Download Conjunctival-associated lymphoid tissue: evidence for a role

ConjunctivQl-ossociQted Lymphoid Tissue: Evidence for
a Role in the Secretory Immune System
Rudolph M. Franklin and Luis E. Remus
A specialized lymphoid tissue in rabbit conjunctiva was studied by various histologic and immunologic
techniques and compared with similar structures along other mucosal surfaces. The flattened conjunctival
lymphoepithelium overlying the lymphoid follicles was devoid of goblet cells. This lack of goblet cells
is characteristic of epithelium overlying similar lymphoid collections in gut and bronchus. The lymphoid
follicles demonstrated neither intra- nor extracellular immunoglobulin, and the lymphocytes in these
follicles were composed of B-cells and T-cells, when studied by various immunologic techniques. A
high proportion of these lymphocytes showed surface immunoglobulin A (IgA), and a high proportion
of IgA precursors were determined by pokeweed mitogen (PWM) stimulation in 4-day cultures. The
morphologic and immunologic results are similar to those obtained from gut and bronchus, tissues
known to disseminate lymphoid cells to other mucosal sites already committed to antigen and IgA
isotype. It is speculated that conjunctival associated lymphoid tissue of rabbit is part of a generalized
system of secretory immunity capable of sampling conjunctival applied antigen, and then disseminating
cells committed to IgA antibody production to other mucosal sites. Invest Ophthalmol Vis Sci 25:181—
187, 1984
Lacrimal gland has been demonstrated to contain
a high proportion of both immunoglobulin A (IgA)
plasma cells and adjacent acinar epithelial cells, that
contain secretory component (SC), in humans 1 ' 2 ' 3 and
rabbits.4 These findings are consistent with observations
in other mucosal-associated tissues5 and suggest that
lacrimal gland constitutes part of a common mucosal
immune system.6 The IgA plasma cells associated with
various mucosal tissues originate from B-lymphocytes
committed to IgA production found in gut-associated
lymphoid tissue (GALT) and bronchus-associated
lymphoid tissue (BALT) and migrate as committed
B-lymphocytes to the various mucosal-associated tissues.7'8
Specialized lymphoid structures located adjacent to
small intestine epithelial cells (Peyer's patches [PP], a
component of GALT)9 and to bronchus epithelial cells8
apparently encounter luminal antigen, initiating a sequence of events that results in a commitment to antigen-specific B-cells of the IgA isotype. Morphologic
observations have suggested that a similarly specialized
structure exists adjacent to conjunctival epithelium in
the rabbit.10 This structure is composed of lymphoid
follicles devoid of plasma cells with an overlying lymphoepithelium. The present experiments confirm several of the previously described morphologic characteristics of this conjunctival-associated lymphoid tissue
(CALT) and add morphologic as well as functional
characteristics of this tissue consistent with studies on
PP and BALT.
From the Lions Eye Research Laboratories, LSU Eye Center,
Louisiana State University School of Medicine, New Orleans, Louisiana.
Presented in part at the Association for Research in Vision and
Ophthalmology meeting in Sarasota, Florida, May, 1982.
Supported in part by Grant RO1 EY03028, from the National
Eye Institute.
Submitted for publication: December 13, 1982.
Reprint requests: to R. M. Franklin, MD, LSU Eye Center, 136
South Roman, New Orleans, LA 70112.
Tissue Preparation and Histologic Technique
Materials and Methods
Rabbits
New Zealand white outbred rabbits of both sexes,
at least 16 weeks of age and weighing greater than 2.5
kg, were used in these studies. By hand light examination, all animals contained easily identifiable CALT
in the inferior cul de sac of the conjunctiva and, in
most instances, contained smaller patches of similar
nodules in the superior conjunctival cul de sac." The
rabbits were killed by an overdose of intravenous sodium pentobarbital.
Tissues from six rabbits were immediately removed
after sacrifice and placed into 10% neutral buffered
formalin. Each tissue was sectioned with a razor blade.
One-half of each tissue was embedded into paraffin,
sectioned, and stained with hematoxylin and eosin,
periodic acid-Schiff, and methyl green-pyronin.
The remaining half of each tissue was prepared for
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / February 1984
examination by the fluorescent antibody technique.
Tissue was removed from the 10% neutral buffered
formalin after 4 hr and placed into 30% sucrose for
18 hr. Serial sections were cut at 4-6 fi in a cryostat
(-30°C) and then stained individually for rabbit a, y,
and n heavy chains by the direct method.12
Fluorescent Antibody Reagents and
Fluorescent Microscopy
Fluorescein-conjugated goat anti-a, anti-7, and antiH rabbit heavy chains were obtained from a commercial
source (Cappel Laboratories, West Chester, PA) and
were tested for specificity by immunoelectrophoresis
and demonstration of accepted proportions of isotypespecific plasma cells in normal rabbit spleen, lymph
node, and ileum. End-point dilutions were obtained
for each reagent, and dilutions of reagents were selected
for use that were two serial dilutions less than the endpoint, usually 1:4 or 1:8. Afluoresceinatedgoat antihuman IgG serum (Cappel) was used as a fluoresceinated control serum.
Immunofluorescent stained tissue—CALT, PP,
ileum, spleen, and lymph node—from each rabbit were
examined separately with a Zeiss microscope equipped
for epifluorescence. Each tissue was examined at multiple levels with adjacent serial sections. A xenon light
source was used with filters specific for fluorescein.
Preparation of Single Cells
PP, spleen (SP), preauricular lymph nodes (PLN),
and CALT were excised immediately after sacrifice
from 22 normal rabbits, teased apart at 4°C in Hanks
balanced salt solution (HBSS) with 10 mM HEPES
buffer (pH 7.4) and 10 mM glucose. The suspended
cells were then centrifuged and resuspended at 4°C in
HBSS with HEPES and glucose two times. Red blood
cell lysis was accomplished by suspending the cell pellet
in a 10-fold volume excess of HEPES-buffered ammonium chloride, (nine parts 0.83% NH4Cl:one part
0.2 M HEPES, pH 7.0), for 10 min at 24°C. The cells
were rewashed and then suspended in 5 ml of HBSS
at 4°C. The suspended cells were centrifuged over a
Hypaque-Ficoll medium (5 parts 32.8%: 12 parts
14%).13 Viable cell counts were obtained by trypan
blue exclusion, and generally ranged between 85
and 95%.
Mitogens and Cell Culture Techniques
Concanavalin A (Con A; Difco Laboratories, Detroit, MI), and pokeweed mitogen (PWM; GIBCO
Laboratories, Grand Island, NY) were reconstituted
to manufacturer's specifications with sterile, deionized
water to form stock solutions, and then stored
at -20°C.
Vol. 25
Stimulation of DNA synthesis in cells from various
lymphoid tissues of six rabbits was evaluated in tissue
culture plates with 96flat-bottomedwells (Linbro, New
Haven, CT). Cultures were prepared using 25 /A of
RPMI-1640 medium (GIBCO) containing mitogen,
25 fd of 25% heat-inactivated normal rabbit serum in
RPMI-1640 medium, and 25 n\ RPMI-1640 medium
containing cells. All cultures contained 2-mercaptoethanol at a final concentration of 5 X 10~5 M. The
mitogen solutions were added to the wells at three
times the final concentration. Dose response curves
were obtained for each mitogen, and the optimal dose
per well selected was 10 /ig Con A and 10 ^1 of a 1:50
PWM solution. Ten to 12 wells were prepared per
mitogen per dilution for SP, PP, and PLN, and four
culture wells were prepared for CALT, the variability
dependent upon cell recovery from each animal's tissues. Thefinalconcentrations of cells from each tissue
was adjusted to equal the concentration of CALT cells
for each experiment involving tissues from 6 rabbits.
The cell concentrations were 5-8 X 104 cells/well (23 X 105 cells/ml). Control experiments consisted of
an equal number of identically prepared wells, but
treated with HBSS instead of mitogen. The culture
plates were covered and incubated in a humidified
atmosphere of 5% CO2/95% air at 37 °C.
At 84 hrs, 10 n\ of tritiated thymidine (specific activity 24.7 Ci per mM; New England Nuclear, Boston,
MA) in RPMI-1640 medium was added to each well.
The cultures were terminated at 96 hrs and the cells
were isolated from individual wells using a cell harvester (Hiller Co., Madison, WI). The individual filter
papers representing individual wells were counted in
a liquid scintillation counter (Beckman LS-7500;
Beckman Instr., Irvine, CA), and the results were expressed in counts per min for mitogen and non-mitogen
stimulated (control) cultures. The mean counts were
calculated for each tissue of each animal, then a final
mean was obtained for each tissue of the six rabbits.
The ability of PWM to effect differentiation of some
rabbit lymphoid cells to plasma cells has been previously described.14 Mononuclear cells from either PLN,
PP, or CALT were placed into microtiter wells at a
final concentration of 1 X 105 viable cells/well in 20%
FCS with PWM at 7.5 ^I/well (30 /tl/ml). Tissues from
four rabbits were examined separately. The culture
plates were covered and incubated as described above.
After 4 days, individual wells were pooled for each of
the three tissues, washed with HBSS, and resuspended
in 5% FCS at 4 X 105 cells/ml.
Cytoplasmic immunofluorescence was used to examine cultures for plasma cells. The cells were deposited on glass slides using a cytocentrifuge (Shandon
Southern Instruments, Inc., Sewickley, PA). The cells
were fixed in acetone-methanol (1:2) for 10 min at
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EVIDENCE FOR ROLE OF CALT / Franklin and Remus
—30°C These slides were then air dried and stored at
—20°C until stained. Cytoplasmic immunoglobulin
was detected usingfluorescein-conjugatedantisera by
placing one drop onto the fixed cells and incubating
for 30 min in a humid chamber. The slides were then
washed twice in PBS for 10 min at room temperature.
A cover slip was applied using mounting medium
(Aquamount; Lerner Laboratories, New Haven, CT),
and the slides were examined using the fluorescence
microscope described above.
Membrane Immunofluorescence of Cells from
Normal Lymphoid Tissue
Cells were suspended influorescein-conjugatedgoat
anti-rabbit a, y, or fi heavy chain specific reagents
diluted with HBSS (1:4 or 1:8) for 12 min at 37°C. A
fluorescein-conjugated goat anti-human IgG was used
as a control serum. The cells were washed once and
resuspended in HBSS supplemented with 5% FCS at
4 X 105 cells/ml. The cells were then deposited on
glass slides using a cytocentrifuge andfixedin acetonemethanol (1:2) for 10 min at —30°C. A cover slip was
applied, and the slides were examined using the flu-
orescent microscope described above. Cells were
counted at X160 magnification in eight separate fields
for each of the three reagents. A fourth slide, stained
with Wright-Giemsa stain, was similarly counted under
bright field conditions to determine the total number
of mononuclear cells per eight -separate X160 fields.
Results
Histology
The CALT tissue was composed of numerous lymphoid follicles immediately beneath and often encompassing the overlying conjunctival epithelium. The
normal stratified conjunctival epithelial cells were often
difficult to distinguish because of the intense infiltration
of small lymphocytes. Goblet cells were not seen in
the lymphoepithelium over the follicles with PAS stain,
but could be seen in normal proportions immediately
adjacent to this area (Fig. la). Occasional follicles had
the appearance of secondary follicles with a central
area of larger, less mature lymphocytes. No plasma
cells were identified within any follicles. With methyl
green-pyronin stain, it was possible to identify occasional plasma cells immediately adjacent to follicles,
Fig. la. CALT tissue from conjunctiva of a normal rabbit. Goblet cells are present {arrows) in the epithelium (E) but are not observed in
the epithelium central to the edges of the follicle (F). (Periodic acid-Schiff stain, X100)
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INVESTIGATIVE OPHTHALMOLOGY & VI5UAL SCIENCE / February 1984
Vol. 25
Fig. lb. Higher magnification of central portion of Fig. la. The epithelium contains numerous lymphocytes and no goblet cells. (Periodic
acid-Schiff stain, XI60)
especially near the edge of a follicle and adjacent to
the normal conjunctival epithelium.
Fluorescent Microscopy of CALT
Immunofluorescent examination of rabbit CALT
showed only occasional plasma cells adjacent to the
follicles, and both IgA and IgG plasma cells could be
identified with a slightly greater proportion of IgA
staining cells. Immediately adjacent to follicles and
under the normal conjunctival epithelium, IgA plasma
cells were prominent, with few cells staining for IgG.
Only a rare IgM cell could be detected adjacent to
follicles or epithelium. The follicle failed to show fluorescence with any of the antisera.
Cell Yields Obtained from CALT and PP
CALT tissue from both eyes of 22 separate rabbits
yielded 1.2-4.0 X 105 cells, with a viability range between 80 and 90%. When cell suspensions were isolated
in HBSS without HEPES and glucose, the viability of
CALT cells ranged from 50 to 80%. Wright-Giemsastained smears of the CALT cell suspensions showed
greater than 90% small lymphocytes, with the re-
mainder being macrophages and epithelial cells. Usually, five PP were isolated from each rabbit and, occasionally, as many as eight. Between 3.0-8.0 X 107
lymphoid cells were isolated per animal, the variability
dependent upon PP number and size per rabbit, with
a viability of 85 to 95%. By Wright-Giemsa stain,
greater than 95% of these cells were shown to be small
lymphocytes and the remainder was a mixture of macrophages and other mononuclear cells.
Proliferative Responses to Mitogens
Dose-response curves were obtained for CALT, PP,
SP, and PLN for each mitogen. Con A doses were 2,
10, and 50 jig/ml, and PWM doses were 10 A*1 of a
1:10, 1:50, and 1:150 dilution of the stock solution.
Since so few cells were available from CALT, individual
rabbits were used for each mitogen along with control
wells. The maximum proliferation was consistently
obtained in each tissue with 10 ^g/ml of Con A and
1:50 dilution of PWM.
Considerable variation was found in the maximum
proliferation between animals, ranging from very high
responses for each tissue with each mitogen to low
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responses. This variation is evident in the standard
error of the mean (SEM) as shown in Table 1.
Con A responses were generally high but varied
greatly from rabbit to rabbit and among the four tissues
examined. PWM stimulation also varied among the
individual rabbits, but the best responses were obtained
with PP and PLN. SP and CALT consistently showed
much lower responses than with Con A.
Table 1. Comparison of responses to mitogens of
normal rabbit tissues in four-day cultures
Con A
Membrane Immunofluorescence
CALT, PP, and SP cells from 12 rabbits were examined for surface immunofluorescence. PP consistently yielded the highest percentage of IgA staining
cells, with fewer IgA B-cells in CALT, but significantly
more than SP (Table 3).
Discussion
These present experiments examine a lymphoid
structure observed consistently in rabbit conjunctiva
(CALT) 1 0 " and provide additional morphologic and
lymphocyte functional observations, consistent with
findings on GALT and BALT tissue. In the present
studies, the lymphoepithelium overlying CALT was
Control
CALT
92,000*
(23,500)f
15,500
(6,800)
600
(150)
PP
140,000
(46,000)
73,400
(17,100)
900
(450)
PLN
145,000
(77,000)
109,400
(39,600)
500
(70)
SPL
87,000
(31,300)
19,900
(8,600)
1,500
(332)
PWM Induced Differentiation of Lymphoid Cells
Cytocentrifuge preparations of cells were examined
for cytoplasmic staining at the beginning and after 4
days of culture. In addition, controls consisted of cells
cultured with HBSS in place of PWM for 4 days. Prior
to culture, CALT and PP showed less than 0.35% cytoplasmic staining cells equally distributed among the
three immunoglobulin isotypes. Spleen cells showed
1.0-1.5% cytoplasmic staining cells prior to stimulation, with a majority of IgG and IgM staining cells.
Viable cell yields following four days of culture with
PWM were 50-80% when compared with the cultures
at Day 0. Control cultures (unstimulated) showed a
recovery of less than 20%. The mean percentage of
cytoplasmic staining cells in PWM-stimulated cultures
after 4 days was 4.5% for PP, 18% for SP, and 12%
for CALT. Four-day cultures not stimulated with PWM
consistently showed fewer positively stained cells. The
isotype distribution presented in Table 2 is a ratio of
PWM stimulated to unstimulated cells from 4-day cultures performed separately on tissues from four rabbits.
Both PP and CALT showed an increase (4.7-fold) in
IgA staining cells as well as a slight increase in IgA
staining cells in SP. The 4.7-fold increase in IgA cells
in PP and CALT compared with SP was significant
for CALT (P = 0.048) but less significant for PP (P
= 0.06) by Student's /-test. Large increases in IgM were
also seen in SP and PP with a slight increase in CALT.
PWM
* CPM, mean of responses for six experiments, 10-12 wells/experiment.
jSEM.
Four wells/experiment for CALT (for details, see Materials and Methods).
found devoid of goblet cells, and the CALT follicles
contained no intra- or extracellular immunoglobulin.
Additionally, CALT tissue contained both T- and Bcells, with a large proportion of IgA-committed B-cells.
Finally, these experiments demonstrated a proportion
of B-cells in CALT capable of differentiation to IgA
plasma cells after mitogen stimulation, similar to previous studies on PP 14 and confirmed in the present
studies.
The response of rabbit lymphocytes to Con A and
PWM have shown that both mitogens stimulate Tcells; however, PWM acted on only a sub-population
of T-cells in addition to stimulating some B-cells.15
The present experiments demonstrated a high response
of all four lymphoid tissues with Con A and consistently
lower responses with PWM. Similar observations have
been seen in most rabbit lymphoid tissues by other
investigators, and the decreased PWM responses were
shown by them to represent a combination of responses
from both B-cells and a sub-population of T-cells.15
Therefore, the response of CALT tissue to Con A and
PWM (Table 1) indicates the presence of T-cells, although the PWM-sensitive component is less than in
PP. Other studies have demonstrated thymus-specific
antigens on a population of rabbit PP and BALT lymphocytes,16 which is similar to observations in other
species.5 In the present studies using mitogens, rabbit
Table 2. Differentiation of lymphoid cells after four
days of culture (ratio of PWM stimulated
to nonstimulated)*
IgA
IgG
IgM
SP
pp
CALT
1.2t
4.7
1
6.9
4.7
1
2.1
1
4.4
* Mean or ratio of four separate experiments (four rabbits).
f Ratio of cytoplasmic staining cells from PWM stimulated to nonstimulated.
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / February 1984
186
Table 3. Percentages of B-cell surface isotypes in
tissues from normal adult rabbits
CALT
PP
SPL
IgA
IgG
IgM
28* (2.3)f
43 (2.6)
17 (1.0)
18(3.1)
19 (2.9)
24 (3.5)
55 (3.8)
39(1.9)
59 (3.0)
* Calculated as percentage of total IgA + IgG + IgM surface staining cells.
fSEM, N = 12.
CALT has been shown to contain a component of Tcells.
PP and BALT have been shown to be an enriched
population of IgA precursor lymphocytes.7'8 Membrane
Ig studies on these tissues have demonstrated a higher
proportion of cells with membrane IgA in BALT (41%)
and PP (38%) as opposed to SP (17%).16 The present
experiments agree with these observations (Table 3)
and also demonstrate a high proportion of IgA B-cells
in CALT. A more forceful demonstration of the potential for CALT lymphocytes to provide IgA precursors was obtained by 4-day cultures with PWM (Table
2). CALT and PP, but not SP cells, responded with a
4.7-fold increase in IgA plasma cells. In a similar series
of experiments that employed the cell sorter in addition
to PWM stimulation, a B-cell fraction from PP containing surface IgA was shown to be the direct precursor
of IgA plasma cells.17 The experiments presented here,
then, demonstrate that CALT is also a source of IgA
precursors similar to PP and BALT.
It has been suggested that all mucosal surfaces are
interrelated to some degree.618 Migration of antigencommitted IgA B-cells from the surface, where antigen
encounter occurred to other surfaces, can proceed independently from prior antigen exposure at the second
mucosal surface.19 Although antigen may play some
role in the homing of the committed IgA B-cell, it is
certainly not absolutely necessary.20 The mechanism
is best understood for GALT, where a specialized epithelium covering the PP dome has been described.
The PP dome epithelium is flattened with surface microfolds, lacks microvilli and contains no goblet cells.21
This specialized epithelium can transfer luminal antigen by pinocytosis into the lymphoid portion of the
PP,22 and then cells committed to antigen and IgA
isotype can migrate to other mucosal surfaces.23 Recently, antigen-specific secretory IgA (slgA) has been
demonstrated in tears,24 suggesting that ocular adnexal
structures participate in this system of mucosal immunity, at least in their ability to accept IgA-committed
B-cells.
Whether CALT can likewise sample antigens and
then disseminate cells committed to antigen and IgA
isotype to other mucosal (sites has yet to be established.
One experiment, however, suggests this possibility.25
Vol. 25
Therightcorneas of rabbits were immunized with heatinactivated Herpes Simplex Virus, Type I (HSV). Neutralizing antibody of the IgA isotype was subsequently
demonstrated in the tears of both eyes, although at
much higher concentrations in the immunized right
eyes. One interpretation of this HSV experiment is
that HSV-committed IgA B-cells migrated from the
right to left ocular adnexa.
Previous morphologic studies demonstrated that
rabbit CALT contained specialized epithelial cells,
identical to PP dome cells, as well as a lack of plasma
cells in CALT, similar to GALT and BALT.910 The
present experiments demonstrate further similarities
among CALT, GALT, and BALT. The evidence suggests that CALT could function as a site of antigen
absorption, followed by dissemination of B-cells to
other mucosal surfaces where they can differentiate to
antigen-specific IgA plasma cells.
Key words: Peyer's Patch, conjunctival-associated lymphoid
tissue (CALT), mitogen, immunoglobulin A, slgA, plasma
cells
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