Selective Malformation of the Splenic White
Pulp Border in L1-Deficient Mice
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J Immunol 2000; 165:2465-2473; ;
doi: 10.4049/jimmunol.165.5.2465
http://www.jimmunol.org/content/165/5/2465
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References
Shih-Lien Wang, Michael Kutsche, Gino DiSciullo, Melitta
Schachner and Steven A. Bogen
Selective Malformation of the Splenic White Pulp Border in
L1-Deficient Mice1
Shih-Lien Wang,* Michael Kutsche,† Gino DiSciullo,* Melitta Schachner,† and
Steven A. Bogen2*
M
ononuclear cells enter lymphoid organs via two distinct routes. Although the blood vascular route has
been a major focus of interest over the last decade,
there is a paucity of information about mononuclear cell passage
across sinusoidal walls. Specifically, mononuclear cells, such as
macrophages, lymphocytes, and dendritic cells, travel from the site
of Ag contact to a draining lymphoid organ. In the context of a
lymph node (LN),3 mononuclear cells drain into the subcapsular
sinus, enter the cortical sinusoids, and then migrate into the lymphoid parenchyma by crossing the relatively poorly defined boundary of the sinusoidal lining. This lining comprises a single layer of
flattened cells associated with collagen fibrils and extracellular matrix proteins, such as laminin (1, 2). Comparatively little is known
about the physiological properties of this lining, such as the expression of adhesion molecules, mechanisms of locomotion of
mononuclear cells along the lining surface, or whether the sinusoidal wall is porous or relatively impermeable to the free flow of
macromolecules and cells (3).
In the spleen, the most analogous structure to the LN sinusoid is
the marginal sinus. The marginal sinus lining cells are a thin layer
of flattened cells that envelop the white pulp. These flattened cells
form the inner layer of the marginal sinus, a space that commu*Department of Pathology and Laboratory Medicine, Boston University School of
Medicine, Boston, MA 02118; and †Zentrum für Molekulare Neurobiologie, Universität Hamburg, Hamburg, Germany
Received for publication December 22, 1999. Accepted for publication June 13, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant 1R29AI34562 to
S.A.B.
2
Address correspondence and reprint requests to Dr. Steven A. Bogen, Department of
Pathology and Laboratory Medicine, Boston University School of Medicine, Boston,
MA 02118. E-mail address: [email protected]
3
Abbreviations used in this paper: LN, lymph node; FDC, follicular dendritic cell;
KO, knockout; MMM, marginal metallophilic macrophage; NRIgG, normal nonimmune rat IgG; RT, room temperature; DAB, 3,3⬘-diaminobenzidine; SLC, sinus lining
cell; RC, reticular cell; MAdCAM-1, mucosal addressin cell adhesion molecule-1;
PECAM-1, platelet endothelial cell adhesion molecule-1.
Copyright © 2000 by The American Association of Immunologists
nicates, directly or indirectly, with the blood vascular system (4 –
6). Ultrastructurally, the marginal sinus wall is more analogous to
the LN sinusoidal lining cells than blood vascular endothelium.
Namely, both types of sinusoidal lining cells associate with a similar type of protein matrix, do not assume the tall morphology often
associated with postcapillary venules, and do not have a known
intercellular junction capable of regulating fluid and solute flow. It
is across this boundary that mononuclear cells must cross to enter
the splenic white pulp.
We are particularly interested in identifying the underlying adhesion molecules that mediate structural features of sinusoidal linings. To this end, we have focused our attention on the L1 adhesion molecule. L1 is a 1260-aa-long Ig superfamily adhesion
molecule (7). It has been implicated in several important neurobiological processes, including neurite outgrowth, neurite fasciculation, axon-Schwann cell interaction, myelination, neuronal cell migration, and synaptic plasticity (8 –12). L1 acts homophilically and
heterophilically (8, 13, 14). The importance of L1 in neuronal development is reflected by the fact that mutations in the human L1
gene lead to a group of neurological syndromes (15, 16).
Although L1 was initially identified in the nervous system, L1 is
also expressed in nonneuronal tissues. Specifically, L1 is expressed by cells of hematopoietic origin (17, 18), intestinal epithelial cells (19), epithelium of the male urogenital tract (20), and
other cells of epithelial origin (21). The functional role of L1 on
these cells is largely unexplored. L1 has been implicated in an in
vitro cell binding assay between lymphocytes and bend 3 endothelioma cells, raising the possibility of a potential role in lymphocyte-endothelial cell interactions (22). It is also involved in
kidney morphogenesis (23).
Previous data from our laboratory implicated the Ig superfamily
adhesion molecule L1 in maintaining normal sinusoidal structure
in LNs during immune hypertrophy (24). Specifically, we found
that in vivo administration of an L1 mAb disrupted the normal
remodeling of the cortical sinusoidal lining cells of LNs during an
immune response. The L1 mAb did not disrupt static, quiescent
sinusoidal lining cells. Rather, it interfered only with sinsusoidal
0022-1767/00/$02.00
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Lymphocytes enter the splenic white pulp by crossing the poorly characterized boundary of the marginal sinus. In this study, we
describe the importance of L1, an adhesion molecule of the Ig superfamily, for marginal sinus integrity. We find that germline
insertional mutation of L1 is associated with a selective malformation of the splenic marginal sinus. Other splenic structures
remain intact. Immunofluorescence analysis of the extracellular framework of the spleen, using an Ab to laminin, reveals that L1
knockout mice have an irregularly shaped, discontinuous white pulp margin. Electron microscopic analysis shows that it is
associated with bizarrely shaped marginal sinus lining cells at the periphery of the white pulp. These abnormalities correlate with
the localization of L1 in normal mice in that L1 is normally expressed on marginal sinus lining cells at the white pulp border. These
L1-immunopositive lining cells coexpress high levels of mucosal addressin cell adhesion molecule-1 and vimentin, indicating that
they are of fibroblastic lineage and express a well-characterized addressin. Our findings are the first to implicate L1 in splenic
lymphoid architectural development. Moreover, these findings help define the poorly characterized sinusoidal boundary across
which mononuclear cells cross to enter the splenic white pulp. The Journal of Immunology, 2000, 165: 2465–2473.
2466
MALFORMATION OF THE WHITE PULP BORDER IN L1 KO MICE
lining cells mediating the process of matrix remodeling induced by
immune stimulation. A limitation with the in vivo L1 Ab study was
that it required a xenogenic rat anti-mouse L1 mAb. Consequently,
a mouse (host) anti-rat IgG immune response developed 10 –14
days after L1 mAb administration. This model was therefore useful
only in short term studies, such as acute hypertrophic responses to
immune stimulation.
Because of the structural similarities between the splenic marginal sinus and LN cortical sinusoids, we hypothesize that the
splenic marginal sinus may also be reliant on the L1 adhesion
molecule for structural integrity. To elucidate the role of L1 in the
development and maintenance of the splenic marginal sinus and
the white pulp boundary, we have studied lymphoid matrix development in an L1-null mutant mouse.
Materials and Methods
Animals
Antibodies
Normal nonimmune rat IgG (NRIgG) and the clone 324 rat anti-L1 mAb
(IgG) were prepared and purified by HPLC as previously described (24).
Other Abs used for indirect immunofluorescence studies were diluted in
PBS, 0.1% BSA: rabbit anti-mouse laminin IgG, 1/500 (Sigma, St. Louis,
MO); normal rabbit serum, 1/1000 (Covance Research Products, Denver,
PA); goat anti-rabbit IgG-FITC, 1/1000 (Organon Teknika, West Chester,
PA); rat anti-mouse mucosal addressin cell adhesion molecule-1 (MAdCAM-1), MECA367, 4 ␮g/ml (PharMingen, San Diego, CA); MOMA-1
(for identifying marginal metallophilic macrophages), 1/25 (Serotec, Raleigh, NC); rat anti-mouse IgM-FITC, 4 ␮g/ml (Serotec); rat anti-mouse
B220 mAb clone TIB146 culture supernatant, rat anti-mouse CD4 mAb
clone TIB207 culture supernatant, rat anti-mouse CD8 mAb clone TIB105
culture supernatant (all from the American Type Culture Collection, Manassas, VA); NLDC145 (for identifying interdigitating dendritic cells),
hybridoma culture supernatant (25); rat anti-mouse IgD (clone JA12.5),
3 ␮g/ml, a gift of Dr. Fred Finkelman (26); FDC-M1 (for identifying follicular dendritic cells), a gift of Dr. Marie H. Kosco-Vilbois (27); rabbit
anti-rat IgG-FITC, 1/600 (Vector Laboratories, Burlingame, CA); donkey
anti-rat IgG (Fab⬘)2-biotin, 1/1000 (Jackson ImmunoResearch, West
Grove, PA); donkey anti-rat IgG-Texas Red, 1/75 (Jackson ImmunoResearch); donkey anti-rabbit IgG-Texas Red, 1/500 (Jackson ImmunoResearch); avidin-FITC, 1/1000 (Vector Laboratories).
Immunofluorescence microscopy
For immunofluorescence studies, spleen tissue was embedded in OCT
(Miles, Elkhart, IN), snap frozen in liquid nitrogen, cryosectioned, and
fixed for 30 s in cold acetone. For the detection of L1, we used the tyramide
signal amplification system (NEN Life Science, Boston, MA). Sections
were first incubated in 1% newborn calf serum, PBS for 30 min at room
temperature (RT). This both hydrated the tissue sections and masked sites
of nonspecific protein adsorption.
We masked endogenous peroxidase activity by incubating the sections
with 3,3⬘-diaminobenzidine (DAB) 0.6 mg/ml in 50 mM Tris buffer, pH
7.6, supplemented with 0.003% hydrogen peroxide for 20 min at RT. This
treatment covers sites of endogenous peroxidase with DAB precipitate,
physically encasing the enzyme to prevent further activity and thus effectively neutralizing the activity of the peroxidase enzyme. It also largely
occludes the excitation and emission light (in a fluorescence detection system, which we used). We found this method to be more effective than
quenching with high concentrations of hydrogen peroxide, especially because tyramide signal amplification is highly sensitive in detecting even
trace levels of residual peroxidase activity. In the system that we describe,
even if a small amount of peroxidase activity was left unquenched, there
Plastic embedding for light and transmission electron
microscopy
Organs were immersed in Karnovsky’s half-strength fixative for 30 min
and then cut with a razor blade into smaller pieces (⬃8 mm3). The tissue
fragments were further fixed in Karnovsky’s half-strength fixative for ⬎1
day. Tissues were rinsed with 0.1 M sodium cacodylate with 5% sucrose,
postfixed with 1% OsO4, stained with 2% uranyl acetate and 2% lead
citrate, and then dehydrated through an ethanol gradient. The tissue was
then embedded in Epon 812 and cured at 60°C overnight. Semithin sections (1 ␮m) were stained with 1% toluidine blue in 1% sodium borate. The
0.06-␮m ultrathin sections were examined and photographed in an electron
microscope (model 300, Philips Electronics, Eindhoven, The Netherlands).
Flow cytometry
Splenocyte suspensions were made by dissociating the spleens through a
steel mesh. The RBC were lysed in Tris-buffered ammonium chloride (0.14
M NH4Cl, 0.017 M Tris-HCl, pH 7.2) for 1 min at 37°C. Cells were
resuspended in PBS at a concentration of 20 ⫻ 106/ml. Splenocytes (1 ⫻
106/tube) were stained with anti-CD4-FITC, anti-CD8-PE (both from
PharMingen), or anti-IgM-FITC (Serotec). Cell suspensions were incubated for 45 min at 4°C. After staining, the cells were washed, fixed in 2%
paraformaldehyde, PBS, and stored at 4°C until assayed. The lymphocyte
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Female L1 heterozygous mice of C57BL/6 background were generated as
previously described (8). They were bred with either wild-type C57BL/6
(The Jackson Laboratory, Bar Harbor, ME) or 129/SvEv (Taconic Farms,
Germantown, NY) males. The male offspring were genotyped by PCR for
detection of the L1 deletion as described (8). Male L1 knockout (KO) mice
and their wild-type littermate controls were used at ⬃8 –15 wk of age.
Normal 8- to 12-wk-old male BALB/cByJ mice were purchased from The
Jackson Laboratory. Animals were housed at the Laboratory Animal Science Center, Boston University School of Medicine (Boston, MA), and
cared for in accordance with the Institutional Animal Care and Use Committee of Boston University School of Medicine.
was no opportunity for confusion to arise. The brown DAB precipitate
denoting endogenous peroxidase is easily distinguished on bright-field optics from the fluorescent signal identifying the location of Ags of interest.
The 324 (anti-L1) mAb or NRIgG control were applied at 0.1 ␮g/ml,
followed by donkey anti-rat IgG (Fab⬘)2-biotin at a 1/1000 dilution. All
incubations were performed at RT for 40 min. Subsequently, streptavidinperoxidase and tyramide-FITC were applied and incubated as per the manufacturer’s recommendations. Between each step, the sections were
washed three times for 5 min in PBS, 0.1% Tween 20.
For two-color staining of L1 and MAdCAM-1, we modified the procedure to distinguish the two rat mAbs with two distinct fluorochromes (fluorescein and Texas Red). L1 was first detected as described above, except
that the 324 mAb was applied at a 10-fold lower than normal concentration
(0.01 ␮g/ml). This ultralow Ab concentration can easily be detected using
the highly sensitive tyramide signal amplification system (fluorescein signal) but is below the level of detection for the standard immunofluorescence detection system using Texas Red. Therefore, this helps ensure the
fidelity of the two-color discrimination. After completing the first color
(fluorescein) stain, we blocked immunoreactive sites on the donkey anti-rat
IgG-biotin conjugate with NRIgG (60 ␮g/ml). This step blocks unoccupied
Ig binding sites on the donkey anti-rat IgG-biotin conjugate reagent. Immunoreactive sites on the L1 rat IgG mAb were blocked with a 40-min RT
incubation of rabbit anti-rat IgG Fab fragment (50 ␮g/ml). The rabbit antirat IgG Fab was prepared by digestion with papain. Fab fragments were
subsequently purified by HPLC. For the second color, sections were
stained with anti-MAdCAM-1 (4 ␮g/ml) for 40 min at RT. The antiMAdCAM-1 mAb was detected with donkey anti-rat IgG-Texas Red and
30 min incubation at RT.
For immunocytochemical identification of T and B lymphocytes, T cells
were stained with a combination of anti-CD4 and anti-CD8 mAbs, whereas
B cells were stained with anti-B220 mAb. The anti-CD4 and anti-CD8
mAbs were detected with a rabbit anti-rat IgG-FITC. The anti-B220 mAb
was detected with a donkey anti-rat-IgG-Texas Red. Because all the primary mAbs are of rat origin, the same previously described procedure
modifications (above) were used for ensuring accurate two-color fluorescence discrimination.
For the double staining of laminin and marginal metallophilic macrophages (MOMA-1 Ab), tissue sections were initially stained with
MOMA-1. The MOMA-1 Ab was detected with a donkey anti-rat IgG
(Fab⬘)2-biotin conjugate and visualized with avidin-FITC. Then, a rabbit
anti-mouse laminin Ab was applied and detected with donkey anti-rabbitTexas Red conjugate. The concentrations and incubation time for each
were as previously specified.
For the detection of IgMhighIgDlow marginal zone B cells, the sections
were stained with anti-IgD and detected with donkey anti-rat IgG-Texas
Red. After incubation with NRIgG (60 ␮g/ml) to block the unoccupied
Ig-binding sites on the donkey anti-rat IgG-Texas Red reagent, the sections
were stained with anti-mouse IgM-FITC.
For the detection of germinal center cells, tissue sections were stained
with biotin-conjugated peanut agglutinin 1/100 (Vector Laboratories) and
detected with avidin-FITC 1/1000 (also from Vector Laboratories).
For the single-color detection of all other Ags, the specific primary Abs
were applied and detected with appropriate FITC-conjugated secondary Abs.
The Journal of Immunology
2467
FIGURE 1. Expression pattern of laminin in wild-type and L1 KO spleens. Splenic sections from wild type (left) or L1 KO (right) mice were stained
with rabbit anti-mouse laminin Ab. The double arrows outline the margin of white pulp. Opposing arrows depict the outer and inner boundaries of the
marginal sinus. Opposing arrowheads depict the outer and inner boundaries of the marginal zone. The L1 KO section has a fragmented and discontinuous
pattern of laminin staining (right). MS, marginal sinus; MZ, marginal zone; RP, red pulp; WP, white pulp. Bar, 50 ␮m.
subsets were quantified by flow cytometry (Coulter Profile, Coulter,
Miami, FL).
The mice were immunized with 5 ⫻ 108 SRBC by i.p. injection on day 0
and then rechallenged with 3 ⫻ 108 SRBC i.p. on day 18. Sera were
collected by tail vein bleed on days 7, 14, and 25.
Soluble SRBC proteins were extracted using 0.5% Triton X-100 in 300
mM NaCl, 50 mM Tris-Cl. The SRBC protein extract was coated onto
polyvinyl chloride (PVC) microtiter plates (Costar, Cambridge, MA) at 20
␮g/ml overnight at 4°C. The plates were then blocked with 5% BSA for 1 h
at RT. Between steps, the plates were washed 5– 6 times with PBS, 0.2%
Tween 20 (PBS/T). Serum, 50 ␮l, diluted either 1:30 or 1:100 in PBS/T,
was incubated for 1 h at RT. Mouse anti-SRBC protein Abs were detected
with an alkaline phosphatase-conjugated rabbit anti-mouse IgM or IgG
(Sigma) after rinsing out unbound mouse serum from the PVC microtiter
wells with PBS/T. The alkaline phosphatase-conjugated rabbit anti-mouse
IgM or IgG was then incubated for 1 h at RT at a dilution of 1:1000 in
PBS/T. Colorimetric development was performed with the alkaline phosphatase substrate, p-nitrophenyl phosphate (Sigma) and was read at 405 nm
in a microplate reader (Bio-Tek Instruments, Winooski, VT).
Results
Matrix abnormalities in L1 knockout (KO) spleen
As a first step in analyzing L1 KO mice, we examined peripheral
lymphoid organs at necropsy. LNs and Peyer’s patches were unremarkable (data not shown). Splenic architecture, on the other
hand, was abnormal. Specifically, there was a striking and selective abnormality at the red-white pulp border.
To generate a view of the splenic white pulp framework, we
performed immunofluorescence microscopy on frozen sections of
mouse spleen. We stained the spleen sections with a polyclonal
rabbit anti-mouse laminin Ab. Because laminin is a component of
the reticular matrix of the spleen, it outlines the margins of the
white pulp and vasculature (28). As shown in Fig. 1 (left), the
Malformation of the splenic marginal sinus in L1 KO mice
To elucidate the nature of this matrix abnormality, spleens from
both wild-type and L1 KO mice were embedded in plastic and
analyzed by light microscopy (Fig. 2) and transmission electron
microscopy (Fig. 3). In a normal spleen, a flattened layer of sinus
lining cells can be traced along the margin of the white pulp. The
sinus lining cells have ovoid nuclei and elongated slender processes connecting to adjacent lining cells, forming a continuous
lining (Fig. 2, left, and Fig. 3, top). In Fig. 2 (left ), the double
arrows identify the white pulp border. For the most part, it is comprised of elongated slender cytoplasmic processes. A single arrow
(Fig. 2, left) identifies the cell body of a marginal sinus lining cell.
In Fig. 3 (top), arrows denote the cytoplasmic processes of two
flattened reticular cells. The arrow adjacent to the letters SLC identifies a sinus lining cell. The other arrow, adjacent to RC, denotes
a reticular cell defining the outer boundary of the marginal sinus.
In contrast, the splenic marginal sinus lining cells in L1 KO
mice were difficult to find. Marginal sinus lining cells in L1 KO
mice did not regularly contact other sinus lining cells to form a
continuous white pulp border. Rather, they displayed an abnormal
stellate, rather than a flattened, linear cell shape. Their cytoplasmic
processes were shorter, oriented in random directions, and often
FIGURE 2. Disruption of marginal sinus in L1 KO spleen. In the wild-type spleen (left), the double arrows outline the margin of the white pulp. A pair
of opposing arrowheads indicates the inner and outer boundaries of the marginal sinus. In the L1 KO spleen (right), the white pulp margin is difficult to
identify. The series of arrowheads identifies what we believe is the probable location of white pulp margin. MS, marginal sinus; MZ, marginal zone; RP,
red pulp; SLC, sinus lining cell; WP, white pulp. Bar, 20 ␮m.
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Immunization and ELISA
laminin staining of wild-type spleen outlines the white pulp margins as a smooth and continuous line. Laminin staining also outlines some fine matrix material in the marginal zone. As a result,
the border of the marginal zone (MZ, arrowheads) and marginal
sinus (MS, opposing arrows) can be clearly distinguished (Fig. 1,
left). By contrast, the laminin staining pattern of the L1 KO spleen
reveals an irregular, fragmented, and discontinuous white pulp
border. The marginal sinus and marginal zone appear to be malformed as well (Fig. 1, right).
2468
MALFORMATION OF THE WHITE PULP BORDER IN L1 KO MICE
shapen sinus lining cells, and a series of arrowheads outline the
presumed white pulp border. In L1 KO mice, this border is often
difficult to identify. The marginal sinus is absent. One of the stellate-shaped sinus lining cells is shown at higher magnification in
Fig. 3 (bottom) (“SLC”).
Normally, the marginal sinus is delimited by sinus lining cells of
the white pulp and the opposing reticular cells adjacent to marginal
zone. Few RBC are found in the marginal sinus (Fig. 2, left, and
Fig. 3, top). However, in the L1 KO spleen, this anatomic distinction is absent. The reticular cells of the marginal zone are rare, and
the marginal sinus is no longer distinguishable as a structure distinct from the marginal zone. As a result, RBC migrate right up to
the white pulp border (Fig. 2, right, and Fig. 3, bottom). These data
demonstrate that the splenic marginal sinus in L1 KO mice is abnormal. Moreover, the cellular abnormality spatially correlates
with the abnormal pattern of laminin immunofluorescence staining
as previously shown in Fig. 1.
L1 expression on sinus lining cells
discontinuous from the adjacent sinus lining cells. These features
are readily apparent in Fig. 2 (right) and Fig. 3 (bottom). In Fig. 2,
the red and white pulp can be readily distinguished by the location
of pale blue-staining erythrocytes. Arrows (“SLC”) identify mis-
FIGURE 4. L1 expression on the margin of
the white pulp. Splenic sections 32 ␮m thick
(left) or 6 ␮m thick (right) from BALB/c mice
were stained with the rat anti-mouse L1 mAb
324. Double arrows outline the margin of the
white pulp. L1 is strongly expressed on cells at
the white pulp margin and cells around the central artery (left). Also note that some lymphocytes
weakly expressed L1 (left). CA, central artery;
RP, red pulp; WP, white pulp. Bar, 200 ␮m (left)
or 50 ␮m (right).
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FIGURE 3. Transmission electron micrographs of the splenic marginal
sinus. Top, wild-type spleen. The marginal sinus, a space delineated by the
SLC and the RC, is clearly identified. Bottom, L1 KO spleen. The marginal
sinus cells and reticular cells that delimit the borders of the marginal sinus
are not readily identified. The sinus lining cell is abnormally shaped. Also
note that RBC come very close to the margin of white pulp. MS, marginal
sinus; MZ, marginal zone; RC, reticular cell; SLC, sinus lining cell; WP,
white pulp. Bar, 5 ␮m.
We then examined whether L1 expression in wild-type mice spatially correlates with the abnormally formed marginal sinus lining
cells in L1 KO mice. We expect that if the absence of L1 is the
cause of the structural abnormality, then those cells at the white
pulp border will likely express L1 under normal circumstances. By
examining the splenic structure of normal BALB/cByJ mice, we
localized L1 expression to the edge of the white pulp by immunofluorescence staining using an L1 mAb. L1⫹ lining cells are
present in the periphery of the white pulp, at the same approximate
location as the marginal sinus (Fig. 4). We sometimes found that
L1⫹ staining almost completely circumscribed the entire white
pulp (Fig. 4, left). However, most of time, only part of white pulp
margin was stained (Fig. 4, right, and Fig. 5, top). L1 staining is
denoted by double arrows at the white pulp periphery. We also
noted intense staining around the central artery in the center of the
white pulp. Two-color staining for tyrosine hydroxylase demonstrated that the periarteriolar staining was due to the expression of
L1 by sympathetic neurons innervating the spleen (data not
shown). However, such neurons did not innervate the marginal
sinus. In addition, lymphocytes within the white pulp also weakly
expressed L1 (Fig. 4, left). Low levels of lymphocyte L1 expression have been described previously (17).
The area of the marginal sinus/marginal zone contains a variety
of specialized cell types. To pin down the precise location of L1
expression within this area, we performed colocalization studies
using two-color immunofluorescence. L1 was stained using fluorescein while other cell type-specific markers were stained with
Texas Red. Optical filters were used that do not allow spillover
into the other respective color. Because the sinus lining cells of the
The Journal of Immunology
2469
splenic marginal sinus are described to express MAdCAM-1 (29),
we compared the localization of L1 and MAdCAM-1. Fig. 5 shows
the staining pattern of L1, MAdCAM-1, and a two-color overlay of
both (top, middle, and bottom, respectively). As seen in the bottom
panel, L1 and MAdCAM-1 colocalize along the margin of the
white pulp, indicating that the L1⫹ cells at the margin of white
pulp are sinus lining cells. Moreover, these L1⫹ sinus lining cells
sometimes expressed high levels of vimentin (data not shown),
often associated with a mesenchymal, probably fibroblast-like origin (30). Marginal metallophilic macrophages (MMM) that express MOMA-1, situated at the margin of white pulp, did not express L1 (data not shown). A separate macrophage population in
the marginal zone and red pulp areas that expresses CD11b also
did not express L1 (data not shown).
These results indicate that L1 is expressed by MAdCAM-1⫹
sinus lining cells. Moreover, the location of L1 expression in wild
type mice correlates with the location of the structural abnormalities (marginal sinus) in L1 KO mice.
FIGURE 5. The expression of L1 on MAdCAM-1⫹ sinus lining cells.
Splenic sections (6 ␮m) were stained with 324 mAb (FITC, green color)
and anti-MAdCAM-1 (Texas Red, red color). The pictures were taken
using an FITC filter (top), a Texas Red filter (middle) or a double exposure
of both filters (bottom). The arrows indicate the margin of white pulp and
the staining of both L1 and MAdCAM-1. Some nerve fibers around the
central arteriole and inside the white pulp also express L1 (top, N). L1 and
MAdCAM-1 staining colocalize at the margin of white pulp (bottom). RP,
red pulp; WP, white pulp; N, nerve fiber. Bar, 50 ␮m.
FIGURE 6. Cellular analysis of L1 KO spleens.
Splenic sections from wild-type (A and C) or L1
KO mice (B and D) were stained to highlight cell
subsets within the marginal zone. MMMs are
stained green (fluorescein) in A and B and are counterstained with rabbit anti-mouse laminin (Texas
Red). In A and B, the arrows indicate the white pulp
border and the arrowheads denote the MMMs in
the marginal zone. The poorly defined white pulp
border in L1 KO mice (B). IgMhigh IgDlow marginal
zone B lymphocytes are highlighted in C and D.
These cells are bright green after staining with for
IgD (Texas Red) and IgM (FITC) (C and D). In C
and D, opposing arrows point out the IgMhighIgDlow
marginal zone B cells. BF, B cell follicle; RP,
red pulp; WP, white pulp. Bar, 50 ␮m.
We then examined whether cell populations inside the white pulp
or around the marginal sinus were affected by the malformation in
the marginal sinus and sinus lining cells in L1 KO mice. The
individual cell types were identified with cell type-specific Abs,
either in situ or by flow cytometry. Because the abnormalities we
found were concentrated in the marginal sinus and marginal zone,
we examined two different cell populations known to inhabit this
microanatomic region. Namely, we determined whether MMMs
and marginal zone B lymphocytes were present and, if so, whether
they are located in their normal anatomic location in L1 KO mice.
As previously mentioned, there is a rim of MMMs situated at the
margin of the white pulp, in the marginal zone. MMMs in L1 KO
mice were examined by double staining of splenic sections with
anti-laminin and MOMA-1 (Fig. 6, A and B). The laminin counterstain helps identify the white pulp border. In contrast to the
wild-type mice (and as previously described), L1 KO mice display
an irregular, poorly defined white pulp border (Fig. 6, A and B).
Nonetheless (and similar to wild-type mice), L1KO mice demonstrate a rim of MMMs located along the white pulp border (Fig. 6,
A and B). This finding suggests that L1 is not solely responsible for
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Cellular analysis of L1 KO spleens
2470
MALFORMATION OF THE WHITE PULP BORDER IN L1 KO MICE
Immune response of L1 KO mice
To test whether the immune response is impaired in L1 KO mice,
we injected L1 KO mice and littermate controls with SRBC i.p. on
days 0 and 18. The anti-SRBC IgM and IgG immune responses
were measured by ELISA using extracted soluble SRBC protein as
Ag. As shown in Fig. 7, there is no significant difference in the
IgM and IgG titers of both primary and secondary responses. This
result indicates that L1 is not required for a T cell-dependent immune response.
Discussion
In this report, we investigate the role of L1 in the development of
normal splenic structure using L1-deficient mice. Using these
mice, we were able to analyze the role of L1 in lymphoid organ
development over longer time periods than otherwise possible using L1-specific mAbs. Therefore, these data provide a molecular/
structural correlation, implicating L1 in the development of the
white pulp border. Specifically, we conclude that L1 serves a critical and highly selective role in the development of the splenic
marginal sinus. These data help define a poorly characterized sinusoidal boundary that defines the border of the splenic white pulp,
and across which mononuclear cells transmigrate to enter the
white pulp.
Our most important finding is that L1 serves an important role
in the structural integrity of this microanatomic region. Specifically, L1 is expressed on the flattened sinus lining cells at the white
pulp border. The congenital absence of L1 results in nearly a complete absence of the marginal sinus and disorganization of RCs in
the marginal zone. There were obvious gaps in the white pulp
boundary, as discerned by light and electron microscopy. Nonetheless, the white pulp mononuclear cells still maintained their
cohesion to each other and the ability to exclude other blood cellular elements, such as erythrocytes and granulocytes. Although
the boundary was damaged, there was still a distinction between
the white and red pulp in L1 KO mice. These observations lead us
to conclude that other factors besides the structural integrity of the
boundary also regulate cellular traffic into the white pulp.
The marginal zone, and possibly the marginal sinus, are the
major sites of termination for the branches of central arteriole (4 –
6). Consequently, an important function of this region is to selectively channel certain cell types into the white pulp. Namely, the
direction of blood flow is toward the red pulp, as indicated by the
left-facing arrow (Fig. 8). Representative blood cells such as erythrocytes and granulocytes are abundantly found in the red pulp and
marginal zone. Selected mononuclear cells, on the other hand, migrate into the white pulp. As a result, the marginal sinus (the area
immediately adjacent to the white pulp) is rich in mononuclear
cells but has few other blood cells, such as erythrocytes or granulocytes. The mechanisms underlying this selective migration are
poorly understood. One possibility is that most blood cells are
passively steered along with the flow of blood toward the splenic
red pulp. To enter the splenic white pulp, mononuclear cells probably must actively locomote, possibly under the influence of chemoattractant agents such as chemokines. If this hypothesis is correct, then partial defects in the reticular lining of the splenic white
pulp might have minimal effect on the coalescence of mononuclear
cells forming a white pulp.
What role does L1 serve in the development and maintenance of
the normal integrity of the marginal sinus lining? We envision two
likely possibilities. First, L1 may be involved in the adhesion of
sinus lining cells to their associated reticular fibers. Laminin is a
component of the extracellular matrix at the white pulp periphery
(28) and is a known ligand for L1 (31). Second, L1 may be involved in the intercellular interaction between adjacent sinus lining
cells. This interaction may be mediated by a homotypic interaction
of L1 on adjacent sinus lining cells.
The biological significance of marginal sinus lining cells lies in
the fact that it is the final physical barrier that mononuclear cells
Table I. Cellular analysis of L1 KO spleen
Analysis
Subset
Quantity/Location
Histological
T/B cell segregation
FDC
Germinal center development
Marginal zone B cells
MMM
Interdigitating dendritic cells
Normal
Normal
Normal
Normal
Normal
Normal
Flow cytometricb
CD4⫹ cells
CD8⫹ cells
IgM⫹ cells
a
Wild Type (%)
L1 KO (%)
20.4 ⫾ 2.8
15 ⫾ 2.4
45.5 ⫾ 3
18.5 ⫾ 2.7
17.8 ⫾ 3
46.9 ⫾ 4.8
a
Single-color or two-color immunofluorescence microscopy were performed on splenic sections of both L1 KO mice and
wild-type littermate controls. The specific cell subsets were stained with cell type-specific Abs as described in Materials and
Methods. For all subsets, “normal” refers to both approximate quantity and tissue location.
b
For flow cytometric analysis, splenocytes from three L1 KO mice and three wild-type littermate controls (in C57BL/6
background) were stained with fluorochrome-conjugated mAbs. The numbers shown respresent the mean ⫾ SD.
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their localization in the marginal zone. Similarly, the location and
the approximate number (as estimated using immunofluorescence
microscopy) of IgMhighIgDlow marginal zone B cells in L1 KO
mice were comparable with those of wild-type mice (Fig. 6, C and
D). These cells are those that stain bright green (IgM) along the
edge of the follicle in Fig. 6, C and D. B lymphocytes that coexpress high levels of IgM and IgD are stained orange, from the
combination of green and red. These results indicate that cell subsets associated with the marginal zone are not noticeably affected
by the splenic structural abnormality in L1 KO mice.
Table I summarizes our findings regarding follicle development
and segregation of T/B lymphocytes in L1 KO mice. We find these
parameters to be indistinguishable from wild-type mice. Follicular
dendritic cells (FDC) and interdigitating dendritic cells were also
present in their normal locations (B and T cell zones, respectively)
and approximate number, as estimated visually. After immunization, germinal centers (highlighted using a peanut agglutinin-biotin
conjugate) also developed normally in L1 KO spleens (data summarized in Table I). Flow cytometric analysis also showed that the
major T and B lymphocyte subsets in the L1 KO spleen were
present in proportions comparable with those of wild-type littermates (Table I).
The Journal of Immunology
must traverse before entering the white pulp (32). This “transmigration” across the white pulp sinusoidal border is as yet poorly
characterized. Certain features suggest that novel mechanisms, distinct from those in blood vascular transmigration, are involved. For
example, the shear force and blood flow velocity in the marginal
sinus may be lower than those in postcapillary venules. This supposition is based on the fact that the marginal sinus and marginal
FIGURE 8. Schematic representation of microanatomy of white-red pulp border. PMN,
polymorphonuclear leukocyte.
zone are relatively open spaces as compared with a postcapillary
venule. Consequently, it is likely that the initial rolling step, negotiating initial contact between a leukocyte and endothelium, is
absent in the marginal sinus.
Fig. 8 is a schematic representation of the microanatomy at the
red-white pulp border of wild-type mice. The white and red pulp
boundaries, marginal sinus, and marginal zone are delineated by
flattened, elongated reticular cells. These reticular cells comprise
the marginal sinus lining cells and reticular cells of the marginal
zone. They are distinct from endothelium, because they do not
assume a tall, activated morphology and do not express CD31
(platelet endothelial cell adhesion molecule-1) (PECAM-1), a
marker for endothelial cells. Also present at the red-white pulp
border are some rather unique cell types, including MMMs, a subset of B lymphocytes expressing the IgMhighIgDlow immunophenotype, and a subset of marginal zone macrophages. Of minor note
is that we observed the MMMs to be located just outside the white
pulp boundary. A previous description of MMMs placed them just
inside the white pulp boundary (5). This location was discerned
using two-color immunofluorescence; MOMA-1 Abs identified
MMMs whereas laminin identified the extracellular matrix of the
white pulp boundary.
Apart from L1, the only other well-characterized adhesion molecule expressed by marginal sinus lining cells is MAdCAM-1 (29).
MAdCAM-1 is also an Ig superfamily adhesion molecule. It is
expressed by mucosal venules and is responsible for the homing of
lymphocytes to Peyer’s patches and the intestinal lamina propria
(33, 34). Its function on marginal sinus lining cells is not known.
In this regard, L1 is the only adhesion molecule that is demonstrated to directly serve a function in the development and preservation of the splenic white pulp boundary.
In this paper, we describe that L1 expression on marginal sinus
lining cells is somewhat heterogeneous. Namely, marginal sinus
lining cells do not express L1 all the time. We believe that L1 is
temporally regulated, in response to as yet unidentified signals.
One of these signals may the cytokine TNF-␣. Pancook et al. (18)
identified TNF-␣ as capable of up-regulating L1 expression on
human dendritic cells. In addition, our previously published findings suggest a temporal functional role for L1. We previously described our findings that L1 antagonism (with an L1 mAb) disrupted lymph node sinusoidal architecture, but only in LN
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FIGURE 7. Immune response of L1 KO mice to SRBC. Four male L1
KO mice and their littermate controls (in a C57BL/6 ⫻ 129 background)
were immunized with 5 ⫻ 108 SRBC i.p. on day 0 and rechallenged with
3 ⫻ 108 SRBC i.p. on day 18. Sera were collected at days 7, 14, and 25.
The titers of anti-SRBC IgM (A) and anti-SRBC IgG (B) were determined
by ELISA. Mean ⫾ SE is represented. For both IgM and IgG titers, there
are no statistically significant differences between the L1 KO mice and
littermate controls.
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MALFORMATION OF THE WHITE PULP BORDER IN L1 KO MICE
tionally redundant molecules. Without such redundancy, the L1null genotype is lethal or nearly so. With increasing levels of
redundant molecule(s), the L1-null genotype has little to no phenotypic effect. There is ample precedent for such functional redundancy, such as among the selectins in mediating initial contact
between leukocytes and vascular endothelium.
We previously reported that administration of an L1 mAb during an immune response disrupted the normal remodeling of the
fibroblastic reticular system in LNs. In these L1 KO mice, we did
not find any obvious abnormalities in LNs. This discrepancy might
be due to functional compensation by other, as yet unidentified,
L1-like molecules in LNs of L1 KO mice. Several L1 homologues
have been identified in the nervous system (9, 39, 40), but their
expression outside the nervous system is largely unexplored.
Moreover, our previous report utilized a distinct model involving
acute hypertrophic responses after immunization in normal mice. It
is possible that the sudden disruption of L1 function by L1-specific
Abs in a normal mouse may have greater structural consequences
than congenital absence of L1.
Several genetically targeted mutant strains of mice, notably the
family of lymphotoxin-␣, lymphotoxin-␤, TNF, and their receptors, have been reported to have structural abnormalities in peripheral lymphoid organs (41). For example, the lymphotoxin-␣ KO
mice developed structural abnormalities of the spleen, and a complete absence of LNs and Peyer’s patches (42). The TNF KO mice
had a decreased number of Peyer’s patches and a defect in FDC
development (43). We have observed that TNF and TNF receptor
I/II KO mice have a similar irregularity of the white pulp border as
that of L1 KO mice (data not shown). As expected, these mice also
show an abnormal distribution of L1⫹ cells at the white pulp border. A naturally mutant mouse strain, aly/aly, also demonstrated
severe developmental abnormalities in all peripheral lymphoid organs (44). Of particular interest, aly/aly mutant mice also had developmental abnormalities in the splenic marginal sinus (45).
However, unlike all these other mutant strains, our L1 deficient
mice had a selective defect on splenic marginal sinus development;
no other structures were affected.
In summary, our findings indicate that L1 serves a crucial role
in the proper development of the architecture at the white pulp
border. The most notable defect resulting from this improper formation of white pulp lining was the near complete absence of the
marginal sinus. These structural abnormalities correlate with the
location of L1 expression in normal mice. By characterizing the
molecular and functional features of the white pulp lining, we believe it may help shed light on the function of the splenic marginal
sinus.
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