In this work we provide several lines of evidence that support a role for mast cells in the pathology of EGFR+ peripheral nerves. First, the number of mast cells increases in EGFR+ nerve at the same time as axon – Schwann cell interactions are disrupted and fibrosis develops. Second, genetic ablation of mast cells through W41 mating prevents pathologies associated with transgenic expression of EGFR. Third, reconstitution of blood-derived cells, including mast cells, to W41/W41/EGFR+ peripheral nerve via marrow engraftment restores pathologies. Fourth, treatment of EGFR+ mice with cromolyn inhibits mast-cell recruitment to peripheral nerves and pathological progression. Based on these experiments, we hypothesize that mast-cells that are normally resident in peripheral nerve are activated in response to the EGFR+ environment, possibly in response to increased Rantes, SCF and VEGF present at 4 and 5 weeks of age, and recruit additional mast-cell progenitors from bone marrow, which exacerbates EGFR+-initiated pathologies ().
Model for the development of EGFR+ pathology
mice are Kit hypomorphs, and Ryan et al.
showed that Kit is expressed in an MPNST cell line derived from an NF1 patient, but not in normal human Schwann cells (Ryan et al., 1994
). Therefore the loss of the nerve pathology phenotype in W41
might result from effects on Schwann cells. Restoration of the phenotype by bone marrow reconstitution, which restores normal mast cells but not Schwann cells, makes this explanation unlikely.
Mast cells arrive in wild-type and EGFR+
nerve at the same time in development (4 weeks) (); although mast cells are known constituents of peripheral nerve, to our knowledge, this is the first study that has documented when they arrive at this tissue. Mast-cell numbers do not increase in EGFR+
nerves until 6 weeks of age. We have shown previously that mRNAs encoding several potential mast-cell chemoattractants are upregulated in adult EGFR+
nerve (Ling et al., 2005
), and show in this study that mRNAs encoding Rantes, SCF and VEGF are transiently upregulated in EGFR+
nerve at 4 and 5 weeks of age. Yang et al.
showed that SCF is secreted by Nf1
−/− Schwann cells and that it is a potent migratory stimulus for Nf1
+/− mast cells (Yang et al., 2003
). SCF also inhibits mast cell chemotaxis, thereby contributing to their accumulation and enhancement of function (Sawada et al., 2005
). A role for SCF in recruitment of mast cells to EGFR+
nerve is consistent with our data, but other factors might also play a role.
SCF was reported to directly trigger mast-cell degranulation (Taylor et al., 1995
), although recent studies call this result into question (Gilfillan and Tkaczyk, 2006
). Our data showing that cromolyn treatment prevents nerve pathology supports the idea that mast-cell degranulation, mediated by SCF or other factors, is crucial in the EGFR+
phenotype. Cromolyn also caused a profound decrease in the number of mast cells recruited to EGFR+
peripheral nerves. Because cromolyn is thought to block mast-cell degranulation, we suggest that mast cells in EGFR+
nerves degranulate in response to the EGFR+
environment, and that the mast-cell mediators that are released contribute to increased recruitment of mast cells to nerves (). However, additional work is necessary to definitely demonstrate mast-cell degranulation in this model and identify factors that might trigger degranulation.
We can exclude a cromolyn effect on other bone marrow-derived cells that contain granules because eosinophils, neutrophils and basophils were not observed in nerves. It is also unlikely that cromolyn affects nerve cells directly because the nerves of wild-type, control animals treated with cromolyn were morphologically similar to the nerves of either untreated or carrier-injected wild-type animals. However, because the exact mechanism of action of cromolyn is not known, we can not completely rule out effects on other cell types. It is also possible that other cell types are necessary intermediates between Schwann cells and mast cells.
Mast cells can release mediators from preformed granules by exocytosis; these mediators include proteases, granule amines, proteoglycans, heparin, NGF and serotonin (Leon et al., 1994
; Gurish and Austin, 2001
; Maurer et al., 2003
; Crivellato et al., 2004
). Any of the >20 molecules released from mast-cell granules might be responsible for disruption of EGFR+
nerves. Although NGF and serotonin are attractive candidates because Schwann cells express their receptors (Taniuchi et al., 1986
; Gaietta et al., 2003
), in preliminary experiments we found that blocking serotonin receptors did not affect EGFR+
nerve pathology (not shown). The local environment can regulate the composition of mast-cell granules (Galli et al., 2005
), and it is possible that the Schwann cell environment in EGFR+
mice results in the de novo
synthesis of mast-cell mediators that are responsible for the pathological progression in EGFR+
nerves. We also cannot exclude piecemeal degranulation in the EGFR+
This work describes novel roles for mast cells in nerve homeostasis and pathology. A pivotal link between inflammation and carcinogenesis is being unraveled (Coussens et al., 1999
; Balkwill and Coussens, 2004
; de Visser et al., 2005
), and the classical view of mast cells as effectors in allergy and IgE-mediated immune response is becoming outmoded as essential, non-immunological roles for mast cells are identified (Crivellato and Ribatti, 2005
; Galli et al., 2005
). Mast cells might have roles in the development of other nerve pathologies in which Schwann cells lose contact with axons, including peripheral nerve tumor formation in NF1, Guillain-Barré syndrome and Wallerian degeneration (Tanzola et al., 2003
; Dines and Powell, 1997
). Our findings contribute to this newly emerging body of work and support a vital role for mast cells in the development of Schwann cell-mediated nerve pathology.