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From the Faculty of Health Science,*
the Department of
Dermatology,
and the Department of
Pathology,
Okayama University Medical School,
Okayama, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B cells enter LN via the same high endothelial venules (HEV) as do T cells, and thus transverse T cell areas before entering follicles.3,4 It has been suggested that the site of primary B cell activation is within the T-cell areas of secondary lymphoid tissues.5-7 Ag-specific activated CD4+ T cells stimulate Ag-specific naive B cells to proliferate and differentiate into germinal center founder cells or into short-lived plasma cells essentially producing IgM.8 MacPherson et al 9-11 recently suggested that IDC act as matrix on which Ag-specific T and B cells interact efficiently. In addition, they suggested that IDC can deliver naive forms of antigens, which are essential for the initial activation of B cells, to Ag-specific resting naive B cells.10,11 In recent years, several investigators have demonstrated that IDC are directly involved in regulating T-cell-mediated humoral immune responses in humans.12,13 Bjorck et al 13 have demonstrated the morphological evidence indicating that naive B cells actually interact with DC in the human tonsils. However, little attention has been paid to the direct interactions between IDC and B cells, and little is known about this subject, particularly in the human immune system. In the present study, we investigated the direct morphological interactions of the human secondary lymphoid tissue IDC with T and B cells in situ and in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of leukocyte cell surface markers was assessed by direct immunofluorescent staining using the following fluorescent isothiocyanate (FITC) or phycoerythrin (PE)-conjugated murine monoclonal antibodies (mAbs): anti-CD1a, anti-CD3, anti-CD4, anti-CD8, anti-CD11c, anti-CD14, anti-CD20, anti-CD22, anti-CD40, anti-CD54 (ICAM-1), anti-CD80 (B7/B7–1), anti-CD86 (B70/B7–2), all of which were purchased from DAKO (Glostrup, Denmark). FITC- and PE-conjugated anti-HLA-DR (class II MHC) were purchased from Becton Dickinson (Mountain View, CA). The PE-conjugated anti-CD83 was purchased from Immunotech (Marseilles, France), and PE-conjugated anti-IL-2R (CD25) and anti-CD40-ligand (CD40L; CD154) were purchased from DAKO. For three-color flow cytometry, PerCP-conjugated anti-HLA-DR was purchased from Becton Dickinson.
For the detection of immunoglobulin classes, we performed indirect immunofluorescent staining using FITC- or PE-congugated anti-rabbit IgG (DAKO) as a secondary antibody, and nonconjugated rabbit polyclonal antibodies to human IgA, IgD, IgG, or IgM (DAKO) as primary antibodies. For the detection of S-100b protein, we performed indirect immunoperoxidase method using rabbit anti-S-100 ß subunit prepared as described previously,14 and horseradish peroxidase (HRPO)-conjugated goat anti-rabbit IgG (DAKO).
Tissue Preparation
The mesenteric lymph nodes, which were devoid of cancer metastasis, were obtained with informed consent from five patients with colon cancer at the time of surgery. The age range of these patients was 55 to 75 years (mean age 62). Some LN were cut, and the fragments were immediately frozen in liquid nitrogen and stored at -80°C until cryostat sectioning. 7-mm-thick sections were placed on slides and air-dried at room temperature for 1 hour, then fixed with cold acetone L for 10 minutes. Sections were stored at -80°C until use.
Cell Preparation and Culture Conditions
LN were cut into 2-mm3 cubes and put into a glass homogenizer (Pyrex, Tokyo, Japan) into which was added 10 ml of RPMI 1640 medium (Nissui, Tokyo, Japan) containing 10% heat-inactivated, low endotoxin (<6 pg/ml) fetal calf serum (Irvine Scientific, Santa Ana, CA). A single cell suspension was obtained by gentle pressure with a glass homogenizer without the addition of digestive enzyme. After the removal of dead cells and erythrocytes by Ficoll-Hypaque density gradient centrifugation, the cells were suspended in RPMI 1640 containing 10% fetal calf serum (FCS). The cells were then cultured at a concentration of 106 cells/ml in 5% CO2 in humidified air, and the culture media were half-changed every 7 days. Fresh or cultured LN cells (n = 105) were smeared on glass slides with a Cytospin (Sakura, Tokyo, Japan) at 1000 rpm for 5 minutes and air-dried. The smeared cells were then fixed with cold acetone for 10 minutes, air-dried, and examined without storage. For immunoperoxidase staining, smeared cells were fixed in 4% paraformaldehyde solution for 10 minutes without drying, rinsed in phosphate buffered saline (PBS), and air-dried.
Two-Color Immunofluorescent Staining
Frozen sections of LN and smeared suspended LN cells were incubated with 1:10-diluted normal goat serum (DAKO) for 15 minutes. The preparations were then incubated with both FITC-conjugated and PE-conjugated mAbs (20 µg/ml) for 30 minutes at room temperature. For the detection of immunoglobulin classes, the preparations were incubated with 1:200-diluted anti-IgD, 1:500-diluted anti-IgM, 1:1000-diluted anti-IgA, or 1:5000-diluted anti-IgG. The preparations were then washed with PBS and incubated with FITC- or PE-conjugated anti-rabbit IgG (1 µg/ml) for 30 minutes. The preparations were then rinsed with PBS and examined by fluorescent microscopy as described previously.15
Immunoperoxidase Staining for S-100 Protein
The smeared LN cells fixed in paraformaldehyde were treated with 0.3% H2O2 in methanol for blocking endogeneous peroxidase activity, and then incubated with normal goat serum diluted at 1:10 in PBS for 10 minutes to block nonspecific staining. The smeared cells were then stained for S-100b protein by indirect immunoperoxidase method as described previously.16 After the development of enzyme activity, the cells were counterstained with methyl green. The percentage of IDC in the smeared LN cells were determined by counting S-100+ dendriform cells/1000 LN cells.
Flow Cytometry
To determine the proportions of T cells and B cells in total LN cells, fresh LN cells prepared as described above or those cultured for 7 days were examined by two-color flow cytometry using FITC-conjugated anti-CD3 and PE-conjugated anti-CD22. Briefly, LN cells (n = 106) were incubated simultaneously with both of these mAbs (1 µg) for 30 minutes at 4°C.
The proportions of naive B cells and non-naive N cells in total LN cells were also determined by two-color flow cytometry. LN cells (n = 106) were incubated simultaneously with FITC-conjugated anti-CD20 (1 µg) and rabbit anti-human IgD (1 µg) for 30 minutes at 4°C. After washing with PBS, the cells were treated with PE-conjugated anti-rabbit IgG at a final concentration of 1 µg/ml for 30 minutes at 4°C.
For the detection of IDC, we performed three-color flow cytometric analysis using FITC-conjugated anti-CD40, PE-conjugated anti-CD83 or anti-CD86, and PerCP-conjugated anti-HLA-DR. As controls, FITC, PE, or PerCP-conjugated class-matched mouse immunoglobulin were used. Mesenteric LN cells (n = 106), which were obtained from one of the patients and cultured after washing with PBS, were fixed in 4% paraformaldehyde solution and examined by a FACScan as described previously.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the frozen sections, IDC were identified as
HLA-DRbright dendriform cells located in the T
cell areas of these LN. Almost all IDC co-expressed CD40, CD54, CD83,
and CD86 strongly, and CD80 weakly, but not CD1a (Figure 1,a–d)
. HLA-DR and CD40 were also
detected weakly on B cells and macrophages; CD54 (ICAM-1) was detected
on macrophages, endothelial cells of HEV, and follicular dendritic
cells (FDC) in the germinal centers; and CD80 and CD86 were detected on
a portion of the macrophages and B cells in the germinal centers.
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IDC usually exhibited extensively irregular contours and closely
contacted numerous T cells, which were mostly CD4+ T cells (Figure 1e)
.
Although a considerable number of CD8+ T cells were present in the T
cell areas, only a small number of them were found to attach to IDC
(Figure 1f)
. Only a small number of T cells attaching to IDC expressed
IL-2R and CD40L (data not shown).
A considerable number of B cells were scattered in the T cell areas.
Two-color immunofluorescent microscopy using FITC-anti-CD40 and
PE-anti-CD22 indicated that approximately 70% of IDC in the T cell
areas attach to a few CD22+ B cells (Figure 1g)
. Interestingly, some of
these attaching B cells were morphologically different from ordinary B
cells in extending one or two cytoplasmic projections, through which
some of B cells attached to IDC. B cells attaching to IDC were positive
for IgD and IgM (Figure 1h)
, but negative for IgA and IgG.
In addition, a small number of IDC, comprised of large dendriform cells
positive for HLA-DR, CD40, CD54, CD83, and CD86, were found in B cell
areas such as the periphery of the primary follicles (Figure 1i)
. They
were surrounded by numerous B cells and morphologically closely
connected with neighboring B cells. However, no IDC were found within
the germinal centers.
IDC in the Fresh LN Cell Suspension
Because S-100b protein is a specific cytoplasmic marker present
for IDC in the human lymphoid tissues,14,16
the proportion
of IDC in the fresh smeared LN cells was determined by S-100b
immunoperoxidase method. As shown in Figure 2,a,b
, IDC were clearly identified as
S-100b+ large dendriform cells, which tended to form complexes with
several small lymphocytes. The percentage of IDC in the fresh smeared
LN cell was 0.6 (mean) ± 0.2 (SD). By immunofluorescent
microscopy, IDC could be clearly distinguished from other types of
cells as HLA-DRbright large cells with irregular
contours co-expressing CD40, CD54, CD80, CD83, and CD86 (Figure 3,a–c)
.
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. IDC often extended narrow slender cytoplasmic projections
between two adjacent attaching B or T lymphocytes (Figure 3, e,f)
. The
immunoglobulin class of the clustered B cells was analyzed by two-color
indirect immunofluorescent microscopy with anti-HLA-DR and anti-IgD,
anti-IgM, anti-IgA, or anti-IgG. The results indicated that the
cultured B cells were mostly IgD+ IgM+ naive B cells, and no IgA+ or
IgG+ B cells were present within the clusters (Figure 3, f–i)
.
Although many T cells were present outside of the clusters, only a
small number of T cells, which were mostly CD4+
IL-2R- CD40L- nonactivated T cells, were found within clusters (Figure 3, j–l)
. IDC in Cultured LN-Cell Suspension
The LN cells were cultured in 10% FCS-supplemented RPMI 1640
medium for 3 to 22 days. On culturing, IDC became larger and
extensively dendritic in shape and formed clusters with several or
sometimes numerous T cells, which were mostly CD4+ helper T cells, and
CD8+ T cells were found in the clusters only in small numbers (Figure 3, m–o)
. B cells within the clusters gradually decreased and almost
completely disappeared within 7 days (Figure 3p)
. These cultured T
cells were mostly CD25+ CD40L+ -activated T cells (Figure 3, q and r)
,
and such IDC/activated T cell-clusters were stable for at least 22
days, whereas other nonclustered cells tended to die in prolonged
culture, although cell death was not evident until day 10 in culture.
The percentage of IDC in the LN cells cultured for 7 days was
approximately 0.6 as determined by indirect immunoperoxidase method for
S-100b protein (data not shown).
Flow Cytometric Analysis
T cells, B cells, naive B cells, and non-naive B cells in the
mesenteric LN cells were identified as CD3+, CD20+, IgD+/CD20+, or
IgD-/CD20+ cells, respectively. The percentages of these subsets in
fresh mesenteric LN cells obtained from the 5 patients were calculated
by a FACScan. The results indicated that T cells were 58 ± 15.7%
and B cells were 31.5 ± 11.7% (Figure 4, 1a
). Approximately 10% were
non-T/non-B cells, which could not be further characterized. The
percentages of naive B and of non-naive B cells were 9.2 ± 1.5 or
18.8 ± 8.8, respectively (Figure 4, 1b)
.
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. The
percentages of naive B cells and of non-naive B cells were
approximately 18 or 20, respectively (Figure 4, 2b)
.
The proportion and immunophenotype of IDC in the mesenteric LN cell
suspension was determined by three-color flow cytometry. Because IDC in
the fresh non-cultured suspension hardly expressed DC-specific antigen
CD83 on the surface, although they expressed this antigen in the
cytoplasm, the proportion of IDC was determined in the mesenteric LN
cells cultured for 7 days. IDC were clearly identified as
CD83+/HLA-DRbright cells in the macrophage/IDC
fraction (Figure 5)
, which co-expressed
CD40. The percentage of CD83+ IDC in total cells was 0.84%. Thus,
CD40+ cells in the macrophage/IDC fraction mostly corresponded to IDC,
which co-expressed CD86 and CD54, but negative for CD40L.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The molecular basis for specific attachment of naive B-cells to DC remains to be clarified, although it has been revealed that LFA-1 is partially involved in the cluster formation of B cells and DC.9 It seems likely that certain cell adhesion molecules other than those specifically expressed on naive B cells mediate this attachment. Further studies should be done to clarify the molecular mechanism of DC/naive B-cell association.
The present finding that freshly suspended IDC clustered preferentially with naive B cells despite the presence of many T cells in the human mesenteric LN, suggests that the affinity of IDC for naive B cells is stronger than that for naive T cells, although this should be confirmed by further studies.
Kushnir et al 9 also demonstrated in experimental animals (rats and mice) that DC/B cell clusters break up within 24 to 48 hours. As shown in this study, in human LN culture, it took as long as 7 days for IDC/B cell-clusters to disappear completely. The present findings that the proportion of B cells, especially the proportion of naive B cells, did not reduce during culture for 7 days, indicate that this disappearance of IDC/B cell cluster was not due to the preferential death of B cells. It seems likely that human resting B cells adhere to IDC nonspecifically for a longer period of time than do rat or mouse B cells. Regardless of such detailed differences, the affinity between human IDC and resting naive B cells is also transient, and IDC never form a stable complex with B cells. The transient nature of the affinity between naive B cells and IDC may explain the fact that IDC are usually surrounded not by B cells but by T cells despite their strong affinity for naive B cells.
As shown in this study, although IDC initially contacted with a small
number of CD4+ T cells, which were mostly IL-2R-CD40L-nonactivated T
cells, some IDC developed into extensively dendriform cells expressing
CD54, CD83, and CD86 strongly, which formed stable clusters with
numerous activated CD4+ T cells expressing IL-2R and CD40L on prolonged
culture. It is probable that these clustered activated T cells happened
to be specific for Ag expressed on the IDC with which they were
interacting, and that they were stimulated by the IDC to express these
antigens. Once activated by DC, T cells express CD40L, which, in turn,
signals the DC to up-regulate co-stimulatory molecules (CD54, CD80,
CD86) and to secrete cytokines (IL-1, IL-6, IL-8, IL-12,
TNF-
).20,21
It is noteworthy that both IDC forming
clusters with CD40L+ activated T cells and these clustered CD40L+
-activated T cells were continuously alive, while non-clustered cells
tended to undergo apoptosis on prolonged culture, although apoptosis
was not evident until day 10 in culture. These findings are consistent
with those in a previous report in which CD40 ligation counteracted the
apoptosis of in vitro-induced DC from CD34+ hematopoietic
precursor cells.22
Moreover, it is suggested that
CD40 ligation also counteracts the apoptosis of the activated T cells.
It has been shown that Ag-specific activated helper T cells assist in the immunoglubulin synthesis and class-switching of Ag-specific resting naive B cells through CD40-CD40L interaction. It is probable that Ag-specific IDC/activated T cell complexes act as highly efficient and stable systems to capture rare Ag-specific resting naive B cells from enormous circulating resting naive lymphocytes, and to stimulate them to differentiate into germinal center founder cells or short-lived plasma cells secreting IgM.
In this study, we investigated the immunophenotype of IDC by two-color immuno-fluorescent microscopy. IDC have been previously examined mainly by single or double enzyme immunostaining, but it is very difficult to detect two different surface antigens on the same cells by double enzyme immunostaining, and the precise surface immunological properties of IDC could not be fully determined. In the present study, we demonstrated that almost all IDC, which are defined as HLA-DRbright large dendriform cells present in the T cell areas, co-express CD40, CD54, CD80, CD83, and CD86. Some IDC express CD4 weakly but do not express CD1a. Several investigators have reported that subsets of DC, such as peripheral blood DC and epidermal LC, express CD40L.23,24 In this study, we demonstrated that almost all IDC in the human mesenteric LN are negative for CD40L. We failed to detect CD40L on the IDC of other LN examined, including axillary, cervical, and mediastinal LN (unpublished data). It seems likely that even though certain immature stages of DC express CD40L, DC lose this antigen during their maturation into IDC.
In our previous study, we detected a considerable number of CD1a+ immature (CD83- CD86-) DC, which are thought to be precursors of IDC derived from epidermal LC, in and/or around the lymph sinuses of the human axillary LN.15 However, such cells were not observed in the human mesenteric LN in the present study. We also found that CD1a+ immature DC are present in superficial LN, but not in the deeply-located LN such as mesenteric, mediastinal, and para-aortic LN (unpublished data). It seems probable that IDC in such deeply located LN are derived from CD1a- precursors, the nature and origin of which remain unclear.
We recently found that the ability of IDC to cluster resting B cells largely depends on their anatomical location and that the ability of IDC in deeply located LN, which usually have well-developed follicles, tends to be strong, while that in superficial LN which usually have poorly developed or no follicles, tends to be weak (unpublished data), and we assume that the architecture of LN partially depends on the ability of IDC to cluster resting naive B cells.
As shown in this study, a considerable number of IDC, which comprised approximately 0.8% of total cells, could be detected in the mixture of LN cells prepared by the simple method using a glass homogenizer. It has been reported that it is very difficult to isolate IDC from the lymphoid tissues, particularly LNs, and we failed to isolate IDC from the mesenteric LNs. It seems likely that the difficulty in the isolation of IDC from the lymph nodes is largely due to their nature to form complexes with lymphocytes rather than their small number. We used the glass homogenizer method, because such simple methods probably minimize the loss of IDC during the preparation processes and the purification of IDC was not required. This method seems to be useful for the immunomorphological investigation of IDC, although it is clear that this method is not suitable for the functional analysis of IDC.
It is, however, noteworthy that the proportion of LN cells including T cells, B cells, and IDC was not significantly changed during 7-day culture. In short term culture, we recently found that if LN cells are cultured in mixture, the viability of LN cells, which included T cells, B cells, and IDC, is much better than the viability of LN cells when they are cultured separately (unpublished data). It seems likely that mixed LN cell culture provided a microenvironment suitable for the survival of LN cells.
Finally, it is noteworthy that a small number of IDC are present in B-cell-rich areas, such as the periphery of the mantle zones or primary follicles. IDC are not found within the germinal centers; they express the mature DC markers CD83 and CD86 but not CD11c and, therefore, are different from germinal center DC, which are reported to be present in the tonsil germinal centers and to express CD4 and CD11c but not CD83 or CD86.25 The functional role of these IDC in B-cell-rich areas remains unclear, but the presence of such cells suggests that IDC are more deeply involved in the regulation of B-cell-mediated immunological responses than has been previously thought. Further studies should be performed to clarify the role of DC in the regulation of the B-cell-mediated immunity.
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Footnotes |
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Accepted for publication March 29, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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