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Published online before print April 13, 2005
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nik-Bergant*,1ka Repnik
Jeras
* Department of Biochemistry and Molecular Biology, Joef Stefan Institute, Ljubljana, Slovenia;
Tissue Typing Center, Blood Transfusion Center of Slovenia, Ljubljana;
Institute of Cell Biology, Medical Faculty, University of Ljubljana, Slovenia; and
Faculty of Pharmacy, University of Ljubljana, and KRKA, d.d., Slovenia
1 Correspondence: Department of Biochemistry and Molecular Biology, Joef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia, EU. E-mail: tina.zavasnik{at}ijs.si
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-), enabling DC maturation. A large increase in intracellular CyC accompanied the differentiation of monocytes to immature DC, also shown by strong immunolabeling of Golgi in immature DC. On DC maturation, intracellular CyC levels decreased, and CyC was mostly absent from the Golgi. On prolonged incubation of mature DC with TNF-, CyC was found located in the proximity of the plasma membrane, indicating that the transport of CyC from Golgi was not blocked as the result of the arrested exocytosis in mature DC. The secretion of CyC ceased, consistent with the peak of the surface expression of phenotypic markers (CD40, CD54, CD80, CD83, CD86, and major histocompatibility complex class II), characteristic for the mature DC stage, whereas the secretion of cathepsins did not correlate with the maturation stage. The difference in localization of CyC and of CatS, -L, and -H in immature and mature DC shows that the regulatory potential of CyC toward CatS, -L, and -H inside DC is limited. However, these interactions may occur extracellularly in lymph, as suggested by the large excess of CyC over secreted CatS, -L, and -H, and they may facilitate DC migration to lymph nodes.
Key Words: human APC • lymph node • cathepsins • protease inhibitor • TNF-
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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APC play a pivotal role in the immune system, and questions about the modulatory effects of CyC on proteolytic enzymes, besides its suggested, direct involvement in developmental control of MHC II presentation [3
, 7
], are relevant to human APC [8
, 9
] and in particular, DC. CyC is, in vitro, a potent inhibitor of human CatS, -L, and -H [10
, 11
], all of which are present and active inside the endocytic pathway of DC [12
]. Fiebiger et al [13
] observed no significant changes in CyC levels or localization in response to proinflammatory cytokine tumor necrosis factor (TNF-) in monocyte-derived human DC. Hashimoto et al. [14
], conversely, showed that the expression of the CyC gene is down-regulated in lipopolysaccharide (LPS)-stimulated, mature DC, and Lautwein et al. [12
] reported no change in protein expression and distribution of CyC on maturation with LPS in monocyte-derived human DC. Furthermore, Greiner et al. [9
] showed selective, late endosomal localization of CyC together with active CatS in isolated peripheral blood monocytes. No secretion profile of CyC and CatS, -L, and -H from human DC, during differentiation from their precursors or during maturation, has been reported, and no connection between intracellular content and secretion profile of these proteins has been described.
To clarify the role of CyC in human DC, we first sought to determine whether CyC content, localization, and secretion change in response to interleukin (IL)-4, which enables differentiation of monocytes to immature DC, and to TNF-, which enables maturation of these DC. Second, if CyC content, localization, and secretion are differentiation- and maturation-dependent in human DC, do they interact differentially with its potential target enzymes CatS, -L, and -H in immature and mature DC, respectively? We have shown that CyC content, localization, and secretion are differentiation- and maturation-dependent and suggest that the secretion of CyC is an additional mechanism for controlling the intracellular CyC content in DC. Conversely, as a result of the different localization of CyC and CatS, -L, and -H, CyC could not be involved significantly in their intracellular regulation in monocyte-derived, immature DC and TNF--matured, human DC.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell cultures of promonocyte U-937 cell line, monocytes, and DC
The Ethics Committee of the Ministry of Health of the Republic of Slovenia approved all investigations concerning human tissues and cells. The human promonocyte U-937 cell line was obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI-1640 medium with 10% fetal calf serum (FCS), as reported [19
]. Peripheral blood mononuclear cells were isolated from buffy coats from healthy donors by Ficoll density centrifugation, and monocytes were obtained by further Percoll density centrifugation [20
]. Isolated monocytes were differentiated to immature DC using IL-4 (400 U/ml, R&D Systems, Minneapolis, MN) and granulocyte macrophage-colony stimulating factor [GM-CSF; 500 U/ml (Leucomax), Novartis Pharma AG, Basel, Switzerland] in RPMI-1640 medium with 10% FCS (both from BioWhittaker, Walkersville, MD) and gentamycin (5 µg/ml, Gibco, Paisley, Scotland, UK). Immature DC were collected after 5 days of differentiation from monocytes. Immature DC were then matured with recombinant human TNF- (15 ng/ml) and GM-CSF (1000 U/ml; both from R&D Systems) for an additional 1–5 days. Viability of cells was checked by Trypan blue staining under the light microscope. Apoptosis of DC during their maturation was checked using Annexin V-staining kit, as recommended by the producer (Becton Dickinson, San Diego, CA). Differentiation and maturation of DC were followed by surface expression of characteristic CD molecules.
Flow cytometry
Nonadherent and nonfixed, immature and mature DC were analyzed by flow cytometry for cell-surface expression of CD1a, CD14, CD40, CD54, CD80, CD83, CD86, and HLA-DR phenotypic markers. Cells were labeled as recommended by BD PharMingen. CyC, CatS, CatL, and CatH were analyzed in fixed U-937 cells, isolated monocytes, and DC. Aliquots of 2 x 105 cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, Steinheim, Germany) in phosphate-buffered saline (PBS), pH 7.2, for 10 min and permeabilized with 0.1% Triton X-100 (Serva, Heidelberg, Germany) for an additional 5 min. Cells were rinsed in PBS, and nonspecific binding was blocked with 3% bovine serum albumin (BSA; Sigma-Aldrich) and 10% goat serum (Sigma Chemical Co., St. Louis, MO). Primary mAb and Alexa FluorTM 488-labeled secondary antibody were added and incubated for 15 min each at room temperature. Isotype-matched negative controls, i.e., mouse IgG1 and IgG2a (Sigma Chemical Co.), were used instead of primary antibody. For triple immunolabeling of DC for CD14, CD86, and CyC, nonfixed cells were first incubated with anti-CD86 mAb (labeled with CyChrome) and anti-CD14 mAb [labeled with phycoerythrin (PE)]. They were then fixed, permeabilized with Triton X-100 (as described above), and incubated with anti-CyC 1A2 mAb (labeled with Alexa FluorTM 488). A total of 10,000 gated cells per sample was evaluated for specific labeling using FACSCalibur (Becton Dickinson), and results were analyzed using CellQuest 3.3 software.
Confocal immunofluorescence microscopy
For cytospin samples, aliquots of 1 x 105 DC, resuspended in RPMI-1640 medium with 10% FCS, were cytocentrifuged (55 g, 4 min) onto poly-L-lysine (Sigma Chemical Co.)-coated slides. Cells were fixed with 4% paraformaldehyde in PBS (pH 7.2) for 1 h and permeabilized with 0.1% Triton X-100 for an additional 5 min. Cells were labeled with antibody and visualized by immunofluorescence microscopy using a confocal laser-scanning microscope, Carl Zeiss LSM 510, as described [21
], unless stated otherwise. Anti-CyC 1A2 mAb was labeled with Alexa FluorTM 488 fluorophore, using ZenonTM One Mouse IgG1 labeling kit, as recommended by the producer (Molecular Probes) prior to colocalization study of CyC with golgin-97 [11
] in immature and mature DC. For labeling with two-mouse mAb, one of which was anti-CyC 1A2 mAb, DC were labeled in three steps. Unlabeled, primary anti-golgin-97 mAb was added first, followed by anti-mouse Alexa FluorTM 546-labeled secondary antibody. DC were then incubated with anti-CyC 1A2 mAb, directly labeled with fluorophore Alexa FluorTM 488 (Zenon). Carl Zeiss LSM image software 3.0 (Correlation Plots) was used to evaluate the colocalization between two labeled proteins (i.e., between red and green fluorescence signals).
Internalization studies
Fixable Alexa Fluor 546TM-labeled dextran with incorporated lysine residues (MW 10,000, Molecular Probes) was used to study the internalization ability of DC during their maturation, using the confocal microscope. Dextran was added (10 µg/ml) to DC in growth medium for 40 min at 37°C. In a parallel experiment, control cells were preincubated for 30 min at 4°C to slow the metabolic uptake of dextran conjugate and then incubated with labeled dextran for another 40 min at 4°C. DC were processed as above for cytospin samples. Fluorescein isothiocyanate (FITC)-labeled dextran 150S-FITC (Sigma Chemical Co.) was also used for internalization studies, and the equivalent experiment was carried out. Immunofluorescence of internalized, FITC-labeled dextran was measured by flow cytometry as described above.
Electron microscopy
For fine structure preservation, DC at 107 cells/ml were fixed with 4% paraformaldehyde in PBS (20 mM Na2HPO4, 150 mM NaCl, pH 7.4) for 3 h at 4°C. Cells were washed with PBS and post-fixed with 1% OsO4 for 1 h at 4°C. Then, cells were dehydrated in ethanol series (30%, 50%, 70%, 90%, 100%) and embedded into Epon resin. Ultra-thin sections were viewed in a Philips CM100 transmission electron microscope.
Immunoelectron microscopy
DC at 107 cells/ml were fixed with 4% paraformaldehyde in PBS (20 mM Na2HPO4, 150 mM NaCl, pH 7.4) for 30 min at 20°C. They were permeabilized with 4% paraformaldehyde plus 0.1% Triton X-100 in PBS for 5 min. Cells were washed with PBS and incubated with 0.02 M glycine in PBS for 5 min. Nonspecific labeling was blocked with blocking buffer (1% BSA, 0.1% gelatin, 0.05% Tween 20 in PBS) for 15 min, and the cells then incubated for 30 min with gentle shaking with primary antibody (rabbit anti-CatS pAb or mouse anti-CyC 1A2 mAb) diluted in the blocking buffer. Cells were then washed with blocking buffer and incubated with anti-rabbit Nanogold®-Fab' or anti-mouse Nanogold®-Fab' conjugates (Nanoprobes, Yaphank, NY), with gentle shaking, for 30 min. Cells were washed with blocking buffer and fixed with 1% glutaraldehyde in PBS for 20 min. Fixative was removed with three PBS washings, and cells were then rinsed with 0.02 M sodium citrate buffer (pH 7.0) and with deionized water. Silver was developed with HQ Silver Enhancement kit (Nanoprobes) for 4 min at 20°C. The reaction was stopped with sodium citrate and subsequent deionized water rinses. Cells were post-fixed with OsO4 for 30 min, dehydrated in ethanol, and embedded in Epon resin. Ultra-thin sections were counterstained with uranyl acetate and lead citrate and viewed in a Philips CM100 transmission electron microscope.
Protein assays
Cell-culture media from promonocyte U937 cells, monocytes, and DC (all at 5x105 cells/ml at the beginning of each cultivation) were analyzed by four specific enzyme-linked immunosorbent assay (ELISA) tests (purchased from KRKA, Novo mesto, Slovenia), as described previously [15
16
17
], to determine the secretion of CyC, CatS, CatL, and CatH. The manufacturer defined the detection limit of each ELISA test. Cells were removed from culture media by centrifugation, and supernatants were diluted in PBS with 2% BSA (pH 7.2) prior to ELISA, i.e., from 1:20 (for medium from U937 cells) to 1:80 (for media from monocytes and DC) for CyC determination and 1:2 for CatS, CatL, and CatH determination (the latter for all the cells tested). Protein concentrations of CyC, CatS, CatL, and CatH, determined by specific ELISA tests, are the mean values ± SE (standard deviation) of three measurements. They are presented as ng of secreted protein per ml culture medium and as molar concentration (nmol/l), respectively. Total protein concentration in culture media supernatants (all diluted 1:50) was determined by the Bradford method using the Bio-Rad protein assay dye reagent and BSA as standard, according to the manufacturer’s microassay procedure (Bio-Rad Laboratories, Hercules, CA).
Immunohistochemical analysis of lymph nodes
Lymph nodes, noninvaded by tumor cells, were obtained from patients undergoing mastectomy and auxiliary dissection for breast carcinoma at the Institute of Oncology (Ljubljana, Slovenia). Tissue sections (5 µm) from formalin-fixed, paraffin-embedded human lymph nodes were prepared [22
] and labeled for CyC, MHC II (HLA-DR), CD68, and FDC. Labeling of lymph node tissue and confocal immunofluorescence microscopy was performed as described previously [21
, 23
].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was selected to achieve slow, but complete, maturation of immature DC, a process that was confirmed by analysis of the characteristic surface CD molecules (Fig. 1
, and see Fig. 3A
). Before labeling surface CD molecules and intracellular CyC and cathepsins, the viability of each particular DC population was controlled using Trypan blue staining (less than 5% dead cells, not shown). Immature DC were determined by flow cytometry to be CD1a+, CD40+, CD54+, HLA-DR+, but CD14– (Fig. 1A
, and see Fig. 3A
) and to be able to internalize labeled dextran (Fig. 2
). Surface expression of CD80 and CD86 costimulatory molecules, together with CD83 and HLA-DR, was highly up-regulated during cell maturation with TNF- and reached a peak after 3 days (Fig. 1B)
. Furthermore, the surface expression of the above-listed phenotypic markers did not increase further after 4 or 5 days after the initiation of maturation in the presence of TNF- (not shown). The distribution of MHC II was shown to differ in immature (Fig. 1A)
and mature (Fig. 1B)
DC, also by using fluorescence microscopy. Translocation of MHC II was observed from intracellular vesicles in immature DC (Fig. 1A)
to the cell surface in mature DC after 3 days of maturation (Fig. 1B)
. Only weak positive staining of MHC II was observed in the perinuclear region of mature DC (Fig. 1B
, arrows).
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and on (Fig. 2C)
.
Intracellular CyC in DC, monocytes, and promonocytes
The level of CyC in a population of CyC+ CD86+ CD14– DC was determined by flow cytometry, as shown for immature DC at the beginning of their in vitro maturation with TNF- (Fig. 3A
and 3B
). Furthermore, the CyC contents of monocytes and promonocyte U-937 cells, determined by immunolabeling, were found to be lower than in immature and mature DC (Fig. 3C)
. During differentiation of monocytes with IL-4, CyC increased and after 5 days, reached a peak in immature DC. Intracellular levels of CyC decreased again during the 3 days of TNF--induced maturation of immature DC to mature DC (Fig. 3C)
. Vesicular labeling of CyC was observed in monocytes (Fig. 4
) and confirmed in promonocyte U-937 cells (not shown). Fluorescence microscopy showed that in a population of isolated monocytes, the majority of cells was weakly labeled, and only a few cells exhibited stronger vesicular labeling (Fig. 4) . The localization of CyC changed during monocyte differentiation to immature DC and during DC maturation with TNF-, as shown in confocal micrographs of labeled, particular DC populations (Fig. 4)
. In immature DC, CyC was strongly labeled in a structure near the nucleus (Figs. 3B
and 4)
, later determined to be the Golgi apparatus (see Fig. 8C
).
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, the immunofluorescence signal of labeled CyC increased again, but now located close to the cell membrane (Figs. 3D
and 4)
. This increase in immunofluorescence after 4 and 5 days in the presence of TNF- was also detected by flow cytometry (Fig. 3C
, last two histograms). When increasing immunofluorescence signal from labeled CyC was observed in the proximity of the cell membrane (Fig. 4
, days 4 and 5), no endocytosis of labeled dextran was observed anymore in these DC (Fig. 2C
, last two histograms).
Secretion of CyC
Extracellular secretion was determined in the cell-culture media during the differentiation of monocytes and maturation of DC (Fig. 5
). The total protein content was the same in all culture media, i.e., 3.0 ± 0.2 mg/ml. During differentiation of monocytes to immature DC with IL-4 (i.e., in 5 days), 399 ± 29 ng CyC per ml (30.7±2.2 nM) was secreted to the culture medium (Fig. 5)
, compared with 66 ± 6 ng CyC per ml (5.1±0.5 nM), secreted from promonocyte U-937 cells during their cultivation for 5 days (Fig. 5)
. In contrast, immature DC secreted more CyC until they reached their mature state, i.e., 256 ± 36 ng/ml (19.7±2.8 nM) after 1 day, 619 ± 38 ng/ml (47.6±2.9 nM) after 2 days, and 1035 ± 15 ng/ml (79.2±1.2 nM) after 3 days. After 3 days of maturation, extracellular levels of CyC no longer increased.
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, 1025 ± 30 ng CyC per ml (78.9±2.3 nM) was observed in the culture medium, 2.4 times more than that after 5 days of differentiation of monocytes to immature DC and 15 times more than that after 5 days of cultivation of promonocyte U-937 cells. CyC concentration in control RPMI-1640 medium (culture medium without cells) was below the detection limit of CyC-specific ELISA, i.e., 0.02 nM.
Intracellular and secreted CatS
Intracellular levels of CatS were determined in populations of immature DC and DC during their maturation with TNF- (Fig. 6A
). CatS decreased over 2 days of maturation and then remained constant over further 3–5 days (Fig. 6A)
, in contrast to intracellular CyC (Fig. 3C)
. After 5 days of differentiation of monocytes to immature DC, the concentration of CatS in the culture medium was 43.0 ± 1.5 ng per ml (1.5±0.05 nM; Fig. 7A
), compared with 76.0 ± 0.6 ng per ml (2.7±0.02 nM) after maturation from immature DC with TNF- for 5 days (Fig. 7A) . DC secreted 28 times less CatS than CyC (2.7±0.02 nM-secreted CatS vs. 78.9±2.3 nM-secreted CyC) during their maturation (Figs. 5
and 7A)
. The molar ratio between secreted CatS during DC maturation and during differentiation of monocytes to immature DC was 1.8, lower than the equivalent ratio of 2.5 for secreted CyC. CatS concentration in control RPMI-1640 medium (without cells) as well as in the cell-culture medium with promonocyte U-937 cells (after 5 days cultivation) was below the detection limit of the CatS-specific ELISA, i.e., 0.2 nM.
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Intracellular and secreted CatH and -L
No change in CatH or CatL content was observed in DC during their differentiation and maturation, as determined by flow cytometry (not shown). During 5 days of differentiation of monocytes to immature DC, however, 18.8 ± 0.5 ng CatH per ml (0.67±0.02 nM) and 4.0 ± 0.5 ng CatL per ml (0.13±0.02 nM) were secreted into the culture medium (Fig. 7A)
. In the next stage, during their maturation induced by TNF- for 5 days (Fig. 7A) , DC secreted 30.8 ± 0.2 ng CatH per ml (1.1±0.01 nM) and 6.7 ± 0.8 ng CatL per ml (0.22±0.03 nM), i.e., 2.5 times less CatH and 12.3 times less CatL than of CatS. Comparing these molar concentrations with those of secreted CyC, 28 times less CatS, 70 times less CatH, and 350 times less CatL were secreted during DC maturation (Figs. 5
and 7A)
. Promonocyte U-937 cells secreted 4.1 ± 0.8 ng CatL per ml (0.14±0.03 nM), whereas CatH levels were below the detection limit of CatH-specific ELISA (Fig. 7A)
. CatH and CatL concentrations in controls were also below the detection limits of 0.1 nM for CatH and 0.056 nM for CatL.
CatS, CatH, and CatL were secreted in much lower amounts (Fig. 7A) than CyC (Fig. 5)
. The secretion of CyC (Fig. 5)
and the increase in surface expression of phenotypic markers (CD40, CD54, CD80, CD83, CD86, and MHC II, Fig. 1B
) stopped simultaneously after 3 days of maturation with TNF-, when DC reached their mature status. In the case of cathepsins, only a low secretion of CatL was observed during differentiation of monocytes to immature DC and ceased after the first day of maturation of DC with TNF-. The secretion of CatH from DC was higher than for CatL and lower than CatS but continued during the 5 days in the presence of TNF-. The secretion of CatS stopped after 4 days of maturation, i.e., 1 day later than the secretion of CyC and compared also to the surface expression of checked, characteristic CD molecules. Thus, the secretion of CyC did not correlate with the secretion profiles of CatS, CatL, or CatH. The secretion of CyC stopped, consistent with the peak of expression of phenotypic markers characteristic for mature DC stage, in contrast to all the measured cathepsins.
The distribution of vesicles, labeled for CatH and CatL, in DC was shown by confocal immunofluorescence microscopy (Fig. 7B and 7C) . For both cathepsins, vesicles were dispersed more evenly in immature DC (Fig. 7B) but concentrated in the perinuclear region of mature DC (Fig. 7C) .
Colocalization studies of CyC in DC
The localization of CyC (Figs. 3
4
8
) was different in immature and mature DC from those of CatS (Fig. 6)
, CatH (Fig. 7)
, and CatL (Fig. 7)
. In immature DC (Fig. 8C)
, but not in mature DC (not shown), CyC was colocalized with human golgin-97, a marker for Golgi apparatus [24
]. It was not possible to preserve a sufficiently good morphology of the Golgi apparatus to prove the exact localization of the observed immunogold particles in anti-CyC-labeled DC under the transmission electron microscope.
A shift from more dispersed vesicular labeling in immature DC to concentrated vesicular labeling in mature DC (after 3 and more days of maturation) was observed for CatS (Fig. 6)
, CatH (Fig. 7)
, and CatL (Fig. 7)
. In contrast, a shift of CyC (Fig. 4 , last two micrographs) from the Golgi apparatus (Figs. 3B
and 8A) toward the cell periphery (i.e., toward the plasma membrane, Figs. 3D
and 8B
) was observed at days 4 and 5 during in vitro DC maturation. We may say that CyC and CatS do not colocalize in immature DC or in mature, human DC generated with TNF- (Fig. 8A and 8B)
. Using Correlation Plots in Carl Zeiss LSM image software 3.0, no significant colocalization between immunolabeled CyC (red fluorescence signal) and CatS (green fluorescence signal) was revealed. Furthermore, no significant colocalization was observed between CyC and MHC II (Fig. 8D)
or CyC and LAMP-2 (Fig. 8E)
in immature DC or in mature DC, respectively.
CyC staining in lymph node tissue
Healthy human lymph node tissue was immunolabeled for CyC. Cells were strongly labeled for CyC in subcapsular and cortical sinuses, indicating the presence of CyC+ cells entering the lymph node with afferent lymph (Fig. 8F
, arrowheads). CyC+ cells were also observed in the paracortex, around the secondary follicles (Fig. 8G
, +). Cells in primary follicles (Fig. 8F
, *) and secondary follicles with germinal centers (Fig. 8G
, the latter located using anti-FDC mAb) inside the lymph node cortex were unlabeled. The ratio between labeled and nonlabeled cells in the paracortex (Fig. 8G)
was lower than in the subcapsular and cortical sinuses (Fig. 8F)
. CyC+ cells in sinuses and paracortex were also positive for MHC II, using anti-HLA-DR mAb in a double-immunolabeling experiment (not shown). As was also evident from double-immunolabeling experiments, the presence of CyC cannot be observed in FDC (Fig. 8G) or in strongly CD68+-tingible body macrophages (not shown) inside germinal centers.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was selected to prolong the maturation process of human monocyte-derived DC (U. Repnik et al., unpublished data). A decrease of CD14 in immature DC as well as an increase of HLA-DR, CD40, CD54, CD80, CD83, and CD86 on maturation were shown to be as reported [4
, 31
, 32
], confirming the validity of the experimental system and providing information on the differentiation and maturation processes. Furthermore, the DC generated were applied successfully in antigen-presentation assays to allogenic T cells, where they secreted IL-12 and elicited T cell proliferation and secretion of interferon-
(U. Repnik et al., unpublished data). After 3 days in the presence of TNF-, the endocytosis of added dextran was completely arrested, and surface expression of CD1a, CD40, CD54, HLA-DR, CD80, CD83, and CD86 no longer increased, confirming that the DC mature stage was attained [31
, 32
].
We showed that in immature DC, CyC content was much higher than in monocytes and promonocyte U-937 cells. The low content of CyC in monocytes and promonocyte U-937 cells was not the result of increased CyC secretion, which was quite low compared with that in immature DC. High CyC secretion in immature DC accords with their strong endocytic activity [33
] but was not observed for promonocyte U-937 cells (T. Zavanik-Bergant et al., unpublished data) or for monocytes [20
]. Therefore, the low CyC content in promonocyte U-937 cells and monocytes most probably resulted from lower CyC expression. Our data indicate that CyC expression and thereby, its content is differentiation-dependent, rising with the differentiation of monocytes to immature DC. Lautwein et al [12
] and Fiebiger et al. [13
] reported the absence of significant differences in CyC levels and its intracellular distribution between human monocyte-derived immature and mature DC, but no analysis by microscopy or quantification of secretion of CyC during differentiation or maturation processes has been reported for human DC. Using flow cytometry, we observed a high CyC content in immature DC, which decreased during maturation to a minimum in mature DC after 3 days of maturation. These results were confirmed by confocal microscopy. CyC was strongly colocalized with the Golgi apparatus of immature, but not of mature, DC. The amount of secreted CyC per day remained constant throughout maturation. The secretion of CyC then stopped completely, in keeping with abolished endocytosis [33
], and was accompanied by high but stable surface expression of the characteristic phenotypic markers (CD1a, CD40, CD54, HLA-DR, CD80, CD83, CD86) and MHC II in mature DC [31
, 32
].
On prolonged incubation in the presence of TNF- for a further 2 days, strong immunofluorescence from labeled CyC was again observed but this time, close to the cell surface, suggesting that transport of CyC from the Golgi apparatus toward the plasma membrane continued in mature DC after 3 days, although secretion stopped. The cells (95%) in these two DC populations were neither apoptotic nor necrotic (T. Zavanik-Bergant et al., unpublished data), confirming the intact structure of the plasma membrane. Furthermore, as the uptake of added FITC-labeled and Alexa FluorTM 546-labeled dextrans was completely arrested after 3 days of maturation, it is also unlikely that the increase in fluorescence from immunolabeled, intracellular CyC, observed in mature DC after 4 and 5 days in the presence of TNF-, would result from the active uptake of CyC from the medium. Expression of the CyC gene was reported to be down-regulated in LPS-stimulated, mature, human DC, relative to that in immature DC [14
]. This is in keeping with the absence of CyC in the Golgi apparatus of mature DC and its presence in immature DC, which we observed. We suggest that elevated secretion could be an additional mechanism by which DC control their intracellular CyC content, differing in immature and mature DC. However, the biological relevance of this proposed dual control of CyC will have to be confirmed in DC inside human lymphatic tissue.
DC, present in afferent lymph, bring antigen from peripheral tissues to the lymph node paracortex to encounter specific CD4+ T cells [34
]. CyC+ MHC II+ DC [35
] were labeled predominantly in sinuses with afferent lymph and in the paracortex of normal human lymph node tissue. We cannot completely exclude that sinus-lining macrophages were not labeled for CyC in addition to DC in afferent lymph, as it has been reported that human macrophages also secrete CyC [36
, 37
]. However, in our study, secondary follicles remained unlabeled for CyC, indicating its absence in another population of lymph node-resident macrophages, i.e., in tingible body macrophages, responsible for the elimination of apoptotic B cells during clonal selection inside germinal centers [38
]. The staining of CyC inside DC in lymph node tissue did not correspond to that in immature DC generated and cultured in vitro, where CyC was labeled predominantly in the Golgi apparatus. These results further confirm that the population of DC, which reaches secondary lymphoid organs, is no longer immature [39
]. Furthermore, the intracellular localization of CyC can be used as an additional marker for the maturation stage of DC in human tissues. We have also checked CyC staining in invaded lymph nodes of several patients with non-Hodgkin B cell lymphoma (T. Zavanik Bergant et al., unpublished data). As the morphology of invaded lymph node tissue changed, the distribution of CyC-positive cells was also fully altered compared with healthy lymph nodes. Furthermore, mainly immature DC were present in invaded tissue.
As the ELISA recognizes free CyC as well as CyC in complex with its target cathepsins and was applied for detecting secreted CyC on nondenaturated samples (culture media from DC and monocytes), we cannot quantitate directly the formation of these complexes. However, the high inhibitor/cathepsin molar ratio determined for all three cathepsins (CatS, CatL, CatH) supports the existence of the complexes in culture media of immature DC and DC during maturation. The microenvironment inside peripheral tissues, afferent lymph, and lymph node paracortex is more complex than in in vitro DC maturation, and the relevance of extracellular regulation of DC-derived cathepsins remains to be elucidated. The question remains as to whether migrating (veiled) DC, during their maturation as a result of the exposure to the inflammatory stimuli and antigen uptake, benefit from the extracellular existence of inhibitor/cathepsin complexes, formed from secreted CyC and CatS, CatL, or CatH. The concept that inhibitors can modulate protease activity and not only abolish it [40 ] has been generally accepted. Outside the DC, where pH is not optimal for lysosomal acidophilic cathepsins such as CatS, CatL, and CatH, reversible complex formation with secreted CyC could preserve their stability and activity. The formation of similar complexes of secreted CyC with elastinolytic protease CatK has been reported in culture media from human monocyte-derived macrophages [35 ]. Another endogenous inhibitor of cysteine proteases, the p41 isoform of Ii (p41 Ii) from mouse LPS-stimulated DC, was shown to interact with CatL, preserving extracellular accumulation of the mature form of the enzyme [41 ]. Local remodeling of extracellular matrix (ECM) with secreted CatS, CatL, and CatH, stabilized by binding to CyC, could be biologically relevant for human DC by facilitating their migration from peripheral tissues to lymph nodes. In the paracortex, where mature DC interact with specific T lymphocytes, tissue remodeling is no longer needed, consistent with the decrease of CyC and cathepsin secretion from mature DC.
To explain the developmental control of MHC II-associated antigen presentation by DC [7
], two main models have been suggested. According to the first model, immature DC are inefficient at loading MHC II with peptides, as processing of Ii is blocked as a result of CyC inhibition of the key enzyme CatS. In mature DC, CatS is active, enabling the degradation of Ii and generation of MHC II-peptide complexes, which then accumulate on the plasma membrane [3
]. In the second model, MHC II-peptide complexes are generated but quickly endocytosed from the plasma membrane back to the endocytic pathway, and the peptide binding is less-dependent on the rate of Ii degradation. Endocytosis is slowed down during maturation, and MHC II-peptide complexes accumulate on the surface of mature DC [4
5
6
]. It was reported that CatS levels in immature and mature DC are the same [3
]; however, in our study, a slight decrease in intracellular CatS was observed on the second day of DC maturation with TNF-. Immunolabeled CyC was reported to be localized in MHC II+ LAMP-2+ vesicles of immature but not of mature mouse DC [3
]. Furthermore, Lautwein et al. [12
] detected CyC in two subcellular fractions from human monocyte-derived, immature and LPS-stimulated, mature DC populations, i.e., in late endosomes and in the Golgi/endoplasmic reticulum/early endosomal fraction but not in lysosomes. On the contrary, in our study, we found no significant presence of CyC in LAMP-2+ vesicles in immature or mature DC. Furthermore, Pierre and Mellman [3
] reported that on LPS-induced maturation, the CyC level decreased, and the inhibitor was located in the perinuclear cytoplasm, reported as Golgi apparatus, whereas MHC II was detected on the cell surface. We have confirmed the location of CyC in the Golgi apparatus in immature DC. Over the 3 days of maturation with TNF-, staining of CyC in the Golgi apparatus decreased. However, after 2 more days in the presence of TNF-, we observed strong CyC staining near the plasma membrane. This suggests that after the initial stage of maturation, CyC is still transported out of the Golgi apparatus toward the plasma membrane. It is possible that on completion of DC maturation, the secretion pathway of CyC was blocked as early as in the Golgi apparatus in LPS-matured DC [3
] but continued from the Golgi apparatus toward the plasma membrane in our experiment with TNF--matured DC. This transport occurred, although the extracellular secretion had already stopped. Our results further showed that CyC was not significantly colocalized with its potential target enzymes CatS, CatL, and CatH in immature or mature DC. In immature DC, cathepsins were found in MHC II+ LAMP-2+ vesicles, whereas CyC was detected in the Golgi apparatus, thus confirming the typical characteristics of a secretory protein [12
]. In mature DC, strong LAMP-2+ and weak MHC II+ (Fig. 1B
, arrows) vesicles with CatS, CatL, and CatH were concentrated in the perinuclear region (also being supported by the low secretion of cathepsins from DC during their maturation), whereas CyC was still directed toward the cell surface, clearly indicating different pathways of CyC and the cathepsins studied during DC maturation. As no significant colocalization between CyC and CatS, CatL, or CatH was observed, measurements of proteolytic activity of these cathepsins were not included in the study.
In conclusion, we have demonstrated that the content, localization, and secretion of CyC are differentiation- and maturation-dependent. Changes in secretion may constitute an additional mechanism of DC for controlling their intracellular CyC content. Our results do not support intracellular interactions among CatS, CatL, and CatH and CyC in immature or mature DC. The suggested differential CyC regulation of CatS activity in mice can therefore not be applied directly to human monocyte-derived DC. However, as supported by the large excess of extracellular CyC over secreted CatS, CatL, and CatH, these interactions may occur extracellularly in lymph and could facilitate DC migration to lymph nodes as a result of the increased local remodeling of ECM around migrating DC. Suggested CyC/cathepsin interactions will be studied further in reconstituted ECM by an in vitro migration assay.
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ACKNOWLEDGEMENTS |
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Received August 11, 2004; revised January 31, 2005; accepted March 3, 2005.
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REFERENCES |
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