaction. Hypothermia was totally abrogated at the 4-hr time point. Diarrhea decreased by 50%. Hypomotility improved although not significantly. This improvement correlated with a major modification in the anti-CD3 pattern of cytokine release. At the 90-min blood withdrawal time point cytokine serum levels showed a 100% decrease for IFN gamma, an 88% decrease for IL-6, and 85% decrease for IL-2, and a 75% decrease for TNF. At 4 hr IL-2 serum levels were diminished by 65%; IL-6, IL-3, and IFN gamma serum levels were comparable to controls; and, interestingly, TNF was still detected, whereas it has already disappeared when 145 2C11 was administered alone. Importantly, when given more than 1 hr prior to anti-CD3 injection, corticosteroids were ineffective. To conclude, high doses of corticosteroids must be given with a precise kinetics--i.e. 1 hr prior to anti-CD3 MoAb--to achieve their maximal beneficial effect in the prevention of the anti-CD3 reaction.

The emergence of resistant strains of Acinetobacter baumannii: clinical and infection control implications.

A prospective study was undertaken to determine colonization rates, susceptibility profiles, and outcomes in patients with clinical isolates of Acinetobacter baumannii. Fifty percent of patients became colonized with A. baumannii, and 29% of these patients had clinical and colonizing isolates with discordant susceptibility profiles, without apparent relation to antibiotic use. Barrier infection control measures are necessary to prevent nosocomial transmission.

Differential expression and tissue distribution of parkin isoforms during mouse development.

Mutations of the parkin gene are a cause of autosomal recessive juvenile parkinsonism. Although the parkin gene has been isolated from mouse, rat, and human, little is known about its expression in neural and nonneural tissues during development. In this study, we used a polyclonal antibody to a peptide downstream of the parkin ubiquitin domain to investigate (1) the differential expression of parkin isoforms in protein extracts from fetal and adult mouse tissues, and (2) the distribution of parkin in mouse fetal tissues at different developmental stages and in adult CNS tissues. By Western blot analyses, at least three isoforms of parkin of 22, 50, and 55 kDa were differentially expressed in mouse tissues. The p22 and p50 isoforms were found in fetal and adult mouse CNS tissues, while the p55 isoform was found only in adult tissues. The p50 isoform is the predominant form in both fetal and adult tissues. Immunolocalization in mouse fetuses showed that parkin was expressed only after neuronal differentiation. Although parkin was localized throughout the cytoplasm, the highest level of parkin was found in the neurites of both fetal and adult neurons.