BACKGROUND
eosinophilic granulomatosis with polyangiitis  is a rare disease characterized by allergic granulomatosis and necrotizing vasculitis developing after the appearance of peripheral and tissue eosinophilia  <cit> .
asthma is present in  <dig> to 100 % of egpa patients and is the cardinal feature of egpa.
asthma may precede systemic vasculitis by approximately 8 years and in some cases by more than 30 years .
eosinophilic infiltrations, such as those found in chronic eosinophilic pneumonia  or eosinophilic gastroenteritis , or both, precede systemic vasculitis in half of all patients with egpa  <cit> .
some patients with asthma complicated by cep are likely to develop egpa if the cep is left untreated  <cit> .
however, some patients in whom asthma is complicated by cep do not develop egpa.
in our retrospective cohort study, we found that the clinical manifestations of asthma occurring in the pre-vasculitic phase of the disease were severe, or the percentage of eosinophils in the peripheral blood of pre-egpa asthmatics at the first hospital visit was high, but bronchial hyperresponsiveness to acetylcholine in these pre-vasculitic patients was slight  <cit> .
we subsequently confirmed that maintenance of regulatory t cell  numbers in asthma patients with cep may inhibit egpa development via the action of cytokines, such as il- <dig> and il- <dig>  produced by cd4+cd25+ and cd4+cd25− t cells, respectively  <cit> .
however, there is no clear mechanism that explains why some cep patients with asthma develop egpa and some do not.
th <dig> cells play important roles in the immune response, depending on their stage of differentiation, be it induction, amplification, or stabilization.
in mice, transforming growth factor beta  induces the development of inducible treg  cells by inducing the expression of the transcription factor forkhead box p <dig>  and the production of interleukin -17a by cd4+ t cells.
il- <dig>  il-1β, tumor necrosis factor alpha, and il- <dig>  together with tgf-β, promote th <dig> differentiation or polarization  <cit> .
some reports have suggested that th <dig> cells are associated with vasculitis, as occurs in giant cell arteritis  <cit> , henoch-schönlein purpura  <cit> , antineutrophil cytoplasmic antibody -associated vasculitis  <cit> , granulomatosis polyangiitis or wegener’s granulomatosis  <cit> , and egpa  <cit> .
intercellular adhesion molecule- <dig>  and vascular cell adhesion molecule - <dig> are expressed predominantly on the surfaces of endothelial cells and play an essential role in local leukocyte recruitment to the vessel wall, adhesion of leukocytes to the endothelium, migration, and extravasation  <cit> .
an enhanced level of soluble vcam- <dig>  is a marker of endothelial cell activation in anca-associated vasculitides  <cit> .
surface expression of icam- <dig>  cd11b, and cd <dig> on eosinophils in the peripheral blood is increased in egpa patients  <cit> .
vascular endothelial growth factor  is secreted by endothelial cells and pericytes in response to hypoxia.
it induces angiogenesis and microvascular hyperpermeability  <cit> .
serum vegf levels are associated with disease activity, for example in anca-associated vasculitis  <cit>  and systemic vasculitis  <cit> .
gastrointestinal tract involvement occurs in  <dig> to 92 % of patients with egpa  <cit> .
characteristic symptoms in egpa patients are abdominal pain, nausea or vomiting, diarrhea, hematochezia or melena, and hematemesis.
gastroduodenal ulceration has been detected endoscopically in  <dig>  of  <dig> patients with systemic vasculitis  and colorectal ulceration in  <dig> , but in one study histologic signs of necrotizing vasculitis or granuloma could not be found in a number of small specimen  <cit> .
patients with egpa who have severe gastrointestinal tract involvement, such as surgical abdomen, can develop peritonitis, bowel perforation, gastrointestinal ischemia or infarction, and intestinal occlusion, which carry a poor prognosis; mesenteric vasculitis may occur, leading to gastrointestinal ulceration, ischemia, and perforation  <cit> .
five-year survival rates in surgical patients with sv  have ranged from  <dig> to 56 %  <cit> .
colonoscopic findings include mucosal erythema  <cit>  and ulceration  <cit> .
however, in egpa specially, the incidence of another minor endoscopic signs such as red flare, and edematous change is not known.
moreover, to our knowledge, there have been no published investigations of the pathology of necrotizing vasculitis with infiltration other than eosinophils or granuloma.
there were various eosinophilic gastrointestinal diseases shown by drugs or parasitic infection, inflammatory bowel disease, reflux esophagitis, coeliac disease, microscopic and infectious colitis, sv , and polyarteritis nodosa  <cit> .
it is not known whether the intestinal endoscopic findings characteristics of eg in the asthmatic phase precede sv in patients with egpa.
moreover, the pathological differences in the colon submucosa between patients with egpa and those with cep or other types of eg have not yet been investigated.
here, we examined the pathological differences related to numbers of eosinophils and amount of edema in the colon submucosa between patients with egpa and those with cep.
we also analyzed the relationship between pathological features and adhesion molecule or growth factor expression and the th <dig> response.
RESULTS
clinical findings and treatment
there was no significant difference between patients with egpa and those with cep in terms of sex, atopic or non-atopic, presence or absence of allergic rhinitis or atopic dermatitis, age at time of onset of asthma, severity of asthma, and daily inhaled corticosteroid dose for treatment of asthma .
in five of  <dig> patients with cep, eosinophilic pneumonitis improved without the patient taking systemic corticosteroids .
however, all patients with egpa received systemic corticosteroids .
the initial dose of prednisolone and the proportion of patients who used immunosuppressants or intravenous immunoglobulin  were significantly higher in patients with egpa than those in patients with cep .
our analysis of organ involvement revealed no significant differences between the two groups in terms of the rates of respiratory system involvement  and the rates of involvement of the liver, gall bladder, and pancreas.
egpa patients had significantly higher rates of multiple polyneuropathy and myocardial, renal, skin, muscle, joint, and cns involvement than cep patients .table  <dig> characteristics and therapies of patients with eosinophilic granulomatosis with polyangiitis  or chronic eosinophilic pneumonia
p
renal involvementc
aza azathioprine, cfc-bdp chlorofluorocarbon-propelled beclomethasone dipropionate, csa ciclosporin, cyc cyclophosphamide, ics inhaled corticosteroid, ivig intravenous immunoglobulin, ns not significant
all values are expressed as means ±  <dig> sd
values of p <  <dig>  were considered statistically significant
atwo-way anova employing a repeated-measures test to explore the significance of differences between any two groups
bchi-squared testing revealed no significant differences in frequency between the two groups
crenal involvement including protein uria or eosinophils in urin or glomerular nephritis or nephrosis or renal dysfunction
asthma severity was according to gina guide lines
gastrointestinal tract characteristics in egpa and cep patients
the incidence of clinical symptoms related to both the upper and lower abdomen did not differ between the two groups .
there were various findings in the colonic mucosa  as well as the gastro-duodenum mucosa  in egpa patients .
in contrast, there were only slight changes in the colonic mucosa, such as red flare, in patients with cep .
however, the overall incidence of positive colonic mucosal findings on endoscopy did not differ between the two groups.
in contrast, positive gastroscopic findings were more common in egpa patients than those in cep patients .fig.
<dig> endoscopic findings in the large intestine in patients with eosinophilic granulomatosis with polyangiitis   or chronic eosinophilic pneumonia  .
arrows show ulcer , erosion , dark red sign , and various red flares .
the number in bright green indicated a region biopsied.
egpa patients had subjective symptoms related to the endoscopic findings in the lower digestive organs , but there were no subjective symptoms in patients with cep
counts and percentages of white blood cells , eosinophils, and th <dig> cells, and serum icam- <dig>  vcam- <dig>  and vegf levels
the numbers of wbcs and eosinophils in the peripheral blood at disease onset were significantly higher in egpa patients than in cep patients .
serum ige radioimmunosorbent assay values did not differ between the two groups.table  <dig> comparison of peripheral blood biomarkers or pathological findings in the large intestinal mucosa between patients with eosinophilic granulomatosis with polyangiitis  and those with chronic eosinophilic pneumonia
p
p
anca antineutrophil cytoplasmic antibodies, icam- <dig> intercellular adhesion molecule- <dig>  mpo myeloperoxidase, ns not significant, pr <dig> proteinase  <dig>  rist radioimmunosorbent, vcam- <dig> vascular cell adhesion molecule- <dig>  vegf vascular endothelial growth factor, wbcs white blood cells
all values are expressed as means ± sd
astatistical comparisons performed with the wilcoxon matched-pairs t-test
btwo-way anova with repeated measures between groups
cchi-squared testing revealed no significant differences between groups
values of p <  <dig>  were considered statistically significant
*egpa at onset vs. cep at onset, p <  <dig>
**egpa at onset vs. cep at onset, p <  <dig>
none of the cep patients was positive for mpo- or pr3-anca.
the serum icam- <dig> level at onset was greater in egpa patients than in cep patients , but this was not the case for the serum vcam- <dig> and vegf levels.
in egpa patients, serum icam- <dig>  vcam- <dig>  and vegf levels were significantly higher at onset than at remission, but those in cep patients did not change between onset and improvement.
the percentage of cd4+ t cells producing il- <dig>  at disease onset was significantly higher in patients with egpa than in those with cep .
the percentage of th <dig> cells in egpa patients was significantly lower at remission than at disease onset , but there was no change in this percentage in cep patients between onset and improvement .
pathological findings in the colon
we could not find any necrotizing vasculitis or granuloma in small size samples of submucosa from patients with either egpa or cep.
at disease onset there were significantly more eosinophils in the colonic submucosa of egpa patients than of cep patients  .
submucosal eosinophil counts in egpa were significantly lower at remission than at onset .
the localization of submucosal edema varied within each patient .
the basement membrane-to-crypt interval and crypt-to-crypt distance reflected the degree of edema in the colonic submucosa.
the basement membrane-to-crypt and crypt-to-crypt distance at disease onset were significantly greater in egpa patients than in cep patients  .
in cep patients, there were few, if any, edematous changes in the submucosa.
the basement membrane-to-crypt and crypt-to-crypt distance in egpa patients were significantly shorter at remission than at onset  .fig.
<dig> pathological findings in the large intestine in patients with eosinophilic granulomatosis with polyangiitis   or chronic eosinophilic pneumonia  .
evidence of bleeding was present in the submucosa .
the number of eosinophils in the submucosa was higher in patients with egpa  than in those with cep .
edematous changes  were more severe in patients with egpa than in those with cep
fig.
<dig> assessment of number of eosinophils and edema in colonic submucosa in a pathology specimen from a patient with eosinophilic granulomatosis with polyangiitis.
blue arrows indicate eosinophils.
red arrows show crypt-to-crypt distance.
yellow arrows show basement membrane-to-crypt distance.
all values are expressed as means ± sd in table 2
correlation between eosinophil counts in peripheral blood and colonic submucosa in patients with egpa or cep
the number of peripheral blood eosinophils in egpa patients at both onset and remission was significantly correlated with the eosinophil count in the submucosa  , but this was not the case in cep patients at onset  .fig.
<dig> correlations between eosinophils count  in the colonic submucosa and eosinophil count in the peripheral blood  or the basement membrane-to-crypt distance  in patients with eosinophilic granulomatosis with polyangiitis .
the basement membrane-to-crypt distance reflected edema in the colonic submucosa.
closed circles: mucous membrane of colon biopsied at the onset of egpa.
open circles: mucous membrane of colon biopsied in remission after treatment with corticosteroids with or without immunosuppressants.
correlation coefficients  and p values were calculated by using spearman’s rank correlation test
correlation between eosinophil count in colonic submucosa and basement membrane-to-crypt interval in patients with egpa or cep
the number of eosinophils in the colonic submucosa of egpa patients at both onset and remission was significantly correlated with the basement membrane-to-crypt distance  , but this was not the case in cep patients at onset  .
similarly, the number of eosinophils in the colonic submucosa of egpa patients was significantly correlated with the crypt-to-crypt distance , but this was not the case in cep patients at disease .
the number of eosinophils in the peripheral blood of egpa patients at onset and remission was significantly correlated with the crypt-to-crypt distance , but again this was not the case in cep patients at disease onset .
correlations between percentage of th <dig> cells in peripheral blood and eosinophil count in submucosa, crypt-to-crypt interval, and basement membrane-to-crypt interval in patients with egpa
the percentage of th <dig> cells in the peripheral blood of egpa patients at onset and remission was significantly correlated with the submucosal eosinophil count  , with the crypt-to-crypt distance  , and with the basement membrane-to-crypt distance  .fig.
<dig> correlations between the percentage of cd4+ t cells producing il- <dig> in peripheral blood and eosinophil count in the colonic submucosa ; the crypt-to-crypt distance ; the basement membrane-to-crypt distance , the icam- <dig> level ; the vcam- <dig> level ; and the vegf level  in the sera of patients with eosinophilic granulomatosis with polyangiitis .
closed circles: mucous membrane of colon biopsied at onset of egpa.
open circles: mucous membrane of colon biopsied in remission after treatment with corticosteroids with or without immunosuppressants.
correlation coefficients  and p values were calculated by using spearman’s rank correlation test
correlations between percentage of th <dig> cells in peripheral blood and serum icam- <dig>  vcam- <dig>  and vegf levels in patients with egpa
the percentage of th <dig> cells in the peripheral blood of egpa patients at onset and remission was significantly positively correlated with the serum icam- <dig> level   and inversely correlated with the serum vegf level  , but it was not correlated with the serum vcam- <dig> level  .
the number of submucosal eosinophils was significantly correlated with the serum icam- <dig> level , but not with the serum vcam- <dig> or vegf level .
the number of eosinophils in the peripheral blood was significantly correlated with the serum icam- <dig> level  and vcam- <dig> level , but not with the serum vegf level .
the basement membrane-to-crypt distance was significantly correlated with the serum icam- <dig> level  but not with the serum vcam- <dig> or vegf level .
discussion
the 5-year survival rate in egpa is  <dig> to 97 % .
the prognosis of egpa patients and the extent of mortality from the disease are associated with disease severity, as assessed by using the five-factor scores developed in  <dig>  <cit>  and  <dig>  <cit> .
in addition, myocardial involvement  <cit>  and the presence of gastrointestinal disease  <cit>  are independent negative prognostic factors.
however, the gastrointestinal pathology of egpa has not yet been fully characterized.
moreover, to our knowledge, differences in eg have not been investigated between patients with egpa at disease onset and patients with asthma preceding the development of sv in the form of egpa.
among the various eosinophilic gastrointestinal diseases  <cit> , there is less submucosal edema in ulcerative colitis, crohn’s disease, and collagenous colitis  <cit>  than we saw here in our egpa patients.
until now there has been a lack of detailed information on pathological findings in the colonic submucosa.
in our egpa patients, we expected to find eosinophilia but were surprised to find no necrotizing vasculitis or granuloma.
the presence of edematous change was also previously unreported.
the eosinophil count in the submucosa was significantly correlated with the eosinophil count in the blood and with the basement membrane-to-crypt interval in patients with egpa , but not in patients with cep .
the lack of correlation in the latter might have been caused by the fact that in these patients the colon was biopsied only once , whereas in egpa patients the colon was biopsied twice .
all patients with asthma did not suffer eosinophilic gastrointestinal disorder.
and there were an infiltration of eosinophils extravascular area in patients with asthma and without any gastrointestinal symptoms.
moreover there was an infiltration of number of eosinophils extra vascular extravascular tissue in patients with having more than  <dig> number of eosinophils in peripheral blood  <cit> .
there were some reports related to type  <dig> cytokine and egpa  <cit> .
however there was little biomarkers distinguishing between egpa and another hypereosinophilic syndrome  <cit> .
we showed here that th <dig> cell production decreased at remission in patients with egpa.
previously, we showed that the count of itreg cells producing il- <dig> was reduced in egpa patients at disease onset and upon relapse, but increased when the disease was inactive  <cit> .
here, we investigated foxp3+/cd4+ t cells as natural treg cells in patients with egpa or cep.
we confirmed that the percentage of th <dig> cells was inversely correlated with the number of foxp3+/cd4+ t cells  .
the number of foxp3+/cd4+ t cells was significantly correlated with the number of eosinophils in the colonic submucosa  , but unlike the th <dig> cell count it was not correlated with the interval between basement membrane and crypt or between crypt and crypt .
this result indicated that the th <dig> cell count but not the treg cell count reflected the occurrence of pathological changes related to vasculitis.
after treatment of egpa patients, the serum icam- <dig> and vcam- <dig> levels decreased significantly, whereas the serum vegf level increased significantly .
the serum icam- <dig> level was significantly correlated with the vcam- <dig> level  .
we found previously that multiple polyneuropathy and cardiac dysfunction improved after ivig administration  <cit> , and the numbers of treg cells  increased for two years  <cit> .
maternal sera obtained after ivig treatment, and polyclonal ivig, decrease constitutive and cytokine-induced icam- <dig> and vcam- <dig> expression on vascular endothelial cells ex vivo  <cit> .
the combination of ivig and methylprednisolone generally has a greater suppressive effect on mrna expression and on the production of vcam- <dig>  il-1β, and vegf on endothelial cells derived from human umbilical vein  <cit> .
ivig-induced inhibition of angiogenesis has an inhibitory effect on vegf-mediated blood perfusion in the ischemic limb in a mouse model  <cit> .
however, the positive clinical effects of high-dose ivig on muscle function in patients with refractory inflammatory active myositis in the form of polymyositis or dermatomyositis are not accompanied by any effects on icam- <dig> and vcam- <dig>  <cit> .
we considered that treatment with various combinations of corticosteroids, immunosuppressants, and ivig induced clinical remission, decreased icam- <dig> and vcam- <dig> levels, and increased vegf levels.
the sicam- <dig> level was high in egpa patients at disease onset, but the svcam- <dig> level varied widely and was relatively high at the time of onset of egpa.
an increase in th <dig> cell numbers likely reflects active vasculitis  <cit> , and an increase in the number of eosinophils or wbcs might induce upregulation of the expression of icam- <dig> or vcam- <dig> on vascular endothelial cells; inflammatory cells such as eosinophils and lymphocytes, including th <dig> cells, then migrate extravascularly into the colonic submucosa.
an increase in serum vegf level might have contributed to neovascularization.
we showed the correlation with th <dig> cells in peripheral blood and icam- <dig> or vegf in serum.
we speculated the decrease of the number of th <dig> cells after the treatment of steroid and immunosuppressant might affect the decrease of icam- <dig> and the increase of vegf.
these results were reflected to an inhibition of migration inflammatory cells and to promote neovascularization.
CONCLUSIONS
in summary, we clarified the pathological and immunological differences between patients with egpa and those with cep.
we expect that this clarification will help us to diagnose egpa in the early stages and to start treatment early, thus giving patients the chance of a good prognosis.

 eosinophilic granulomatosis with polyangiitis is a rare disease characterized by allergic granulomatosis and necrotizing vasculitis developing after the appearance of peripheral and tissue eosinophilia. asthma is present in to 100 % of egpa patients and is the cardinal feature of egpa. asthma may precede systemic vasculitis by approximately 8 years and in some cases by more than 30 years. eosinophilic infiltrations, such as those found in chronic eosinophilic pneumonia or eosinophilic gastroenteritis, or both, precede systemic vasculitis in half of all patients with egpa. some patients with asthma complicated by cep are likely to develop egpa if the cep is left untreated. however, some patients in whom asthma is complicated by cep do not develop egpa. in our retrospective cohort study, we found that the clinical manifestations of asthma occurring in the pre-vasculitic phase of the disease were severe, or the percentage of eosinophils in the peripheral blood of pre-egpa asthmatics at the first hospital visit was high, but bronchial hyperresponsiveness to acetylcholine in these pre-vasculitic patients was slight. we subsequently confirmed that maintenance of regulatory t cell numbers in asthma patients with cep may inhibit egpa development via the action of cytokines, such as il- and il- produced by cd4+cd25+ and cd4+cd25− t cells, respectively. however, there is no clear mechanism that explains why some cep patients with asthma develop egpa and some do not. th cells play important roles in the immune response, depending on their stage of differentiation, be it induction, amplification, or stabilization. in mice, transforming growth factor beta induces the development of inducible treg cells by inducing the expression of the transcription factor forkhead box p and the production of interleukin -17a by cd4+ t cells. il- il-1β, tumor necrosis factor alpha, and il- together with tgf-β, promote th differentiation or polarization. some reports have suggested that th cells are associated with vasculitis, as occurs in giant cell arteritis, henoch-schönlein purpura, antineutrophil cytoplasmic antibody -associated vasculitis, granulomatosis polyangiitis or wegener’s granulomatosis, and egpa. intercellular adhesion molecule- and vascular cell adhesion molecule - are expressed predominantly on the surfaces of endothelial cells and play an essential role in local leukocyte recruitment to the vessel wall, adhesion of leukocytes to the endothelium, migration, and extravasation. an enhanced level of soluble vcam- is a marker of endothelial cell activation in anca-associated vasculitides. surface expression of icam- cd11b, and cd on eosinophils in the peripheral blood is increased in egpa patients. vascular endothelial growth factor is secreted by endothelial cells and pericytes in response to hypoxia. it induces angiogenesis and microvascular hyperpermeability. serum vegf levels are associated with disease activity, for example in anca-associated vasculitis and systemic vasculitis. gastrointestinal tract involvement occurs in to 92 % of patients with egpa. characteristic symptoms in egpa patients are abdominal pain, nausea or vomiting, diarrhea, hematochezia or melena, and hematemesis. gastroduodenal ulceration has been detected endoscopically in of patients with systemic vasculitis and colorectal ulceration in, but in one study histologic signs of necrotizing vasculitis or granuloma could not be found in a number of small specimen. patients with egpa who have severe gastrointestinal tract involvement, such as surgical abdomen, can develop peritonitis, bowel perforation, gastrointestinal ischemia or infarction, and intestinal occlusion, which carry a poor prognosis; mesenteric vasculitis may occur, leading to gastrointestinal ulceration, ischemia, and perforation. five-year survival rates in surgical patients with sv have ranged from to 56 %. colonoscopic findings include mucosal erythema and ulceration. however, in egpa specially, the incidence of another minor endoscopic signs such as red flare, and edematous change is not known. moreover, to our knowledge, there have been no published investigations of the pathology of necrotizing vasculitis with infiltration other than eosinophils or granuloma. there were various eosinophilic gastrointestinal diseases shown by drugs or parasitic infection, inflammatory bowel disease, reflux esophagitis, coeliac disease, microscopic and infectious colitis, sv, and polyarteritis nodosa. it is not known whether the intestinal endoscopic findings characteristics of eg in the asthmatic phase precede sv in patients with egpa. moreover, the pathological differences in the colon submucosa between patients with egpa and those with cep or other types of eg have not yet been investigated. here, we examined the pathological differences related to numbers of eosinophils and amount of edema in the colon submucosa between patients with egpa and those with cep. we also analyzed the relationship between pathological features and adhesion molecule or growth factor expression and the th response.  clinical findings and treatment there was no significant difference between patients with egpa and those with cep in terms of sex, atopic or non-atopic, presence or absence of allergic rhinitis or atopic dermatitis, age at time of onset of asthma, severity of asthma, and daily inhaled corticosteroid dose for treatment of asthma. in five of patients with cep, eosinophilic pneumonitis improved without the patient taking systemic corticosteroids. however, all patients with egpa received systemic corticosteroids. the initial dose of prednisolone and the proportion of patients who used immunosuppressants or intravenous immunoglobulin were significantly higher in patients with egpa than those in patients with cep. our analysis of organ involvement revealed no significant differences between the two groups in terms of the rates of respiratory system involvement and the rates of involvement of the liver, gall bladder, and pancreas. egpa patients had significantly higher rates of multiple polyneuropathy and myocardial, renal, skin, muscle, joint, and cns involvement than cep patients.table characteristics and therapies of patients with eosinophilic granulomatosis with polyangiitis or chronic eosinophilic pneumonia p  renal involvementc aza azathioprine, cfc-bdp chlorofluorocarbon-propelled beclomethasone dipropionate, csa ciclosporin, cyc cyclophosphamide, ics inhaled corticosteroid, ivig intravenous immunoglobulin, ns not significant all values are expressed as means ±  sd values of p <  were considered statistically significant atwo-way anova employing a repeated-measures test to explore the significance of differences between any two groups bchi-squared testing revealed no significant differences in frequency between the two groups crenal involvement including protein uria or eosinophils in urin or glomerular nephritis or nephrosis or renal dysfunction asthma severity was according to gina guide lines gastrointestinal tract characteristics in egpa and cep patients the incidence of clinical symptoms related to both the upper and lower abdomen did not differ between the two groups. there were various findings in the colonic mucosa as well as the gastro-duodenum mucosa in egpa patients. in contrast, there were only slight changes in the colonic mucosa, such as red flare, in patients with cep. however, the overall incidence of positive colonic mucosal findings on endoscopy did not differ between the two groups. in contrast, positive gastroscopic findings were more common in egpa patients than those in cep patients.fig. endoscopic findings in the large intestine in patients with eosinophilic granulomatosis with polyangiitis or chronic eosinophilic pneumonia. arrows show ulcer, erosion, dark red sign, and various red flares. the number in bright green indicated a region biopsied. egpa patients had subjective symptoms related to the endoscopic findings in the lower digestive organs, but there were no subjective symptoms in patients with cep counts and percentages of white blood cells, eosinophils, and th cells, and serum icam- vcam- and vegf levels the numbers of wbcs and eosinophils in the peripheral blood at disease onset were significantly higher in egpa patients than in cep patients. serum ige radioimmunosorbent assay values did not differ between the two groups.table comparison of peripheral blood biomarkers or pathological findings in the large intestinal mucosa between patients with eosinophilic granulomatosis with polyangiitis and those with chronic eosinophilic pneumonia p p anca antineutrophil cytoplasmic antibodies, icam- intercellular adhesion molecule- mpo myeloperoxidase, ns not significant, pr proteinase rist radioimmunosorbent, vcam- vascular cell adhesion molecule- vegf vascular endothelial growth factor, wbcs white blood cells all values are expressed as means ± sd astatistical comparisons performed with the wilcoxon matched-pairs t-test btwo-way anova with repeated measures between groups cchi-squared testing revealed no significant differences between groups values of p <  were considered statistically significant *egpa at onset vs. cep at onset, p <  **egpa at onset vs. cep at onset, p <  none of the cep patients was positive for mpo- or pr3-anca. the serum icam- level at onset was greater in egpa patients than in cep patients, but this was not the case for the serum vcam- and vegf levels. in egpa patients, serum icam- vcam- and vegf levels were significantly higher at onset than at remission, but those in cep patients did not change between onset and improvement. the percentage of cd4+ t cells producing il- at disease onset was significantly higher in patients with egpa than in those with cep. the percentage of th cells in egpa patients was significantly lower at remission than at disease onset, but there was no change in this percentage in cep patients between onset and improvement. pathological findings in the colon we could not find any necrotizing vasculitis or granuloma in small size samples of submucosa from patients with either egpa or cep. at disease onset there were significantly more eosinophils in the colonic submucosa of egpa patients than of cep patients. submucosal eosinophil counts in egpa were significantly lower at remission than at onset. the localization of submucosal edema varied within each patient. the basement membrane-to-crypt interval and crypt-to-crypt distance reflected the degree of edema in the colonic submucosa. the basement membrane-to-crypt and crypt-to-crypt distance at disease onset were significantly greater in egpa patients than in cep patients. in cep patients, there were few, if any, edematous changes in the submucosa. the basement membrane-to-crypt and crypt-to-crypt distance in egpa patients were significantly shorter at remission than at onset.fig. pathological findings in the large intestine in patients with eosinophilic granulomatosis with polyangiitis or chronic eosinophilic pneumonia. evidence of bleeding was present in the submucosa. the number of eosinophils in the submucosa was higher in patients with egpa than in those with cep. edematous changes were more severe in patients with egpa than in those with cep fig. assessment of number of eosinophils and edema in colonic submucosa in a pathology specimen from a patient with eosinophilic granulomatosis with polyangiitis. blue arrows indicate eosinophils. red arrows show crypt-to-crypt distance. yellow arrows show basement membrane-to-crypt distance. all values are expressed as means ± sd in table 2 correlation between eosinophil counts in peripheral blood and colonic submucosa in patients with egpa or cep the number of peripheral blood eosinophils in egpa patients at both onset and remission was significantly correlated with the eosinophil count in the submucosa, but this was not the case in cep patients at onset.fig. correlations between eosinophils count in the colonic submucosa and eosinophil count in the peripheral blood or the basement membrane-to-crypt distance in patients with eosinophilic granulomatosis with polyangiitis. the basement membrane-to-crypt distance reflected edema in the colonic submucosa. closed circles: mucous membrane of colon biopsied at the onset of egpa. open circles: mucous membrane of colon biopsied in remission after treatment with corticosteroids with or without immunosuppressants. correlation coefficients and p values were calculated by using spearman’s rank correlation test correlation between eosinophil count in colonic submucosa and basement membrane-to-crypt interval in patients with egpa or cep the number of eosinophils in the colonic submucosa of egpa patients at both onset and remission was significantly correlated with the basement membrane-to-crypt distance, but this was not the case in cep patients at onset. similarly, the number of eosinophils in the colonic submucosa of egpa patients was significantly correlated with the crypt-to-crypt distance, but this was not the case in cep patients at disease. the number of eosinophils in the peripheral blood of egpa patients at onset and remission was significantly correlated with the crypt-to-crypt distance, but again this was not the case in cep patients at disease onset. correlations between percentage of th cells in peripheral blood and eosinophil count in submucosa, crypt-to-crypt interval, and basement membrane-to-crypt interval in patients with egpa the percentage of th cells in the peripheral blood of egpa patients at onset and remission was significantly correlated with the submucosal eosinophil count, with the crypt-to-crypt distance, and with the basement membrane-to-crypt distance.fig. correlations between the percentage of cd4+ t cells producing il- in peripheral blood and eosinophil count in the colonic submucosa; the crypt-to-crypt distance; the basement membrane-to-crypt distance, the icam- level; the vcam- level; and the vegf level in the sera of patients with eosinophilic granulomatosis with polyangiitis. closed circles: mucous membrane of colon biopsied at onset of egpa. open circles: mucous membrane of colon biopsied in remission after treatment with corticosteroids with or without immunosuppressants. correlation coefficients and p values were calculated by using spearman’s rank correlation test correlations between percentage of th cells in peripheral blood and serum icam- vcam- and vegf levels in patients with egpa the percentage of th cells in the peripheral blood of egpa patients at onset and remission was significantly positively correlated with the serum icam- level and inversely correlated with the serum vegf level, but it was not correlated with the serum vcam- level. the number of submucosal eosinophils was significantly correlated with the serum icam- level, but not with the serum vcam- or vegf level. the number of eosinophils in the peripheral blood was significantly correlated with the serum icam- level and vcam- level, but not with the serum vegf level. the basement membrane-to-crypt distance was significantly correlated with the serum icam- level but not with the serum vcam- or vegf level. discussion the 5-year survival rate in egpa is to 97 %. the prognosis of egpa patients and the extent of mortality from the disease are associated with disease severity, as assessed by using the five-factor scores developed in and. in addition, myocardial involvement and the presence of gastrointestinal disease are independent negative prognostic factors. however, the gastrointestinal pathology of egpa has not yet been fully characterized. moreover, to our knowledge, differences in eg have not been investigated between patients with egpa at disease onset and patients with asthma preceding the development of sv in the form of egpa. among the various eosinophilic gastrointestinal diseases, there is less submucosal edema in ulcerative colitis, crohn’s disease, and collagenous colitis than we saw here in our egpa patients. until now there has been a lack of detailed information on pathological findings in the colonic submucosa. in our egpa patients, we expected to find eosinophilia but were surprised to find no necrotizing vasculitis or granuloma. the presence of edematous change was also previously unreported. the eosinophil count in the submucosa was significantly correlated with the eosinophil count in the blood and with the basement membrane-to-crypt interval in patients with egpa, but not in patients with cep. the lack of correlation in the latter might have been caused by the fact that in these patients the colon was biopsied only once, whereas in egpa patients the colon was biopsied twice. all patients with asthma did not suffer eosinophilic gastrointestinal disorder. and there were an infiltration of eosinophils extravascular area in patients with asthma and without any gastrointestinal symptoms. moreover there was an infiltration of number of eosinophils extra vascular extravascular tissue in patients with having more than number of eosinophils in peripheral blood. there were some reports related to type cytokine and egpa. however there was little biomarkers distinguishing between egpa and another hypereosinophilic syndrome. we showed here that th cell production decreased at remission in patients with egpa. previously, we showed that the count of itreg cells producing il- was reduced in egpa patients at disease onset and upon relapse, but increased when the disease was inactive. here, we investigated foxp3+/cd4+ t cells as natural treg cells in patients with egpa or cep. we confirmed that the percentage of th cells was inversely correlated with the number of foxp3+/cd4+ t cells. the number of foxp3+/cd4+ t cells was significantly correlated with the number of eosinophils in the colonic submucosa, but unlike the th cell count it was not correlated with the interval between basement membrane and crypt or between crypt and crypt. this result indicated that the th cell count but not the treg cell count reflected the occurrence of pathological changes related to vasculitis. after treatment of egpa patients, the serum icam- and vcam- levels decreased significantly, whereas the serum vegf level increased significantly. the serum icam- level was significantly correlated with the vcam- level. we found previously that multiple polyneuropathy and cardiac dysfunction improved after ivig administration, and the numbers of treg cells increased for two years. maternal sera obtained after ivig treatment, and polyclonal ivig, decrease constitutive and cytokine-induced icam- and vcam- expression on vascular endothelial cells ex vivo. the combination of ivig and methylprednisolone generally has a greater suppressive effect on mrna expression and on the production of vcam- il-1β, and vegf on endothelial cells derived from human umbilical vein. ivig-induced inhibition of angiogenesis has an inhibitory effect on vegf-mediated blood perfusion in the ischemic limb in a mouse model. however, the positive clinical effects of high-dose ivig on muscle function in patients with refractory inflammatory active myositis in the form of polymyositis or dermatomyositis are not accompanied by any effects on icam- and vcam-. we considered that treatment with various combinations of corticosteroids, immunosuppressants, and ivig induced clinical remission, decreased icam- and vcam- levels, and increased vegf levels. the sicam- level was high in egpa patients at disease onset, but the svcam- level varied widely and was relatively high at the time of onset of egpa. an increase in th cell numbers likely reflects active vasculitis, and an increase in the number of eosinophils or wbcs might induce upregulation of the expression of icam- or vcam- on vascular endothelial cells; inflammatory cells such as eosinophils and lymphocytes, including th cells, then migrate extravascularly into the colonic submucosa. an increase in serum vegf level might have contributed to neovascularization. we showed the correlation with th cells in peripheral blood and icam- or vegf in serum. we speculated the decrease of the number of th cells after the treatment of steroid and immunosuppressant might affect the decrease of icam- and the increase of vegf. these results were reflected to an inhibition of migration inflammatory cells and to promote neovascularization.  in summary, we clarified the pathological and immunological differences between patients with egpa and those with cep. we expect that this clarification will help us to diagnose egpa in the early stages and to start treatment early, thus giving patients the chance of a good prognosis. 
we also examined the numbers of submucosal eosinophils and the basement membrane-to-crypt and crypt-to-crypt distance to evaluate edema in the colon submucosa at onset and remission in egpa and at onset in cep. chronic eosinophilic pneumonia or eosinophilic gastroenteritis, or both, with asthma precede the onset of eosinophilic granulomatosis with polyangiitis in half of all egpa patients. churg-strauss syndromeeosinophilic granulomatosis with polyangiitisintercellular adhesion molecule-1vascular cell adhesion molecule-1vascular endothelial growth factorissue-copyright-statement© the author 2015 nine of cep patients had symptoms or endoscopic findings. in egpa, peripheral blood th cells were significantly correlated with serum icam- level, colonic submucosal eosinophil count, and degree of edematous change; inversely correlated with serum vegf level; but not correlated with vcam- level. the mechanism of vasculitis in egpa appears related to increases in serum th cell numbers and icam- levels and decreases in vegf levels. we studied egpa patients and patients with cep but without egpa.
 chronic eosinophilic pneumonia or eosinophilic gastroenteritis, or both, with asthma precede the onset of eosinophilic granulomatosis with polyangiitis in half of all egpa patients. it is not known what determines whether patients with cep or with eg following asthma will develop egpa. methods we studied egpa patients and patients with cep but without egpa. we assayed serum icam- vcam- and vegf, and the percentage of peripheral blood cd4+ t cells producing il-, at both onset and remission. we also examined the numbers of submucosal eosinophils and the basement membrane-to-crypt and crypt-to-crypt distance to evaluate edema in the colon submucosa at onset and remission in egpa and at onset in cep.  nine of cep patients had symptoms or endoscopic findings. colonic submucosal eosinophil counts and edema in egpa at onset were greater than at remission or in cep at onset. th cells and serum icam- levels at onset were greater in egpa than in cep. in egpa, peripheral blood th cells were significantly correlated with serum icam- level, colonic submucosal eosinophil count, and degree of edematous change; inversely correlated with serum vegf level; but not correlated with vcam- level.  eosinophilia and colonic submucosal edematous change were greater in egpa than in cep. the mechanism of vasculitis in egpa appears related to increases in serum th cell numbers and icam- levels and decreases in vegf levels. keywords churg-strauss syndromeeosinophilic granulomatosis with polyangiitisintercellular adhesion molecule-1vascular cell adhesion molecule-1vascular endothelial growth factorissue-copyright-statement© the author 2015 