1953

J. Exp. Med.



The Rockefeller University Press • 0022-1007/98/06/1953/11 $2.00
Volume 187, Number 12, June 15, 1998 1953–1963
http://www.jem.org

L1 Antibodies Block Lymph Node Fibroblastic
Reticular Matrix Remodeling In Vivo

By Gino Di Sciullo,

*

Tim Donahue,

*

Melitta Schachner,

‡§

and Steven A. Bogen

*

From the

*

Department of Pathology and Laboratory Medicine, Boston University School of Medicine,
Boston, Massachusetts 02118; the

‡

Department of Neurobiology, Swiss Federal Institute of
Technology, CH-8093 Zurich, Switzerland; and

§

Zentrum für Molekulare Neurobiologie, Universität
Hamburg, D-20246 Hamburg, Germany

Summary

L1 is an immunoglobulin superfamily adhesion molecule highly expressed on neurons and in-
volved in cell motility, neurite outgrowth, axon fasciculation, myelination, and synaptic plas-
ticity. L1 is also expressed by nonneural cells, but its function outside of the nervous system has
not been studied extensively. We find that administration of an L1 monoclonal antibody in
vivo disrupts the normal remodeling of lymph node reticular matrix during an immune re-
sponse. Ultrastructural examination reveals that reticular fibroblasts in mice treated with L1
monoclonal antibodies fail to spread and envelop collagen fibers with their cellular processes.
The induced defect in the remodeling of the fibroblastic reticular system results in the loss of
normal nodal architecture, collapsed cortical sinusoids, and macrophage accumulation in mal-
formed sinuses. Surprisingly, such profound architectural abnormalities have no detectable ef-
fects on the primary immune response to protein antigens.
Key words: lymph node • L1 • cell adhesion molecule • fibroblastic reticular system •
architecture

L

ymph nodes (LNs) can undergo rapid and profound
hypertrophy during an immune response. However,
the mechanisms underlying lymphoid tissue matrix remod-
eling remain poorly understood. Previous studies of the
lymphoid reticular matrix in immune animals have largely
been descriptive in nature, as it has not been possible to
selectively disrupt this feature in an otherwise normal,
healthy animal.
Under normal immune conditions, the integrity of the
lymphoid tissue is maintained because of the adaptive na-
ture of the reticular matrix. This matrix, termed the fibro-
blastic reticular system (FRS),

1

is comprised of a fibrous
reticulum and associated fibroblasts (1). The reticular fibers
are primarily made up of type III collagen, but also contain
small amounts of elastic fibers (2), microfibrils (3), type IV
collagen (4), and laminin (5). The elastic components, ad-
mixed with the collagen bundles, confer resiliency to the
fibers during organ distention.
Anchored to the reticular fibers are reticular fibroblasts
(RFs), which ensheathe the fibers by wrapping around
them in a manner analogous to Schwann cell enclosure of
axons (1, 6). The enclosure of the reticular fibers by RF cy-
toplasmic processes is sealed by desmosome-like junctional
complexes at the juxtaposed RFs’ membranes (7).
The reticular fibers and associated fibroblasts together
form a three-dimensional cellular reticulum that forms the
walls of sinuses and delimits compartments within the
node. Based on these structural features, the FRS is be-
lieved to function primarily as (

a

) a mechanical supportive
scaffolding, (

b

) a substratum for immune cell motility, and
(

c

) partitions that establish immune microenvironments. It
has also been hypothesized that the extracellular space
formed by the RF enclosure of the reticular fibers may fa-
cilitate antigen transport between different compartments
within the node (8–10).
Our studies of LN architecture began when, in the
course of studying the function of the L1 cell adhesion
molecule in the murine immune system, we observed
structural abnormalities in LNs from mice injected with an
L1 mAb. L1 is a type I transmembrane glycoprotein with
an apparent molecular mass of 200 kD that is highly ex-
pressed by neural cells and tumors of neural origin (11). Al-
though L1 was isolated and purified initially from mouse

1
Abbreviations used in this paper:

ANOVA, analysis of variance; CLSM,
confocal laser scanning microscopy; DTH, delayed type contact hyper-
sensitivity; FRS, fibroblastic reticular system; M

f

, macrophage; NRIg,
normal rat immunoglobulin; RF, reticular fibroblast; RT, room tempera-
ture; TEM, transmission electron microscopy.

1954

L1 Is Important for Lymph Node Matrix Remodeling

brain extracts, it is also expressed by a variety of nonneu-
ronal cells. Cells of hematopoietic origin express L1 (12), as
do epithelial cells of the intestine (13) and male urogenital
tract (14). Of special relevance to this study, L1 expression
has also been demonstrated in mouse fibroblast L cells by
Western blot analysis of membrane preparations (15).
In this report, we examine the in vivo consequences of
influencing the effect of L1 during an immune response in
mice, using a previously described blocking rat anti–mouse
L1 mAb designated 324 (11). We find that in vivo adminis-
tration of the 324 anti-L1 mAb during the evolution of an
immune response prevents normal remodeling of the retic-
ular matrix of the draining popliteal LN.

Materials and Methods

Animals and Immunizations.

In vivo experiments were con-
ducted using 8–10-wk-old female BALB/CByj mice according
to the following protocol. On day 0, 25

m

g of KLH/CFA was in-
jected into the right footpad of each mouse. For all structural ex-
periments, 250

m

g of antibody was injected intraperitoneally each
day during days 2–5. Other studies testing the role of L1 during
the early phases of in vivo lymphocyte sensitization involved in-
jection with 300

m

g of antibodies on days 0–5 and killing on day
7. In these latter experiments, antibody injection on day 0 pre-
ceded KLH/CFA immunization by 3 h. Mice were killed either
on day 6 or day 7, and popliteal LNs were harvested. Animals
were cared for in accordance with The Institutional Animal Care
and Use Committee of Boston University School of Medicine.

mAb Preparation.

Normal rat immunoglobulin (NRIg) was
purified from normal rat serum (The Jackson Laboratory, Bar
Harbor, ME) by HPLC using a J.T. Baker ABx semipreparative
column (16). Likewise, the 324 and TIB 126 (American Type
Culture Collection, Rockville, MD) hybridomas were grown as
ascitic tumors in NCR/nude mice (Taconic Farms Inc., German-
town, NY), and mAbs were purified from ascites by HPLC.

Transmission Electron Microscopy.

For transmission electron mi-
croscopy (TEM) studies, whole LNs were fixed in Karnovsky’s 1/2
strength fixative at 4

8

C for

.

1 d. Tissues were washed with 0.1 M
phosphate buffer, postfixed in 1% OsO

4

in 0.1 M phosphate
buffer, stained with 2% uranyl acetate in 60% ethanol, and dehy-
drated through an ethanol gradient and embedded in Araldite
(E.F. Fullam Inc., Latham, NY). 0.055-

m

m sections were exam-
ined and photographed in an electron microscope (model 300;
Philips Electronics, Eindhoven, The Netherlands).

Immunohistochemistry and Immunofluorescence Microscopy.

For con-
focal microscopy and immunohistochemical studies, LNs were
embedded in OCT (Miles Inc., Elkhart, IN), snap frozen, cryo-
sectioned, and acetone fixed. L1 expression in LN sections was
detected using anti–mouse L1 hyperimmune rabbit serum, or
preimmune rabbit serum diluted 1:2,000 (Covance Research
Products Inc., Denver, PA). Rabbit anti–mouse laminin 1:100
was detected with biotin-conjugated goat anti–rabbit IgG diluted
1:100 (both from Sigma Chemical Co., St. Louis, MO) followed
by ABC-HRP (Vector Laboratories, Inc., Burlingame, CA).
Laminin staining was detected colorimetrically using 3,3

9

-diami-
nobenzidine (Sigma Chemical Co.). Macrophages were immu-
nostained with biotin-conjugated anti–MAC-1 mAb (5C6) at 1

m

g/ml.

Confocal Laser Scanning Microscopy.

Confocal laser scanning
microscopy (CLSM) was carried out on a CLSM microscope (Leica
Microsystems, Wetzlar, Germany) equipped with an argon ion
laser. The objective used was a 50

3

long working distance water
immersion lens. Continuous series of 2 scans/

m

m were recom-
bined to produce three-dimensional reconstructions of the entire
thickness of the LN tissue. Anti–vimentin-Cy3 1:50 (V-9 clone;
Sigma Chemical Co.) was used to stain frozen sections cut 48

m

m
thick, which had a post-acetone fixation thickness of 16

m

m.

Secondary In Vitro Lymphoproliferation.

LNs from KLH/CFA-
immune mice treated with either anti-L1 or control antibodies
were harvested on day 7. Lymphocytes were collected and
washed twice in ice-cold HBSS, resuspended in complete media,
and plated (5

3

10

5

cells/well) in flat-bottomed 96-well plates
(Corning Glass Works, Corning, NY) containing media alone or
with varying concentrations of soluble KLH (Calbiochem Corp.,
La Jolla, CA). Cells were cultured for 4 d, pulsed for 8 h with 1

m

Ci
of [

3

H]thymidine per well, and harvested using a PHD cell har-
vester (Cambridge Technology, Inc., Cambridge, MA). Counts
were determined using a liquid scintillation counter (model 1414;
Wallac, Gaithersburg, MD).

ELISA.

Day 7 serum was tested for KLH-specific IgM and
IgG antibodies by ELISA. Flexible PVC microtiter plates (Costar
Corp., Cambridge, MA) were coated overnight at 4

8

C with 20

m

g/ml KLH, and blocked with 4% BSA for 1 h at room temper-
ature (RT). Between each step, plates were washed five times
with PBS/0.2% Tween 20 (PBS/T). 50

m

l of serum, diluted 1:
100 in PBS/T, was incubated for 1 h at RT. Alkaline phos-
phatase–conjugated rabbit anti–mouse IgM and IgG gamma-spe-
cific antibodies (Sigma Chemical Co.) were diluted 1:1,000 in
PBS/T and incubated for 1 h at RT. Colorimetric development
of AP substrate (Sigma Chemical Co.) was read at 405 nm in a
plate reader (Bio-Tek Instruments, Inc., Winooski, VT).

Flow Cytometry and LN Cell Enumeration.

The total number
of cells in the draining LNs was quantified by trypan blue dye ex-
clusion with a hemocytometer. Lymphocyte subsets were quanti-
fied by flow cytometry (Coulter Profile; Coulter Corp., Miami,
FL) using the following biotin-conjugated rat anti–mouse mAbs:
ant-B220 (3A1/6.1 ATCC, TIB 146), anti-CD8 (ATCC, TIB
105), anti-CD4 (GK1.5), and anti-CD11b (5C6). FITC-avidin D
cell sorter grade was used at 600 ng/ml (Vector Laboratories,
Inc.). Each incubation (both the primary antibody and fluores-
cein-conjugated avidin) was for 45 min, at 4

8

C. Afterwards, cells
were washed, fixed in 2% paraformaldehyde/PBS, and stored at
4

8

C until assayed.

Results

Model of LN Hypertrophy.

To study the function of L1
in the immune system in vivo, we used a well-character-
ized immune response model in which subcutaneous injec-
tion of 25

m

g of KLH emulsified in CFA to the hind foot-
pad of a mouse elicits a vigorous immune response. In the
preimmune animal, the draining popliteal LN is modest in
size, averaging 1.1 mm in diameter (Table 1). However, by
day 6 after immunization, the mean greatest-width diame-
ter increases to 4 mm (Table 1). Since LNs are roughly
spherical in shape, this change in dimension represents a
63-fold increase in organ volume (i.e., 4

3

–1.1

3

). Such ex-
treme and rapid increases in organ volume must necessarily
be accompanied by extensive matrix remodeling, regulated
through poorly characterized mechanisms.
To elucidate the role of L1 in immune responses, we