Deleterious Effects of IL-9–Activated Mast Cells and Neuroprotection by Antihistamine Drugs in the Developing Mouse Brain
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0031-3998/01/5002-0222 PEDIATRIC RESEARCH Vol. 50, No. 2, 2001 Copyright © 2001 International Pediatric Research Foundation, Inc. Printed in U.S.A. Deleterious Effects of IL-9–Activated Mast Cells and Neuroprotection by Antihistamine Drugs in the Developing Mouse Brain JULIANA PATKAI, BETTINA MESPLES, MARIE-ALIETTE DOMMERGUES, GAËLLE FROMONT, ELISABETH M. THORNTON, JEAN-CHRISTOPHE RENAULD, PHILIPPE EVRARD, AND PIERRE GRESSENS INSERM E-9935 and Service de Neurologie Pédiatrique, Hôpital Robert-Debré, 75019 Paris, France [J.P., B.M., M.A.D., P.E., P.G.], Service d’Anatomopathologie, Institut Mutualiste Montsouris, 75014 Paris, France [G.F.], Department of Veterinary Studies, University of Edinburgh, Edinburgh, EH8 9YL, U.K. [E.M.T.], and Ludwig Institute for Cancer Research and Experimental Medicine Unit, Université Catholique de Louvain, 1200 Brussels, Belgium [J.C.R.] ABSTRACT Elevated mean IL-9 serum levels have been observed in mice. IL-9 pretreatment had no significant effect on ibotenate- human neonates who will later develop cerebral palsy. In earlier induced excitotoxic brain lesions in mast cell–deficient P5 pups studies, using a newborn mouse model of excitotoxic lesions (WBB6F1/J kitW/W-v), whereas IL-9 exacerbated these lesions in mimicking those described in human cerebral palsy, we found the control littermates with normal mast cell populations. Finally, that IL-9 pretreatment exacerbated brain damage produced by cromoglycate or antihistamine drugs significantly reduced ibote- intracerebral injections of the glutamatergic analog ibotenate. nate-induced brain lesions in IL-9–treated Swiss pups. Taken to- Among its different cell targets, the Th2 cytokine IL-9 is a mast gether, these data suggest that recruitment of cerebral mast cells cell growth and differentiation factor that can cause mast cells to with histamine release may contribute to the exacerbation of neo- release various substances including histamine. In the present natal excitotoxic brain lesions produced by IL-9. Neuroprotective study, we sought to determine whether the deleterious effects of strategies targeting mast cells may be useful in some neonates at risk IL-9 in our mouse model were mediated by mast cells through for cerebral palsy. (Pediatr Res 50: 222–230, 2001) histamine release. All mouse pups were pretreated with intra- peritoneal injections of IL-9 or saline between postnatal days (P) P1 and P5. Immunohistochemistry for murine mast cell pro- Abbreviations tease-1 performed on P5 showed an increased density of labeled mMCP, murine mast cell protease cells in the neopallium of IL-9–treated Swiss pups as compared NMDA, N-methyl-D-aspartate with controls. Western blot analysis confirmed the increased P, postnatal day murine mast cell protease-1 brain content of IL-9–treated Swiss TNF-␣, tumor necrosis factor-␣ Pathophysiologic concepts for cerebral palsy have changed limited, levels of proinflammatory cytokines (IL-1-, IL-6, IL-8, over the last decade (1–7). Recent hypotheses have implicated and TNF-␣) were associated with one another, but no correlation multiple preconceptional and prenatal factors, such as hypoxia– was detected between IL-9 levels and the levels of the other perfusion failure, genetic factors, growth factor deficiency, and cytokines tested. Although further studies will be necessary to excess production of cytokines. In a striking retrospective study, confirm this initial observation, these data could suggest the mean levels of perinatal circulating cytokines—including IL-1, existence of a subpopulation of cerebral palsy patients character- ␣ IL-6, IL-8, IL-9, and TNF- —were higher in patients with sub- ized by increased levels of circulating IL-9 at birth. A better sequent occurrence of cerebral palsy than in control subjects (8). understanding of the mechanisms bridging IL-9 blood level ele- In that study, although the numbers of included patients were vation and brain damage might open up new therapeutic possi- bilities for preventing cerebral palsy in infants with high circulat- Received September 6, 2000; accepted December 12, 2000. ing IL-9 levels. Correspondence and reprint requests: Pierre Gressens, M.D., INSERM E 9935, Hôpital Robert-Debré, 48 Blvd Sérurier, F-75019 Paris, France; e-mail: [email protected] Intracerebral injection of ibotenate, a glutamate analog, to Supported by the INSERM, the Fondation pour la Recherche Médicale, the Fondation newborn mice produces histologic lesions mimicking the brain Grace de Monaco, and the Société d’Etudes et de Soins pour les Enfants Paralysés et lesions frequently associated with cerebral palsy, such as cystic Polymalformés. J.C.R. is a research associate with the Fonds National de la Recherche Scientifique (Belgium). periventricular leukomalacia and hypoxic-ischemic or ischemic- 222 MAST CELLS AND EXCITOTOXIC BRAIN LESIONS 223 like cortical damage (9–10). The thickness of the murine periven- Excitotoxic brain lesions. Ibotenate (Sigma Chemical Co, tricular white matter is much smaller than in humans, making St. Louis, MO, U.S.A.) is a glutamate analog with effects on some inferences between rodents and human potentially difficult. NMDA and metabotropic receptors, but not on ␣-3-amino- However, ibotenate-induced white matter lesions in newborn hydroxy-5-methyl-4-isoxazole or kainate receptors. Ibotenate mice mimic several aspects of the human cystic periventricular was diluted in PBS containing 0.02% acetic acid. The follow- leukomalacia, including their periventricular location, their initial ing protocol was used for ibotenate administration to mouse cystic appearance, their secondary evolution toward a glial scar, pups. All ibotenate injections were administered on P5. One and the discrete ontogenetic window of sensitivity of the periven- hour after the last injection of cytokines or PBS, intracerebral tricular white matter to ibotenate-induced damage (9). In this injections of 10 g of ibotenate were performed as previously well-characterized mouse model, pretreatment with proinflamma- described (9–11, 21–23). Briefly, after ether anesthesia of the tory cytokines (IL-1, IL-6, or TNF-␣) or with IL-9 increased the pups, the injections were given under a warming lamp using a extent of the excitotoxic brain lesions (11). These deleterious 26-gauge needle on a 50-L Hamilton syringe mounted on a effects of proinflammatory cytokines were mediated in part by the calibrated microdispenser. The needle was inserted 2 mm recruitment of brain macrophages/microglial cells. However, IL-9 under the external surface of the scalp skin in the frontoparietal had no detectable effects on this cell population, suggesting a area of the right hemisphere, 2 mm from the midline in the separate mechanism of action. lateral-medial plane, and 3 mm from the junction between the IL-9 is produced by activated Th2 lymphocytes and is active sagittal and lambdoid sutures in the rostrocaudal plane. Two on various immune cells including mast cells, B lymphocytes, 1-L boluses were injected 30 s apart. In all cases, the tip of T thymocytes, eosinophils, erythroid progenitors, and myeloid the needle reached the periventricular white matter (some progenitors (12). Mast cells are present in the brain, including animals injected with toluidine blue confirmed the correct the neopallium, where their numbers increase during early positioning of the needle). After the injections, the pups were postnatal development (13–17). Mast cells are derived from the returned to their dams. pluripotent hematopoietic stem cells in the bone marrow. Their Five days after intracerebral ibotenate injection (e.g. on differentiation is dependent on tissue production of stem-cell P10), the pups were decapitated, and their brains were fixed in factor and the expression of the corresponding KIT receptor on formaldehyde for 5 d. After embedding in paraffin, 15-m mast cell precursor cell surface. Mast cells can produce a sections were cut in the coronal plane, from the frontal to the variety of potentially toxic factors including histamine, sero- occipital pole. Every third section was stained with cresyl tonin, neutral proteases, cytokines, chemokines, and free rad- violet. This permits accurate and reproducible determination of icals (18). Mast cells have been implicated in the pathophysi- the maximal sagittal frontooccipital diameter (which is depen- ology of brain lesions associated with multiple sclerosis, dent on the number of sections containing the lesion and the Guillain-Barré syndrome, and Gayet-Wernicke encephalopa- width of each section) of both the cortical plate and periven- thy (19). Techniques for detecting mast cells include toluidine tricular white matter lesions. Here, we defined the maximal blue staining (heparin-containing granule labeling) and immu- diameter as the length of the lesion along the sagittal fronto- nohistochemistry with antibodies to various types of mast cell parietal axis. We used this measure as an index of lesion size. proteases. Immature mast cells enter the tissues, where they Throughout the study, two investigators blinded to the treat- differentiate, taking on phenotypic features (predominance of ment groups of the animals determined the size of the lesion in heparin-containing or serotoninergic granules, predominant ex- each pup. The numbers of pups in each experimental group pression of some proteases, etc.) dependent on tissue type (20). (see below) are shown in Table 1. In aggregate, these data suggest that mast cells may mediate Effects of IL-9 pretreatment on excitotoxic lesions. Mouse the deleterious effects of IL-9 on the neonatal brain. recombinant IL-9 (Endogen, Woburn, MA, U.S.A.), in a dose The goal of the present study was to investigate whether mast of 40 ng diluted in 5 L of PBS, was injected intraperitoneally cells mediate the ability of IL-9 pretreatment to exacerbate ibote- twice a day (between 0800 h and 1000 h and between 1800 h nate-induced lesions in our mouse model of neonatal excitotoxic and 2000 h) on P1 to P4 and once a day (between 0800 h and brain damage. Accordingly, we studied the effects of IL-9 pre- 1000 h) on P5 to Swiss mice (Janvier, Le Genest-St-Isle, treatment on density and phenotype of brain mast cells.