RESEARCH ARTICLEOpen Access
B Lymphocyte intestinal homing in inflammatory
Caterina Defendenti1*, Piercarlo Sarzi-Puttini2, Silvia Grosso1, Annamaria Croce3, Olivia Senesi, Simone Saibeni3,
Simona Bollani4, Piero Luigi Almasio5, Savino Bruno3and Fabiola Atzeni2,6
Background: Inflammatory bowel disease (IBD) is thought to be due to an abnormal interaction between the host
immune system and commensal microflora. Within the intestinal immune system, B cells produce physiologically
natural antibodies but pathologically atypical anti-neutrophil antibodies (xANCAs) are frequently observed in
patients with IBD. The objective is to investigate the localisation of immunoglobulin-producing cells (IPCs) in
samples of inflamed intestinal tissue taken from patients with IBD, and their possible relationship with clinical
Methods: The IPCs in small intestinal, colonic and rectal biopsy specimens of patients with IBD were analysed by
means of immunofluorescence using polyclonal rabbit anti-human Ig and goat anti-human IgM. The B cell
phenotype of the IPC-positive samples was assessed using monoclonal antibodies specific for CD79, CD20, CD23,
CD21, CD5, l and ? chains. Statistical correlations were sought between the histological findings and clinical
Results: The study involved 96 patients (64 with ulcerative colitis and 32 with Crohn’s disease). Two different
patterns of B lymphocyte infiltrates were found in the intestinal tissue: one was characterised by a strong to
moderate stromal localisation of small IgM+/CD79+/CD20-/CD21-/CD23-/CD5±IPCs (42.7% of cases); in the other
(57.3%) no such small IPCs were detected in stromal or epithelial tissues. IPCs were significantly less frequent in the
patients with Crohn’s disease than in those with ulcerative colitis (p = 0.004).
Conclusion: Our findings suggest that different immunopathogenetic pathways underlie chronic intestinal
inflammation with different clinical expressions. The presence of small B lymphocytes resembling B-1 cells also
seemed to be negatively associated with Crohn’s disease. It can therefore be inferred that the gut contains an
alternative population of B cells that have a regulatory function.
Keywords: Inflammatory bowel disease, inflammation, mucosal immunity, lymphocytes, B1 cells, lymphocyte
Crohn’s disease (CD) and ulcerative colitis (UC) are
idiopathic inflammatory bowel disorders  attributable
to an abnormal immune response to bacterial antigens.
Deficiencies in anti-inflammatory and immunosuppres-
sive mechanisms are important to the development of
the disease, but the basic pathogenetic mechanisms are
still largely unknown .
Recently collated evidence supports the view that IBD
consists of disorders with distinct genetic, microbial and
environmental determinants that cluster into an UC or
CD phenotype . IBD is polygenic, and experimental
data suggest that a number of not mutually exclusive
pathways may contribute to the inflammatory cascades.
CD has been attributed to the mediation of Th1,
whereas UC shows a modified Th2 cytokine response
Recent findings suggest that tissue injury in IBD is
mediated by novel effector pathways, the most promi-
nent of which is the interleukin-23/Th17 axis .
* Correspondence: email@example.com
1Laboratory Unit, Fatebenefratelli Hospital, Milan, Italy
Full list of author information is available at the end of the article
Defendenti et al. BMC Immunology 2011, 12:71
© 2011 Defendenti et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
In both UC and CD, leukocyte recruitment is
increased and this provides a potential target for thera-
peutic inhibition . Effective defence against enteric
pathogens requires leukocytes to be appropriately
recruited and positioned in the gut to form an effective
mucosal immune system.
The majority of in vivo studies of mouse intestinal B
cells have shown that immunoglobulin-producing cells
(IPCs) participate in the intestinal immune system by
producing physiological intraluminal IgA and natural
antibodies . Pathologically atypical anti-neutrophil
antibodies (xANCAs) may also be detected during the
course of IBD .
Although > 80% of the B cells in the murine model
are found in gut lymphoid tissue, it is actually unknown
whether these derive from activated or recirculating B
cells, or if they include populations of naïve B cells
residing in the periphery .
However, it is known that the mammalian immune
system contains a B-1 cell subset strategically positioned
in the peritoneal and pleural cavities. These cells might
migrate from the peritoneal cavity to a distant inflam-
matory lesion. Moreover they do not circulate through
the lymph nodes, but migrate directly to the site of
These B-cells play a role in defending against infection
during the period between activation of phagocytic cells
(innate immunity) and T and B cells (adaptive immu-
nity), and they also demonstrate the “promiscuous”
expression of both myeloid and lymphoid characteristics
. In addition, B1 cells produce low-affinity antibodies,
called natural antibodies, with limited diversity in the
absence of infection.
The aim of this study was to evaluate the morphology,
phenotype and tissue distribution, of IPCs in a substan-
tial number of IBD patients in order to gain further
insight into B cell pathobiology.
Small intestinal, colonic and rectal tissue samples were
obtained from 96 patients undergoing complete colono-
scopy at Fatebenefratelli Hospital in Milan, Italy. Biopsy
specimens were taken from the inflamed mucosa and
for each biopsy three sections were analysed. Informed
consent was obtained from all of the patients before the
procedure. The diagnosis of each case was confirmed
using standard endoscopic and histological criteria
(additional hematoxylin and eosin staining of each sam-
ple), and clinical data were obtained from clinical
records. Sulphasalazine was used as a maintenance of
treatment in the majority of the cases. If disease reacti-
vation occurred, corticosteroids i.v. and infliximab were
used. Control specimens were taken from ten patients
(30 sections) with normal endoscopic findings and no
macroscopic evidence of inflammatory or neoplastic dis-
ease. The biopsy sites were selected to obtain informa-
tion from all parts of the intestinal tract.
After deparaffinisation (72°C) and pre-treatment to
enhance antigenicity (95°C × 36 min), we used indirect
immunofluorescence (IFI) to detect the tissue expression
distribution of immunoglobulins in intestinal sections (3
μm). The sections were incubated for 30 minutes with
horse serum at room temperature in order to prevent
non-specific binding. After removing the blocking solu-
tion, the sections were incubated with a polyclonal rabbit
anti-human Ig (Santa Cruz Biotechnology, Santa Cruz,
California, USA) or a goat anti-human IgM (μ chain spe-
cific, Vector Laboratories, Burlingame, California, USA).
While being protected from direct light exposure at 37°C
for one hour, the samples were washed four times for
five minutes in PBS High Salt (NaCL 4 M and PO4Buf-
fer) and mounted. Images were taken from four ran-
domly chosen sites on each sample (Euroimmun, Lubeck,
Germany) at magnifications of 40× and 20×.
Murine monoclonal antibodies (mAbs) CD79, CD20,
CD21, CD23 CD5, l and ? chains were purchased from
Medical Systems (Tucson, Arizona, USA) and used on
formalin-fixed, paraffin-embedded tissue employing a
Ventana automated slide stainer (Medical Systems). Bio-
tinylated secondary antibodies were then added, fol-
lowed by streptavidin-horseradish peroxidase conjugate.
The anti-CD and anti l and ? chains were optimally
diluted for use with Ventana detection kits and auto-
mated slide stainers. Each stage of the staining protocol
included incubation for a precise period of time at a
specific temperature. At the end of each incubation
stage, the sections were rinsed by the Ventana auto-
mated slide stainer to block the reaction and to remove
any unbound material that might hinder desired reac-
tions at subsequent stages. To minimise evaporation of
the aqueous reagents from the specimen-containing
slide, a coverslip solution was applied inside the slide
stainer. The complex was then visualised using a hydro-
gen peroxide substrate and 3, 3’-diaminobenzidine tetra-
hydrochloride (DAB) chromogen.
All patients in the study had an established diagnosis of
CD or UC based on standard criteria (i.e. endoscopy,
histology, barium contrast enema or other recognised
criteria). Disease activity was defined using the Crohn’s
Disease Activity Index (CDAI) score of biological mar-
kers of inflammation or, in the case of UC [10-12], with
Defendenti et al. BMC Immunology 2011, 12:71
Page 2 of 6
the Disease Activity Index (DAI) validated by St Mark’s
Hospital and Academic Institute, England.
The continuous variables were recorded as mean values
and standard deviations and compared using an
unpaired Student’s t-test. The categorical variables were
recorded as absolute and relative frequencies and com-
pared using the Chi-squared test. The data were ana-
lysed using the Statistical Package for Social Sciences
(SPSS 13.0; SPSS Inc., Chicago, IL, USA). All of the
tests were two-sided and p values of < 0.05 were consid-
ered statistically significant.
The study population comprised 96 patients with UC or
CD, and consisted of more males than females. Their
demographic and clinical characteristics are shown in
IPC morphology and localisation can discriminate two
types of cellular homing
Analysis of the morphology and distribution of IPCs
in the biopsy specimens of inflamed tissue identified
two subgroups of patients. 41 patients showed numer-
ous small subepithelial IPCs distributed within the
glands of the analysed specimens, which were charac-
terised by a large central nucleus (Figure 1). These
IPCs were surface IgM positive and monomorphic,
and they often massively infiltrated inflamed tissues
(Figures 2 and 3). Morphologically, they had a low
cytoplasmic/nuclear ratio, unlike conventional plasma
cells (Figure 4).
These were also present as discrete foci on ten normal
small intestinal, colonic and rectal biopsy specimens.,
and seemingly contributed to the physiological intestinal
The remaining 55 patients had no such small IPCs
across a number of different bowel tissue sections. The
typical pattern in these cases was either a complete
absence of IPCs (Figure 5) or evidence of a few typical
plasma cells or IPCs with irregular nuclei (Figure 6).
Characteristically, both types of IPC in this group were
sporadically distributed and did not contribute to mas-
sive tissue infiltrates.
B cell surface phenotype analysis of inflammatory infil-
trates identified a distinct CD79+/CD20-/CD21-/CD23-/
B cell-specific surface molecules were examined in 20
representative samples taken from patients showing a
large infiltrate of small IPCs with a low cytoplasmic/
nuclear ratio. All of these samples originated from
patients affected by UC.
Table 1 Characteristics of the patients included in the
Patients Ulcerative Colitis
Mean age at diagnosis, years
No therapy at diagnosis
44.2 ± 14.2
47.2 ± 16.6
Figure 1 Morphologic aspect of IPC in IFI method
(Magnification 40×). These cells had a low cytoplasmic/nuclear
ratio unlike conventional plasma cells. There is a fluorescent cut into
monomorphic IPC (Magnification 40×). On using fluorescent
antibodies anti chain μ, numerous small IPCs distributed within the
glands, and characterised by a large central nucleus, were observed
in the sections of inflamed mucosa.
Stromaland infraglandulardistribution of
Defendenti et al. BMC Immunology 2011, 12:71
Page 3 of 6
All of the samples were CD79+ and ? and l chain-
positive. This finding was confirmed by IFI, thus sup-
porting the fact that surface Ig expression can be used
as a marker to identify B cells .
All of the samples were negative for three other mar-
kers specific to the B-2 cell subset: CD20 (a mature B
cell-specific molecule), CD23 and CD21. To test
whether the cells belonged to the B-1 subset, we ana-
lysed the surface expression of CD5, which was positive
in 17 of the 20 samples. The CD5-negative samples
were strongly positive for Ig and the symptoms of these
patients were different from those observed in the CD5-
positive group, particularly in terms of neurological
manifestations. One of these patients had type one
spino-cerebral ataxia and two female patients had per-
ipheral neuropathy responsive to corticosteroid treat-
ment. In one of these two cases this was associated with
pluris nodular goiter micro and macro follicular.
The lack of CD79+/CD20-/CD21-/CD23-/CD5±IPCs
within the intestinal specimens was statistically asso-
ciated with a diagnosis of CD (Table 2). The presence of
B-1 like cells had a negative predictive value (NPV) of
82.9% for CD, with a sensitivity of 0.781 and a specificity
of 0.531 (Table 2).
Treatment did not influence CD79+/CD20-/CD21-/
CD23-/CD5±IPC expression (Table 3) as IPCs were
observed with similar frequency in treated and untreated
patients, and there was no significant difference between
the IBD subgroups (Table 3).
Only one of the 41 patients in the CD79+/CD20-/
CD21-/CD23-/CD5±IPC positive group had previously
undergone surgical intervention for his/her disease dur-
ing its clinical history compared to 13 out of 55 in the
CD79+/CD20-/CD21-/CD23-/CD5±IPC negative group.
No significant statistical difference was found between
these two groups in terms of anti-inflammatory and
immunosuppressive drug use.
Figure 3 Massive IPC Invasion seen on the intestinal tissue
(Magnification 20×). In some biopsy specimens B-1 like cells were
so numerous that they occupied all the periglandular space.
Figure 4 Negative sample (Magnification 40×). In other sections
and biopsies of inflamed mucosa taken from the same patient it is
not possible to find the IPC on immunofluorescence.
Figure 5 Typical plasma cells with a “flame of the citoplasma”
aspect/appearance (Magnification 40×). Characteristically this IPC
had a sporadic distribution.
Figure 6 IPC cells with irregular nuclei (Magnification 40×).
These cells had a periglandular distribution and were characterized
by positivity of the chain μ on imnunofluorescence even if the
irregular aspect of the nucleous.
Defendenti et al. BMC Immunology 2011, 12:71
Page 4 of 6
In this study, we identified an IgM+B cell subset in
human intestinal tissue that is characterised by a CD79
+/CD20-/CD21-/CD23-/CD5±surface phenotype, which
is atypical of B2 cells. CD5 (a marker that was initially
used to distinguish B-1 from B-2 cells)  was often,
but not always, co-expressed.
It is well-known that humans have one class of CD5+
B cells that appear to share the phenotype properties of
murine B-1a cells . It has been reported that, like
murine B-1a cells, CD5+human peripheral blood B cells
produce polyspecific autoreactive antibodies [16,17]. It
has been shown that CD5 is expressed on mouse B-1a
but not B-1b cells, and its expression is largely extin-
guished during B-1a differentiation into antibody-secret-
ing cells . It has also been demonstrated that IL-9
restores a B-1 population in Xid mice, but exclusively
with the B-1b surface phenotype; there, however, the
Xid-B-1b cells failed to restore the classical functions of
B-1a cells, thus indicating that this Xid sister population
is functionally distinct from the B-1a cell subset .
Abrahão et al. identified B-1 cells using a colloidal
gold immunocytochemical assay and found that mouse
B-1a or B-1b cells have a single morphology that is dis-
tinct from that of B-2 cells. Mouse B-1 B cells are stra-
tegically positioned in the peritoneal and pleural cavities
, but there is currently no marker or combination of
markers that is expressed uniquely on all B-1 cells .
Shimomura et al.  described a previously unidenti-
fied subset of mouse IgM+B cells that present with an
AA4.1-CD21-CD23-major histocompatibility complex
class II (bright) surface phenotype. These reside within
the normal mucosa of the large intestine and expand in
response to inflammation. This subset did not express
B-1 cell markers (CD43, CD11b and CD5) but it prolif-
erated in vitro in response to B cell receptor ligation
and lipopolysaccharide stimulation. These cells appeared
to exist in a unique pre-activated state and may
originate from AA4.1+transitional B cells in the steady
state. These B cells may be significantly increased in the
inflamed intestine after recruitment from the recirculat-
ing naïve B cell pool.
In our study, we observed that the normal mucosa of
the human intestine contains a CD79+/CD20-/CD21-/
CD23-/CD5±subset of IPCs. In 42.7% of the BD
patients included in this study we observed a relevant,
and sometimes massive, increase in these cells in the
biopsies of inflamed intestinal tissue. Their absence, in
turn, was associated with CD but unrelated to disease
severity or treatment. Moreover, only one of the 41
patients in the CD79+/CD20-/CD21-/CD23-/CD5□IPC
positive group had undergone surgical intervention for
his/her disease during its clinical history, compared to
13 out of 55 in the CD79+/CD20-/CD21-/CD23-/CD5□
IPC negative group. Thus, as shown above, B-1 cells
might be active in intestinal inflammation, but their
massive recruitment seems to be less consequential
than their absence. IL-10 production is a well-known
feature of the peritoneal CD5+ B1a subset, and it is
interesting that a number of studies have shown that
the phenotype of IL-10-producing Breg cells (CD11b-
CD5-IgD+) is similar to that of B-2 (conventional B
cells) but not to B-1a cells (CD11blowCD5+IgD-) .
Experiments using co-cultures of B-1 cells and macro-
phages from Xid mice have demonstrated that B-1
cells down-regulate macrophage activity by means of
IL-10 secretion , which illustrates their involve-
ment in immune regulation.
In the subset of seemingly-negative B-1 cells, CD
patients showed a periglandial infiltration compared
with those patients with UC [Figure 6]. Our partial
results demonstrate the presence of almost three differ-
ent pathogenetic patterns of IPC found during the IBD
evolution. The diagnosis of the patients analysed was
related not only to the presence or absence of ICPs, but
also to their distribution and morphology.
Moreover, because there were extra-intestinal manifes-
tations in three patients with B-1 like cells and a CD5-
phenotype, we could ask whether systemic mechanisms
are involved in this subgroup of patients.
The immune system has developed many different
mechanisms to regulate immune responses, and B cells
Table 3 Distribution of B cells by diagnosis and
B-1 cells highly representedTherapyNo therapyp
Table 2 Distribution of B cells by diagnosis
B-1-like cellsUlcerative colitis (n = 64) Crohn’s disease
(n = 32)
pSensitivitySpecificity PPV NPV
Highly represented 34
0.004 0.781 0.5310.455 0.829
Defendenti et al. BMC Immunology 2011, 12:71
Page 5 of 6
also seem to play a pathogenic role in inflammation. Download full-text
Autoimmune diseases are often characterised by the co-
existence of clinical features compatible with chronic
inflammatory conditions, with a variable prevalence of
different components. The finding that IPCs derived
from innate immunity B cells are present in inflamed
tissues in close contact with several environmental sti-
muli strengthens the hypothesis that microbes can
induce autoimmune responses . However further
studies are needed to clarify the role of cells linking
innate to adaptive immunity, including their homing
potential, to different tissues.
IBD: inflammatory bowel disease; IPC: immunoglobulin-producing cell; IFI:
indirect immuno-fluorescence; xANCA: atypical cytoplasmic and perinuclear
anti-neutrophil cytoplasmic antibody; TLR: Toll-like receptor; NOD:
nucleotide-binding oligomerisation domain; UC: ulcerative colitis; CD:
Crohn’s disease; TNF: tumour necrosis factor; IL: interleukin; mAb: murine
monoclonal antibody: DAB: 3,3’-diaminobenzidine tetrahydrochloride; PPV:
positive predictive value; NPV: negative predictive value.
We would like to thank Mario U. Mondelli, Research Laboratories,
Department of Infectious Disease, Fondazione IRCCS Policlinico San Matteo
and the University of Pavia, Italy, for his helpful comments and critical
editing of this manuscript.
This project was supported by our Department.
1Laboratory Unit, Fatebenefratelli Hospital, Milan, Italy.2Rheumatology Unit,
L. Sacco University Hospital, Milan, Italy.3Division of Gastroenterology,
Fatebenefratelli Hospital, Milan, Italy.4Division of Pathology, Fatebenefratelli
Hospital, Milan, Italy.5GI & Liver Unit, DIBIMIS, Policlinico, University of
Palermo, Palermo, Italy.6Experimental Medicine, Queen Mary University,
CD and SG carried out the Immunofluorescence and Immunoperoxidase
method and drafted the manuscript. OS, AC, SS and SB participated in the
coordination of the study. CD, FA, PSP participated in the design and
coordination of the study and helped to draft the manuscript. MA
participated in the study and performed the statistical analysis. SB
participated in the design of the study and performed the statistical analysis.
PLA conceived of the study, and participated in its design and coordination.
All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Received: 23 July 2011 Accepted: 30 December 2011
Published: 30 December 2011
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Cite this article as: Defendenti et al.: B Lymphocyte intestinal homing in
inflammatory bowel disease. BMC Immunology 2011 12:71.
Defendenti et al. BMC Immunology 2011, 12:71
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