The Neutrophil Respiratory Burst and Bacterial Digestion
in Crohn’s Disease
Bu’Hussain Hayee•Farooq Z. Rahman•
Jane Tempero•Sara McCartney•Stuart L. Bloom•
Anthony W. Segal•Andrew M. Smith
Received: 12 March 2010/Accepted: 9 September 2010/Published online: 9 October 2010
? Springer Science+Business Media, LLC 2010
immune defence against microbes, using the respiratory
burst (RB) to optimise killing and digestion. Previous
studies of the neutrophil RB in Crohn’s disease (CD) have
yielded conflicting results.
Superoxide production in response to phorbol-
myristyl acetate (PMA) was measured in neutrophils from
100 patients with CD compared to 50 healthy controls
(HCs) and 50 patients with ulcerative colitis (UC). A fur-
ther 22 CD and 10 HCs were studied using f-Met-Leu-Phe
(fMLP), and digestion of E. coli by neutrophils was also
The mean ± SEM PMA-stimulated RB (nmol
O2/106cells/min) was 10.86 ± 0.26 in HCs, 9.76 ± 0.23
in CD (P=0.02) and 10.04 ± 0.28 in UC (P=0.09 vs HC
and 0.47 vs CD). No significant effect of age, gender or
medication was observed. The RB in three patients with
presumed CD was found to be in the range expected in
patients with inherited neutrophil disorders. Stimulation
with fMLP was calcium dependent and attenuated in
patients on 5-ASA. Digestion of E. coli by neutrophils was
not different in HC vs CD (21.6 vs 20.53%, P=0.60).
The significant reduction in neutrophil RB in
CD does not appear to result in defective bacterial diges-
tion and is therefore unlikely play a major role in
Neutrophils are a key part of the innate
pathogenesis. Three patients in this cohort of patients with
presumed idiopathic CD were found to have a profound
defect of the neutrophil RB. A high index of suspicion for
such patients is prudent, as their prognosis can be improved
by altering or augmenting the conventional treatment reg-
imens employed for CD.
Digestion ? Crohn’s disease
Neutrophil ? Respiratory burst ? Superoxide ?
Neutrophil phagocytes are essential in the mounting and
resolution of the acute inflammatory response—acting as
the first line of cellular defense against invading microor-
ganisms . Their antimicrobial function is dependent on
the presence and action of digestive enzymes [2, 3], the last
being optimized by the respiratory burst (RB). Generated
by the membrane-bound NADPH oxidase enzyme com-
plex, the RB results in the production of superoxide (O2-).
The function of this enzyme complex is attenuated in
chronic granulomatous disease (CGD)  and glycogen
disorders associated with intestinal inflammation that is
indistinguishable from Crohn’s disease (CD), in 30–50% of
cases , reflecting a pathogenic role for innate immune
dysfunction in CD. A number of other monogenic disorders
of neutrophil function exist, but are less prevalent than CGD
and GSD-1b, do not generally affect NADPH oxidase
function, and are less commonly associated with intestinal
inflammation (with the notable exception of Chediak–
Given the prevalence of inflammatory bowel disease in
patients with inherited neutrophil disorders, it is possible
B. Hayee (&) ? F. Z. Rahman ? J. Tempero ?
A. W. Segal ? A. M. Smith
Department of Molecular Medicine, University College London,
WC1E 6JJ London, UK
S. McCartney ? S. L. Bloom
Department of Gastroenterology, UCLH NHS Foundation Trust,
London NW1 2BU, UK
Dig Dis Sci (2011) 56:1482–1488
that such patients present to Gastroenterology services
without a prior diagnosis of their underlying disorder, thus
having their intestinal inflammation classified as idiopathic
CD, and then treated accordingly.
Although the pathogenesis of CD still remains to be
fully elucidated, there is now increasing evidence that
innate immune dysfunction, and more specifically defec-
tive acute inflammation, plays a primary role [7, 8].
Impaired neutrophil influx to, and subsequent clearance of,
a bacterial stimulus in CD has been attributed to defective
secretion of pro-inflammatory cytokines by macrophages
[7, 9, 10]. When isolated in vitro, chemotaxis and migra-
tion in neutrophils from patients with quiescent CD appears
preserved [11, 12].
Previous studies of the neutrophil RB in CD, however,
have yielded conflicting results. Using a variety of methods
and stimuli, it has been shown either to be increased [13,
14], normal [15, 16], or reduced [17, 18]. In addition, the
ability of neutrophils in CD to kill Candida albicans has
been shown to be either reduced  or near-normal under
physiological conditions , with a normal level of
superoxide production in response to the same organism
. Varying inferences have therefore been made about
the pathogenic contribution of these cells.
There has been increasing interest in the impaired han-
dling of bacteria by antigen-presenting cells of the innate
immune system in CD [21, 22]. However, the ability of
neutrophils in CD to digest bacteria (their primary function
) has not been evaluated.
This study was performed on a large cohort of patients
with CD to determine whether the neutrophil respiratory
burst was attenuated and perhaps related to impaired bac-
Ethical Approval and Patient Selection
Full ethical committee approval was obtained for all
experiments in accordance with National Research Ethics
informed consent was obtained in all cases prior to inclu-
sion (Joint UCL/UCLH committee alpha, ref: 02/0324).
Patients attending the IBD clinics at UCLH NHS Foun-
dation Trust with quiescent inflammatory bowel disease
(aged 18–65) were selected. Quiescent disease was defined
as a Harvey-Bradshaw Index score of 3 or less. Blood tests
were only performed at physician discretion, and therefore
not all had CRP performed. However, where performed,
and in all patients with perianal disease, this test was
normal (\5.0 mg/l). Patients with perianal disease met the
additional criteria of being on no antibiotics within
2 months of inclusion and having no radiological evidence
of perianal sepsis within 2 months (or at time of last
scanning–whichever was the nearer), and no draining fis-
tulae or seton in situ at the time of sampling. In general,
patients on no medication were preferred, but those on
stable doses of mesalamine (5-ASA) (for 3 months prior to
inclusion) were also examined. Doses of immunosuppres-
sants or infliximab, where prescribed, had been stable for at
least 6 months prior to sampling and patients on infliximab
therapy were sampled immediately prior to regular infusion
(within 10 min).
Briefly, neutrophils were isolated from peripheral venous
blood (heparinized 6 U/ml) by gradient centrifugation
(Lymphoprep?, Axis-Shield) and hypotonic lysis to remove
red blood cells. Neutrophils were resuspended at required
concentrations in phosphate-buffered saline (PBS; Gibco).
cytochrome-c (Sigma) in a final volume of 1 ml were
equilibrated to 37?C in a temperature-controlled spectro-
photometer (UV-3,000, Shimadzu Corp). The RB was
induced by the addition of either 1 lg/ml phorbol myristyl
acetate (PMA) or 10 nM f-Met-Leu-Phe (fMLP; both from
Sigma). The color change thus produced at 549 nm was used
cells/min) using a pre-determined formula . With fMLP,
results were compared to a second, paired reaction mixture
containing 2 mM calcium chloride prior to stimulation.
(2 9 106)in PBSwith 0.54 mg/ml
Preparation of Bacteria for Digestion Assay
A clinical reference strain of E. coli (NCTC 10418) was
labeled in culture with35S-methionine, washed to remove
unabound label and killed by UV irradiation. Bacteria were
then counted, aliquoted, snap-frozen, and stored until use at
-70?C. On the day of use, the required amount of bacteria
was opsonized with de-salted pooled human IgG (Octa-
gam; Octapharma, UK) resuspended in PBS (Gibco).
In triplicate reactions, 2 9 106neutrophils in PBS with
5 mM glucose were mixed with opsonized , radiolabeled
bacteria in a 1:2 ratio, and digestion left to occur in a
Dig Dis Sci (2011) 56:1482–14881483
(Corning) at 37?C and 200 rpm. After 60 min, the reaction
was stopped by cooling to 4?C. Undigested bacteria and
cells were removed by centrifugation at 14,000 rpm and
radioactivity counted in the supernatant and pellet. Diges-
tion was calculated from the amount of radioactivity in the
supernatant and expressed as a percentage of the total
counts in each reaction, as an average of the triplicate
reactions. The average variation in each assay around the
triplicate mean was 3.51 ± 0.68 units. Expressed as a
percentage of the triplicate mean, this was 26.0 ± 4.91 %.
Diphenylene iodonium (5 lM) (DPI; an inhibitor of
NADPH oxidase; Sigma) was used to inhibit digestion.
Data were subjected to the Shapiro–Wilk test for normality
and comparisons between two groups were made by Stu-
dent’s t test if normally distributed, and two-tailed Mann–
Whitney U test if not. For multiple group comparisons,
one-way ANOVA with Bonferroni post-test comparisons or
in Prism v4.0 (GraphPad). All results are expressed in tables
and figures as mean ± SEM unless otherwise specified.
Clinical and demographic details are shown in Table 1.
One hundred patients with CD were compared to 50
healthy controls and 50 patients with ulcerative colitis.
Mean values ± SEM for superoxide production (in nmol
O2/106cells/min) after stimulation with PMA (Fig. 1a)
were 10.86 ± 0.26 for healthy controls, 9.76 ± 0.23 for
patients with CD (p = 0.02) and 10.04 ± 0.28 for ulcera-
tive colitis (p = 0.09 vs. healthy controls and 0.47 vs. CD).
Nine CD and one UC patient released superoxide at levels
below the lowest healthy control subject. Of these, three
CD patients had superoxide production less than 30% that
of the healthy control mean, a level that might be expected
from patients with variant CGD  (Table 2).
No significant differences were observed between CD
patients divided according to disease location (Fig. 1b),
although the mean superoxide production in patients with
colonic disease (L2) was significantly lower than healthy
controls (p = 0.008).
Clinical and Demographic Influences
on the PMA-Induced RB
There were no significant differences in mean RB between
patients on no medication (10.31 ± 0.26), those on 5-ASA
(10.49 ± 0.32, p = 0.70), or those on immunosuppressant
or biologic therapy (9.79 ± 0.35, p = 0.24). As patients
with colonic disease had a significantly reduced RB com-
pared to healthy controls, they were analyzed further with
regard to possible influence of anti-inflammatory medica-
tions (which might be expected to attenuate the RB).
Although significantly different to healthy controls (p =
0.02), superoxide production in colonic CD patients on
no medication (9.19 ± 0.49) was no different to those on
5-ASA (10.03 ± 0.47, p = 0.28) or those on immuno-
suppressant or biologic therapy (9.80 ± 0.98, p = 0.56)
(Fig. 2a). Mean superoxide production was also unaffected
by gender, smoking, or age (r2= 0.00003, p = 0.99 for
HC; 0.01, 0.40 for CD) (Fig. 2b–d).
Table 1 Characteristics of study subjects
With PMA With fMLPFor bacterial digestion
HCCD UC HCCD HCCD
n = 50 10050 11221531
Gender (M:F)27:2340:6023:27 4:76:16 9:614:17
Mean age ± SD (years)41.7 ± 7.241.5 ± 15.145.9 ± 15.227.2 ± 4.639.6 ± 14.836.8 ± 8.242.5 ± 4.9
Current smokers9 20 121000
No medication–2714–7– 10
Mesalazine only– 49 25–14–11
L2– 40–– 10–6
IS immunosuppressant (azathioprine or methotrexate)
1484 Dig Dis Sci (2011) 56:1482–1488
Cells from 11 healthy controls and 22 patients with CD
were stimulated with 10 nM fMLP and the rate of super-
oxide measured (Fig. 3a). Neutrophils from CD patients
demonstrated diminished superoxide production compared
to healthy controls (p = 0.03). No significant differences
were observed between CD patients divided according to
disease location (Fig. 3b), although the mean superoxide
production in patients with ileal disease (L1) was signifi-
cantly lower than healthy controls (p = 0.03). There
appeared to be a relationship between an attenuated RB and
therapy with 5-ASA (mesalamine; open symbols Fig. 3b)
and previous studies have demonstrated a calcium-depen-
dent attenuation of the fMLP-induced RB in vitro with this
drug [28, 29]. In keeping with this observation, pre-incu-
bation of neutrophils with calcium corrected the attenuated
RB observed in patients on mesalamine (Fig. 3c).
Diminished neutrophil NADPH oxidase activity in patients
with disorders such as CGD and GSD-1D has been shown
to result in defective bacterial digestion. No significant
difference in bacterial digestion was observed between
healthy controls (n = 15) and patients with intestinal CD
(n = 16; p = 0.18, Fig. 4). Patients with perianal disease
(n = 15) were analyzed as a separate group due to the
strong link between inherited neutrophil disorders and
perianal sepsis  (although the RB was not found to be
abnormal in such patients). In all cases, digestion was
partially inhibited by the addition of 5 lM diphenylene
iodonium (DPI) a known inhibitor of the NADPH oxidase.
Neutrophils from the three patients with an abnormal RB
were found to have markedly diminished digestive capacity
compared to healthy controls and patients with CD.
Patients were subdivided according to phenotype and
medication to determine whether the differences observed
in RB affected bacterial digestion. Neither patients with
colonic disease nor those on 5-ASA demonstrated a sig-
nificant attenuation of digestive capability (Fig. 4b, c).
A profound defect of superoxide production in a number of
rare inherited disorders is strongly associated with intesti-
nal inflammation that is indistinguishable from CD  and
it has been well documented that the loss of NADPH
oxidase activity results in a reduced capacity of neutrophils
to digest phagocytosed microbes [4, 24]. We have recently
shown that CD patients have delayed bacterial clearance in
vivo as the result of diminished neutrophil recruitment .
While this could be linked to attenuated pro-inflammatory
cytokine secretion by macrophages, given previous con-
flicting descriptions of neutrophil dysfunction in CD, it was
important to determine whether a relevant intrinsic neu-
trophil defect could be demonstrated.
Our findings demonstrate that a small but significant
reduction in superoxide production in CD patients (par-
ticularly those with L2 disease) does not have an effect on
the digestive capability of the neutrophil. An interesting
finding arising from this study was the identification of
three patients with superoxide level in the region usually
Fig. 1 The PMA-stimulated
neutrophil RB. (a) The RB was
studied in response to PMA in
patients with CD and UC
compared to HCs (dotted line
denotes 30% of healthy control
mean; Labeled points
correspond to patients 1–3 in
Table 2). (b) Patients with CD
divided into groups according to
disease location (Montreal
classification). * p\0.05,
** p\0.01. All other
comparisons were non-
Table 2 Three patients with an abnormal respiratory burst
Age at diagnosis (years) 22.214.171.124
Current age (years)272321
Oral; L2L2Oral; L3
CN congenital neutropenia,apatient 3 previously treated with aza-
O2/106cells/min, on three separate occasions
Dig Dis Sci (2011) 56:1482–1488 1485
Fig. 2 Demographics and the
PMA-stimulated RB. The effect
of medication (a) gender (b),
and smoking status (c) on the
PMA-stimulated RB is shown in
patients with CD and HCs.
(d) Linear correlation of age and
PMA-stimulated RB with
patients 1–3 removed from
correlation (HC: dotted line;
r2= 0.000004, p = 0.99; CD:
solid line; r2= 0.03, p = 0.18)
Fig. 3 The fMLP-stimulated RB. (a) The RB was studied in response
to fMLP in patients with CD compared to HC. (b) Patients with CD
divided by disease location. Those on mesalamine are shown as open
symbols while those on no medication are denoted by filled symbols.
(c) Paired reactions were carried out with or without pre-incubation
with calcium (Ca2?). * p\0.05, ** p\0.01 (paired t test). All other
comparisons were non-significant
1486 Dig Dis Sci (2011) 56:1482–1488
associated with variant CGD. Subsequently, one patient has
been shown to have GSD-1b and another a congenital
neutropenia (CN) syndrome arising from a mutation in
G6PC3, both of which result in attenuated NADPH oxidase
activity and superoxide production. The third patient had
neither CGD nor the above two conditions and her defect
It is interesting that polymorphisms in NCF4, encoding
the NADPH oxidase component p40phox, have been asso-
ciated with CD, although only with ileal (L1) disease and
not in all Caucasian populations . The small, but sta-
tistically significant reduction, in mean superoxide pro-
duction by CD neutrophils may be the result of a
combination of influences across the group as a whole—
including age, medication, disease location, or underlying
genetic influences. When analyzed alone, however, the
influence of each of these factors (although NCF4 poly-
morphisms were not investigated in this study) is statisti-
The use of fMLP as a stimulus produced differing
results. Formyl peptide receptor (FPR) signaling induced
by fMLP results in activation of the NADPH oxidase
complex via a calcium-dependent pathway involving
phospholipase-C . The correction in RB observed when
neutrophils were pre-incubated with calcium implies that
patients with CD have reduced intracellular calcium stores
or have deficient mobilization of calcium in response to
fMLP. These results would be in keeping with the finding
of reduced intracellular calcium levels in patients with
quiescent CD on maintenance therapy with 5-ASA .
The capacity of 5-ASA and related compounds to inhibit
the fMLP-induced neutrophil RB in vitro [28, 29] also
appear to be reflected in our results. There are many neu-
trophil functions that are associated with or dependent on
the release of intracellular calcium [30, 31]. The ‘‘anti-
bacterial’’ consequences of the diminished fMLP-induced
RB are unclear since bacterial digestion appeared pre-
served. It is unlikely, therefore, that the variable response
to fMLP indicates a primary or pathogenically significant
Defective bacterial handling has been linked to the
development of CD [22, 32], particularly in epithelial cell
lines  or macrophages  from CD patients express-
ing ATG16L1 polymorphisms. While these polymorphisms
were not studied as part of this investigation, our results
demonstrate normal digestion in neutrophils from patients
with CD, suggesting that the CD-associated polymorphism
is more relevant to bacterial processing and antigen pre-
sentation in the above cells  rather than primary anti-
bacterial activity in neutrophils.
Three patients in this large adult cohort were found to
have an RB in the range expected in CGD. Further analysis
of these patients has been submitted elsewhere. Although
the proportion of adult patients with an undiagnosed pri-
mary neutrophil disorder is likely to be small, the advan-
tage of identifying such patients in a cohort with presumed
idiopathic CD is considerable. It may be advantageous to
‘‘screen’’ for neutrophil dysfunction in this cohort—
perhaps concentrating on patients with an early onset of
disease, a history of recurrent bacterial infections, ‘‘unu-
sual’’ (cf. disease location) or therapy-recalcitrant inflam-
matory bowel disease. Oral involvement was a feature of
all three patients with an abnormal RB and no patients with
CD and a ‘‘normal’’ RB had oral involvement at the time of
sampling. However, our method of classification may not
reliably detect patients with a previous history of oral
involvement at some stage in their disease course. A sim-
ilar ‘‘screening’’ study of the RB in patients with oral
involvement is planned.
The impaired clearance of bacteria seen in patients with
CD has been attributed to attenuated neutrophil influx 
and can now be thought of in terms of reduced neutrophil
Fig. 4 Digestion of E. coli by neutrophils. (a) The release of radio-
activity into the supernatant of a reaction with neutrophils and
35S-labeled E.coliwasmeasured inHC,CD, andpatientswithperianal
involvement (P). Patients 1–3 (Table 2) were analyzed as a separate
group. Patients with CD were subdivided according to (b) disease
location and (c) patients on treatment with 5-ASA were compared to
those on no treatment ** p\0.01. All other comparisons were non-
Dig Dis Sci (2011) 56:1482–14881487
number rather than function. Innate immunodeficiency in Download full-text
CD does not appear to involve an intrinsic defect of neu-
trophil antimicrobial function.
review of this manuscript. Neutrophil respiratory burst assays were
performed by BHH, JT, and FZR. Digestion experiments were per-
formed by BHH and AMS. Patients were identified and characterized
by BHH, FZR, SM, and SLB. This work was supported by a project
grant from the CGD Trust. BHH was awarded a project grant from the
Broad Medical Research Program. AWS was the recipient of a pro-
gram grant from the Wellcome Trust.
All authors participated in the drafting and
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