Antibody response of autogenous splenic tissue implanted in the abdominal cavity of mice.

Abstract

There is still controversy about the immunologic function of autotransplanted splenic tissue. In this study, splenic autotransplantation was performed in the abdominal cavity of mice, and the plaque-forming cell (PFC) assay was used to investigate the frequency of antibody-forming cells in response to sheep red blood cell (SRBC) immunization. BALB/c mice were divided into four groups according to the location of the autogenous graft: intraomental (IO), free peritoneal splenosis (FPS), retroperitoneal (RP), and nongrafted control (CT). Thirty days after surgery the mice were immunized intraperitoneally with SRBCs, and 4 days later splenic immunoglobulin M anti-SRBC-secreting cells were determined by counting the number of PFCs. All the immunized mice showed increased numbers of PFCs that were about 2 logs higher than those in the the nonimmunized controls (P < 0.005). The frequencies of anti-SRBC-producing cells in the tissues grafted in various sites of the abdominal cavity (IO, FPS, RP), in the normal spleen from nonoperated controls (CT), or in the sham-operated control group (SCT) were not notably different (5582 +/- 2475 PFC/10(7) cells for IO; 4849 +/- 1856 for FPS; 6604 +/- 2903 for RP; 5940 +/- 5029 for CT; and 6172 +/- 2203 for SCT). Similar histology with small architectural variations was observed in all implants; less white pulp was involved, and there was more congestion in the red pulp, with extensive sinusoids and reticular fiber proliferation. This study shows that the T cell-dependent antibody response in implanted splenic tissues is as efficient as in the intact spleen, with no difference between the graft sites studied. This immune response does not depend on the slight architectural variations observed in the splenic implants.

Full text

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Antibody Response of Autogenous Splenic
Tissue Implanted in the Abdominal Cavity of Mice
Se ´rgio I. Nunes, MD, PhD,1Alice B. Rezende, MD,1Francisco M. Teixeira, MS,1
Ana Paula Ferreira, PhD,1Ma ´rcio M.J. Alves, MD, PhD,2Nelson Jamel, MD, PhD,3
Raimunda V.C. Assis, MD, PhD,4Henrique C. Teixeira, PhD1
1Department of Parasitology, Microbiology and Immunology, Biological Sciences Institute, Federal University of Juiz de Fora,
36036-900 Juiz de Fora, MG, Brazil
2Department of Collective Health, Faculty of Medicine, Federal University of Juiz de Fora, 36036-900 Juiz de Fora, MG,
Brazil
3Department of Surgery, Faculty of Medicine, Federal University of Rio de Janeiro, 21941-590 Rio de Janeiro, RJ,
Brazil
4Department of Pathology, Faculty of Medicine, Federal University of Juiz de Fora, 36036-900 Juiz de Fora,
MG, Brazil
Abstract
There is still controversy about the immunologic function of autotransplanted splenic tissue. In this
study, splenic autotransplantation was performed in the abdominal cavity of mice, and the plaque-
forming cell (PFC) assay was used to investigate the frequency of antibody-forming cells in
response to sheep red blood cell (SRBC) immunization. BALB/c mice were divided into four
groups according to the location of the autogenous graft: intraomental (IO), free peritoneal sple-
nosis (FPS), retroperitoneal (RP), and nongrafted control (CT). Thirty days after surgery the mice
were immunized intraperitoneally with SRBCs, and 4 days later splenic immunoglobulin M anti-
SRBC-secreting cells were determined by counting the number of PFCs. All the immunized mice
showed increased numbers of PFCs that were about 2 logs higher than those in the the nonim-
munized controls (P < 0.005). The frequencies of anti-SRBC-producing cells in the tissues grafted
in various sites of the abdominal cavity (IO, FPS, RP), in the normal spleen from nonoperated
controls (CT), or in the sham-operated control group (SCT) were not notably different
(5582 – 2475 PFC/107cells for IO; 4849 – 1856 for FPS; 6604 – 2903 for RP; 5940 – 5029 for
CT; and 6172 – 2203 for SCT). Similar histology with small architectural variations was observed
in all implants; less white pulp was involved, and there was more congestion in the red pulp, with
extensive sinusoids and reticular fiber proliferation. This study shows that the T cell-dependent
antibody response in implanted splenic tissues is as efficient as in the intact spleen, with no
difference between the graft sites studied. This immune response does not depend on the slight
architectural variations observed in the splenic implants.
T
he importance of the splenic autotransplant tech-
nique is justified by the high incidence of total
splenectomy, the spleen being the most affected organ
during blunt trauma as well as highly susceptible to iat-
rogenic incidents.1,2Since the nineteenth century sple-
nectomy has been the treatment advised after splenic
trauma, although current studies indicate that this pro-
cedure is not exempt from complications.3It is accepted
Correspondence to: Henrique C. Teixeira, PhD, Department of
Parasitology, Microbiology and Immunology, Biological Sciences Insti-
tute, Federal University of Juiz de Fora, 36036-900 Juiz de Fora, MG,
Brazil, e-mail: henrique.teixeira@ufjf.edu.br
? 2005 by the Socie ´te ´ Internationale de Chirurgie
Published Online: 16 November 2005
World J Surg (2005) 29: 1623–1629
DOI: 10.1007/s00268-005-0060-7

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that important alterations in hematologic physiology occur
after splenectomy, compromising the immune system.
The overwhelming postsplenectomy infection syndrome
is much more frequent in patients subjected to splenec-
tomy than in the general population, affecting children
primarily.4,5Moreover, partial or complete maintenance of
the spleen preserves its important capacity for filtration of
cells and particles and can avoid alterations of the lipid
metabolism that occur after splenectomy.6,7There are
still situations where splenectomy is a therapeutic option,
as for portal hypertension, but loss of splenic function is
not desirable.7,8Methods for conserving the spleen have
been described, such as splenorrhaphy, partial splenec-
tomy, subtotal splenectomy, arterial ligatures, and appli-
cation of hemostatic agents.2,8
complete splenectomy is inevitable, autotransplant is an
alternative for preserving a satisfactory immunologic
response.9
Autogenous grafting of the spleen has been shown to
be a viable procedure. Scintigraphic studies have re-
vealed the presence of captive tissue in autotrans-
planted fragments,1,10,11and histopathologic analysis
has shown that the architecture of the autograft is sim-
ilar to that of normal spleen.12–18In addition, preserva-
tion of its phagocytic capacity and the absence of
Howell-Jolly corpuscles in peripheral blood have been
described after autotransplantation of the spleen.1,7,14
Both intraperitoneal and extraperitoneal locations have
been used as implant sites for the experimental tech-
nique of autogenous spleen graft.3,18,19The most fre-
quent scenarioof autotransplantation
involves implanting sections of splenic parenchyma into
pouches created in the greater omentum because of the
favorable revascularization, portal drainage, and omen-
tal bacterial clearance.12,17However, there have been
complicationsassociated
autotransplantation, such as torsion of the implant,
anemia, and subphrenic abscesses due to necrosis of
the implanted tissue.2,19Although many studies have
attempted to elucidate the immunologic function of
autotransplanted splenic tissue, a general consensus
has not been achieved.
To learn more about the immunologic properties of
splenic implants, the present study evaluated the fre-
quencies of antibody-forming cells against sheep ery-
throcytes in immunized BALB/c mice 30 days after
splenic autotransplantation in the peritoneal cavity. Grafts
were located in the greater omentum and retroperitoneum
and as free splenosis in the peritoneal cavity. The
histologic examination of these splenic implants is also
outlined.
In situations where
inhumans
with intraomentalsplenic
MATERIALS AND METHODS
Experiments were performed with 80 isogenic mice of
the BALB/c lineage, all females, 10 to 12 weeks old.
The animals were grouped in the following way: (1)
control group (CT), with the mice not subjected to
splenectomy; (2) intraomental group (IO), with auto-
transplantation in the greater omentum; (3) free peri-
toneal splenosis group (FPS), with autotransplantation
in the peritoneal cavity; and (4) retroperitoneal group
(RP), with autotransplantation in the retroperitoneum.
An additional group of mice was used to study the ef-
fect of laparotomy on anti-sheep red blood cell (SRBC)
plaque-forming cell (PFC) responses, which we call the
sham surgery control (SCT) group. The project was
approved by the Committee for Ethics in Animal
Experimentation of the Federal University of Juiz de
Fora (No. 14/2003).
After anesthesia with phosphate-buffered saline (PBS)-
chloral hydrate (4%, 8 ll/g body weight, injected intraper-
itoneally), a midline laparotomy was performed with sub-
sequent splenectomy and binding of the vascular pedicle
andshort vessels with5.0catgut (Shalon,Goia ˆnia, Brazil).
All operations were done under sterile conditions. The
spleenwascutintosixslicesabout2.5mmthickandkeptin
physiologic saline at room temperature. In the RP group,
the retroperitoneum was opened near the left kidney, and
two pieces were placed close to the large abdominal blood
vesselwithoutfixation.IntheIOgroup,twofragmentswere
placedinpocketsofthegreateromentum,wheretheywere
sutured with 5.0 catgut. In the FPS group, two fragments
were placed loose in the peritoneal cavity without fixation
(splenosis). Skin closure was done using a 4.0 running
polyglactin suture. In the SCT group, midline laparotomy
with subsequent laparorrhaphy was performed.
A direct hemolytic plaque assay was used for estimat-
ing the number of immunoglobulin M (IgM)-producing B
cells.20,21Seven mice of each group were immunized
intraperitoneally with 0.1 ml of a 10% SRBC suspension
(in PBS, pH 7.2) 30 days after splenic autotransplanta-
tion. Four days later the animals were sacrificed, and the
spleen or splenic autotransplants were removed. Tests
were performed in duplicate in solutions containing wa-
shed spleen cells or transplant cells in RPMI medium
(105to 5 · 106cells, 0.1 ml), washed SRBCs (1:3, 20 ll),
fresh guinea pig serum as a complement source (1:10,
50 ll), and 0.5% Difco agar/DEAE-dextran (250 ll). The
mixture was poured into a petri dish to form a thin,
semisolid top layer. The number of PFCs was determined
after 4 hours of incubation at 37?C and expressed as
PFCs/107cells.
1624 Nunes et al.: Antibody Response of Splenic Implants in Mice

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For PFC studies from lymph nodes, single cell sus-
pensions from inguinal and periaortic lymph nodes were
prepared from nonimmunized or immunized mice killed
on day 4 after SRBC immunization. Cell viability was
determined by the trypan blue exclusion test. The
peripheral blood was obtained from the left axillary artery.
Peripheral blood mononuclear cells (PBMCs) were iso-
lated from a heparinized pool (2–3 ml/2 animals) with
Histopaque-1077 (Sigma, St. Louis, MO, USA) by density
gradient centrifugation at 1700 rpm for 30 minutes at
room temperature. PBMCs and lymph node cells were
washed twice in PBS and resuspended in RPMI 1640
medium.
For histologic evaluation, the splenic fragments were
fixed in 10% formaldehyde before processing for 14
hours and then embedded in paraffin. Histologic sec-
tions about 3 lm thick were removed from all the blocks
and dyed with hematoxylin-eosin; a histochemical reac-
tion was also undertaken for reticular fibers using
ammonia silver solution. All material was evaluated by
the same observer who considered: (1) general struc-
tures of the organ and the presence of the capsule; (2)
the morphologic pattern of the white pulp, the existence
of primary and secondary follicles, lymphocyte expan-
sion through reactivity with tingible-body macrophages,
and mitoses; (3) the condition of the red pulp and
sinusoids, evaluated mainly through the reticulum and
determining the diameter; and (4) the presence of giant
cells and epithelioid cells.
The groups tested were compared by nonparametric
analysis using the Kruskal-Wallis test. A value of P < 0.05
was considered significant.
RESULTS
Autogenous Splenic Implants
Several anatomic sites have been described as suit-
able for spleen autotransplant, including the omentum
and the peritoneum (free peritoneal splenosis).3,12In this
study autogenous spleen grafts were prepared in the
retroperitoneum and the results compared with those at
sites more commonly used (IO and FPS).
All animals that had undergone splenic autotransplan-
tation had gross evidence of viable splenic implants
Figure 1. Photograph of splenic implants. Splenic
fragments implanted in the greater omentum (A), free
peritoneal splenosis (B), and retroperitoneal sites
(C, D). Mice were killed by cervical displacement.
Arrow: implanted tissue.
Nunes et al.: Antibody Response of Splenic Implants in Mice1625

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(Fig. 1). The survival rate of the transplanted mice was
97.72% (43/44). The average operating times were sim-
ilar for the groups: 7.3 – 2.1 minutes for IO, 8.6 – 1.5
minutes for FPS, and 7.7 – 2.0 minutes for RP. Adhe-
sions were found in 66.6% (8/12) of the IO animals,
40.0% (4/10) of the FPS animals, and 18.2% (2/11) of the
RP animals.
Histologic Evaluation
The histologic pattern of control spleens and trans-
planted splenic fragments were evaluated 34 days after
autotransplantation. The main alterations found among
the original and transplanted spleens were in respect to
loss of the original capsule and formation of thick fi-
brous tissue on cut surfaces of the fragments as well
as the presence of granulation tissue, giant cells, and
macrophages phagocytosing hemosiderotic pigments in
the periphery (Fig. 2). Animals with implants showed
autotransplanted tissue with a structure similar to that
of the normal spleen, but minor differences were ob-
served in all implanted groups, such as slight volume
reduction of white pulp, congested red pulp, and sinu-
soids with a more extensive but less organized pattern
and with proliferation of the reticular fibers (Fig. 2). No
significant histologic differences were observed be-
tween the original spleen and implanted splenic frag-
ments from mice stimulated with SRBCs. Epithelioid
cells, some giant cells, mitosis, immunoblasts, and
macrophages containing cellular remnants were ob-
served in the periphery of secondary follicles in SRBC-
immunized mice (Fig. 2).
Figure 2. Photomicrograph of a
retroperitoneal splenic implant.
A. White and red pulp stained with
hematoxylin-eosin. B, C. Reticulin
fibers (B) and sinusoids (C) with
proliferation of the reticulin fibers
(long arrows) stained with
ammonia silver solution. D–F.
Note the presence of giant cells
(arrowhead), immunoblasts
(circle), and macrophages
phagocytosing hemosiderotic
pigments (short arrows) in the
periphery of secondary follicles.
(A, B · 40; C · 200; D–F · 400)
1626Nunes et al.: Antibody Response of Splenic Implants in Mice

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Plaque-forming Cell Assay
The hemolytic plaque assay was used to determine if
the antibody response of the autotransplanted tissue is
preserved; this response is due to the activity of the
splenic cells present in the autotransplanted tissue.
Figure 3 shows that SRBC-immunized animals had an
increase in the number of splenic anti-SRBC PFCs of
log 2 in relation to the nonimmunized animals (P <
0.0005). At day 4 after SRBC immunization, the number
of anti-SRBC PFCs per 107splenic cells were similar in
all of the studied groups (P = 0.582): 5582 – 2475 in the
IO group, 4849 – 1856 in the FPS group, 6604 – 2903
in the RP group, and 5940 – 5029 in the control group.
To discard the possibility of any effect of laparotomy on
the PFC responses, a sham surgery group was evalu-
ated. Anti-SRBC PFC responses were evaluated 4 days
after immunization in three groups (34 days after sur-
gery in the groups that underwent operation): the sham
surgery group controls (SCT), the splenectomized/
autotransplanted group (AT), and the nonoperated con-
trol group (CT). As expected, the number of IgM-PFCs
was similar in the three groups studied: 6172 – 2203 in
the SCT group, 5305 – 585 in the AT group, and
4237 – 903 in the CT group. The number of PFC re-
sponses in the nonimmunized animals was also similar
among these groups, at less than 100 PFCs/107cells
(data not shown).
An additional experiment examining IgM anti-SRBC
responses in lymph nodes and peripheral blood was
performed to analyze PFC responses in a splenectom-
ized but not autotransplanted group compared to trans-
planted animals and a control group. The three groups
studied were a splenectomized/autotransplanted in the
retroperitoneum group (AT); a splenectomized/nontrans-
planted control group (SP); and a nonoperated control
group (CT). The PFC numbers were much higher in
spleens or in splenic autotransplanted fragments than in
lymph nodes or peripheral blood. In the three groups
studied, the PFC numbers in lymph nodes were not sig-
nificantly different, although they were slightly higher in
the SP group. The PBMC anti-SRBC response was
measured as an indicator of response from both spleen
and lymph nodes. Both groups in which the spleen was
removed had a dramatic decline in PBMC-PFCs, but it
was even lower in the SP group (Fig. 4).
DISCUSSION
The most important findings of this study are the fol-
lowing: (1) The histologic and immunologic patterns at the
autotransplanted sites do not differ significantly from each
other. (2) Histologic structures observed in the implants
are similar to normal splenic tissue 34 days after surgery,
or 4 days after immunization with SRBCs. (3) The fre-
quency of cells secreting anti-SRBC antibodies in trans-
planted tissues was comparable to that found in the
original organ.
Figure 3. Immunoglobulin M (IgM) plaque-forming cells (PFC)
response in splenic implants. Mice were immunized with 2 · 108
sheep red blood cells (SRBC) 30 days after splenic autotrans-
plantation. The number of spleen cells producing anti-SRBC
antibodies was determined in the immunized (black symbols)
and nonimmunized (white symbols) groups 4 days after
immunization by counting the number of PFCs after 4 hours of
spleen cell culture. CT: control spleen; FPS: free peritoneal
splenosis implant; IO: intraomental implant; RP: retroperitoneal
implant. Bars indicate median values.
Figure 4. The IgM-plaque forming cells’ (PFCs) response in
lymph nodes and peripheral blood. Mice were immunized with 2
· 108sheep red blood cells (SRBCs) 30 days after splenic
autotransplantation. The number of peripheral blood mononu-
clear cells (PBMCs), lymph node cells (LNs), and splenic cells
producing anti-SRBC antibodies was determined 4 days after
immunization by counting the number of PFCs. CT: nonoper-
ated control group; AT: autotransplanted in the retroperitoneum;
SP: splenectomized and nontransplanted group.
Nunes et al.: Antibody Response of Splenic Implants in Mice 1627