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Published online before print May 22, 2003
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reveals an essential role for TNF
Nikolaus-Fiebiger Zentrum für Molekulare Medizin, Erlangen, Germany
Correspondence: Heinrich Körner, Nikolaus-Fiebiger Zentrum für Molekulare Medizin, IZKF NW 1, Glückstrasse 6, 91054 Erlangen, Germany. E-mail: hkoerner{at}molmed.uni-erlangen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in DC maturation, we studied the development of DCs from the BM of B6.TNF-/-, B6.LT-/-, and B6.TNF/LT-/- mice and compared it to B6.WT mice. Although the development of BM precursor cells to the level of immature DCs (CD11c+, MHC class IIlow, CD40low, and CD86low) was equivalent in all genotypes, B6.TNF-/- and B6.TNF/LT-/- cells showed an impaired capacity to differentiate to mature DCs. In contrast, mature BM-DCs generated from LT-negative, immature DCs developed like B6.WT cells. Further studies revealed that once matured, the phenotype of all tested genotypes was comparable. They expressed high levels of CD40 and CD86, were exclusively positive for the chemokine receptor (CCR)7 but negative for CCR5 and CCR2, and were able to enter the paracortex of draining lymph nodes. The limited maturation of TNF-deficient BM-DCs could be restored by mixing TNF-negative with TNF-positive Ly5.1 BM cells, and maturation of B6.WT DCs could be blocked with an anti-TNF monoclonal antibody. The substitution of B6.TNF-/- BM cells with recombinant TNF revealed promotion or suppression of BM-DC maturation depending on the point of time of TNF addition.
Key Words: tumor necrosis factor • mouse • knockout • bone marrow precursors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TNF and a structurally related cytokine, lymphotoxin (LT), bind to two receptors, TNF-receptor 1 and 2, with similar affinities [11
], and an additional role for LT in BM-DC maturation can therefore not be excluded. It has been demonstrated that the number of splenic DCs is markedly reduced in mice deficient for LT [12
]. In contrast, TNF- and TNF-receptor-negative mice exhibit a normal number of DCs [12
, 13
]. This points to a factor that can replace TNF-mediated signaling and argues for a role of LT in DC differentiation. To understand the biological function of TNF and LT in DC development, we therefore compared BM-DCs generated from B6.TNF-/-, B6.LT-/-, and B6.TNF/LT-/- with C57BL/6 (B6.WT) BM-DCs. We provide evidence that the absence of TNF does not affect proliferation and differentiation of myeloid precursor cells to immature DCs in the presence of GM-CSF but is essential for BM-DC differentiation from an immature to a mature state. The deficient maturation can be reconstituted by substitution with recombinant TNF and mixed tissue cultures of TNF-positive and TNF-negative BM cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-/-, B6.TNF/LT-/- [14
, 15
], and congenic B6.WTLy5.1 were bred and housed on a genetically pure B6.WT background under conventional conditions in the animal facility of the Nikolaus-Fiebiger Zentrum für Molekulare Medizin (Erlangen, Germany).
Tissue-culture conditions of BM-DCs
BM cells were harvested from femurs of 6- to 10-week-old mice and were cultured as described previously [7
]. Cultures were supplemented with supernatants (SNs) from Ag8653 myeloma cells transfected with the gene encoding murine GM-CSF (kindly provided by B. Stockinger, NMRI, Mill Hill, London, UK) or recombinant GM-CSF (200 U/ml; Tebu, Peprotec, Frankfurt, Germany). Maturation of TNF-negative BM cells was induced with lipopolysaccharide (LPS; 1 µg/ml, Escherichia coli, serotype O111; B4, Sigma, Taufkirchen, Germany).
To analyze the role of TNF produced by BM cells in BM-DC maturation, gene-deficient, Ly5.1-positive BM cells, cells were mixed (ratio of 1+1) with Ly5.2-positive BM cells generated from B6.TNF-/-, B6.LT-/-, and B6.TNF/LT-/- mice. Furthermore, TNF-deficient BM cells were reconstituted with endotoxin-free, recombinant TNF (BD PharMingen, Heidelberg, Germany) beginning at day 0, 6, or 8 until the end of the culture period. For blocking experiments, purified rat anti-mouse TNF [XT-22, immunoglobulin G (IgG)1] was added on days 3, 6, and 8 of tissue cultures.
Antibodies
The following antibodies were used for flow cytometric analysis of BM-DCs: Armenian hamster anti-mouse CD11c [clone HL3; phycoerythrin (PE)- or allophycocyanin-conjugated], Armenian hamster anti-mouse CD40 [clone HM40-3; fluorescein isothiocyanate (FITC)-conjugated], rat anti-mouse CD86 (clone GL1; unlabeled), rat anti-mouse major histocompatibility complex (MHC) class II (clone AF6-120.01; biotinylated), anti-mouse Ly5.2 (clone 104; FITC-conjugated), and anti-mouse Ly5.1 (cloneA20; PE-conjugated). All monoclonal antibodies (mAb) were purchased from BD PharMingen. The secondary reagents were used for detection of purified or biotinylated, primary mAb: Cy5-conjugated goat anti-rat IgG (Dianova, Hamburg, Germany) and peridinin chlorophyll protein-conjugated streptavidin (BD PharMingen).
Flow cytometry and flow cytometric cell sorting
Multicolor flow cytometry was performed as described [16
]. The cells were electronically gated according to light-scatter properties and the expression of the DC marker CD11c. Data were collected using a FACSCalibur flow cytometer (BD PharMingen) and were analyzed with CellQuest software (BD PharMingen). Alternatively, BM-DCs were subjected to flow cytometric cell sorting using a MoFlo high-speed cell sorter (Dako Cytomation, Hamburg, Germany). The average purity of the sorted populations that were used for mRNA preparation and subsequent reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was over 98%.
RNA extraction and RT-PCR
RNA was extracted from purified cells using the "Perfect RNA mini kit" (Eppendorf, Hamburg, Germany), according to the manufacturer’s instructions. All samples were treated with RNase-free DNase (Promega, Mannheim, Germany) for 15 min followed by chloroform/phenol extraction to remove the enzyme. First-strand cDNA was synthesized from 1 to 2 µg total RNA using Moloney murine leukemia virus RT (Promega) and oligo(dT) primer (Life Technologies, Karlsruhe, Germany). cDNA was amplified with standard Taq-polymerase (PAN, Aidenbach, Germany). The following primers were used: chemokine receptor (CCR)5 (sense 5'-CAT CCG TTC CCC CTA CAA GAG A-3', antisense 5'-TGC AGC ATA GTG AGC CCA GAA T-3'), CCR2 (sense 5'-GAG CCT GAT CCT GCC TCT ACT TGT-3', antisense 5'-CCT GCA TGG CCT GGT CTA AGT GC-3'), CCR7 (sense 5'-ATT TCT ACA GCC CCC AGA GC-3', antisense 5'-TGA GCC TCT TGA AAT AGA TGT ACG-3'), ß-actin (sense 5'-AAT CCT GTG GCA TCC ATG AAA C-3', antisense 5'-CGC AGC TCA GTA ACA GTC CG-3') at a concentration of 200 µM. For detection of gene expression, 35 cycles were used after an initial denaturation step of 94°C for 2 min (each cycle, 20 s 94°C, 20 s 58°C, and 50 s 72°C).
TNF–enzyme-linked immunosorbent assay (ELISA)
SNs were harvested at days 3, 6, and 8 of BM-DC culture and were stored at -70°C. A TNF–ELISA was performed according to the manufacturer’s instructions (R&D Systems, Heidelberg, Germany). The detection limit was 
25 pg/ml.
In vivo migration of BM-DCs
BM-DCs from B6.WT and BM.TNF-/- were harvested at day 9 of culture and labeled with carboxyfluorescein diacetate succinimidylester (CFSE) [17
]. Mature (CD11c+, CD86high) B6.WT and BM.TNF-/- BM-DCs were purified by flow cytometry as described above and labeled with CFSE (1 µM). BM-DCs (1x106) were injected subcutaneously (s.c.) into the hind footpad of B6.WT or BM.TNF-/- mice. Twenty-four hours after injection, the popliteal lymph node (LN) was analyzed by immunofluorescence microscopy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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60% exhibit an immature (CD40low and CD86low) and 40% a mature CD40high CD86high phenotype (Fig. 1A
) [7
]. To analyze the role of TNF in this maturation process in more detail, BM cells from TNF-negative animals were harvested and analyzed after 9 days of tissue culture with GM-CSF. In the absence of TNF, in vitro only, 16% of the cells differentiated spontaneously into the mature CD40high CD86high phenotype (Fig. 1B) . The extension of the tissue-culture period of B6.TNF-/- BM cells up to 20 days did not substantially increase the percentage of matured BM-DCs (data not shown). Tissue culture with recombinant GM-CSF (200 U/ml) also resulted in a spontaneous maturation of DCs (B6.WT BM cells, 32%; B6.TNF-/- BM cells, 14%), which was comparable with the effect of GM-CSF-containing, tissue-culture SNs (data not shown). Addition of LPS 24 h before harvesting and analyzing the DCs pushed the maturation of TNF-negative BM-DCs to approximately 90% and resulted in a homogenous, mature CD40high CD86high phenotype (data not shown). As a result of the use of TNF-receptor 1 and TNF-receptor 2 by TNF and LT, there was the possibility that secreted LT was able to replace TNF and cause the residual maturation. Therefore, BM-DCs derived from BM of B6.LT-/- and B6.TNF/LT-/- mice were analyzed. LT deficiency did not influence the maturation of BM-DCs (33±3%; Fig. 1C
). Cultures of BM-DCs that were negative for TNF and LT showed a retarded maturation comparable with TNF-negative BM-DC (17±2%; Fig. 1D
). This indicates that TNF is solely responsible for spontaneous maturation of immature-to-mature BM-DCs but is not involved in the development of immature BM-DCs from BM precursor cells. The number of living cells was comparable between the tested gene-deficient mice and wild-type controls. (data not shown).
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exhibit the same phenotype as mature B6.WT BM-DCs-/-, B6.TNF/LT-/-, and B6.WT BM-DCs was further investigated. First, the mean fluorescence intensity of MHC class II, CD40, CD80, or CD86 expressed on the surface of this mature BM-DC population (see Fig. 1
) was compared. Matured BM-DCs of all genotypes showed the same cell-surface expression of those molecules (data not shown). Furthermore, we determined the induction of CCR mRNA by RT-PCR. As BM-DCs represent a heterogeneous group of mature and immature BM-DCs (see Fig. 1
), we isolated a mature and immature BM-DC population of all genotypes by flow cytometry. The data demonstrated that CCR7 was expressed exclusively on mature BM-DCs, whereas only immature BM-DCs were positive for CCR5 and CCR2 (Fig. 2
). The comparable expression of these genes in BM-DCs of all genotypes indicated that once matured, the phenotype of all tested gene-deficient BM-DCs was wild-type-like. Third, as the level of expression of CCRs is important for cell migration, the potential of DCs to migrate in vivo was tested. A mixture of mature and immature, nonadherent B6.WT and B6.TNF-/- DCs was labeled with the cell tracker CFSE and transferred s.c. into the hind footpad of B6.WT and B6.TNF-/- mice. Sections of the draining, popliteal LN were analyzed and showed a strong migration of B6.WT DCs to the paracortex of B6.WT mice (Fig. 3A
). Only a few B6.TNF-/- DCs could be detected in the paracortex of B6.TNF-/- mice (Fig. 3B)
. A transfer of B6.TNF-/- DCs to B6.WT mice showed a similar lack of migration (data not shown). As CCR7 is expressed exclusively on mature DCs (see Fig. 2
), the difference in migrating cell numbers could be a result of the different percentage of mature B6.WT and B6.TNF-/- DCs. Therefore, mature DCs were sorted from B6.WT and B6.TNF-/- mice and again transferred s.c. After adoptive transfer of an equivalent cell number of sorted cells, the mature DCs of both genotypes exhibited the same migration pattern (Fig. 3C and 3D)
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-/-, and B6.TNF/LT-/- mice (Ly5.2+). After 9 days of culture, the cells were electronically gated according to their congenic marker, and the percentage of mature DCs (CD11c+, CD86high) was quantified in both populations (Fig. 4A
). The total number of nonadhered BM-DCs was comparable between the genotypes, and the ratio between CD11c-positive Ly5.1 or Ly5.2 cells was unchanged (45–51%, Fig. 4A
). Furthermore, CD86 was expressed at the same level of intensity on all combinations of genotypes (data not shown). This indicated that TNF produced by TNF-positive B6.WTLy5.1 cells restores the maturation deficiency of TNF-negative BM cells (Fig. 4B)
and does not interfere with the maturation process of LT-deficient BM-DCs.
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Reconstitution of normal BM-DC maturation depends on TNF dose and time-point of application and can be blocked with an anti-TNF mAb
To analyze the dose-response and the optimal point of time of cytokine application, BM cells of B6.TNF-/- mice were substituted with recombinant TNF. If TNF were added after 6 or 8 days of tissue culture, a concentration of 500 U/ml induced maturation of B6.TNF-/- BM-DCs (day 6=26±3%; day 8=30±5%), which was comparable with the wild type (40±3%; Fig. 6B
and 6C
). When TNF was added at day 0, a high dose of the cytokine (5000 U/ml) failed to induce the maturation (19.2%±4.4%). A low dose of TNF (5–50 U/ml) added from the beginning did not increase the maturation of BM-DCs (Fig. 6B)
. This demonstrates that soluble TNF in a concentration of 500 U/ml is able to induce BM-DC maturation only when added relatively late. This result is consistent with the ELISA data (cf., Fig. 5A
). After neutralization of TNF with a blocking mAb, the maturation of B6.WT BM-DCs decreased to a level (12%±4%) that was comparable with the level seen in B6.TNF-/- mice (12%±2%; Fig. 7
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-negative BM cells develop like wild-type BM cells. Secreted TNF can be detected in the SN of B6.WT BM cells. It reaches a plateau after 6 days of tissue culture. The TNF expression level can be correlated with the appearance of mature DCs. Moreover, the defective maturation of TNF-negative BM-DCs can be reconstituted using recombinant TNF or with the addition of wild-type BM cells, and conversely, differentiation of wild-type BM cells can be blocked with a neutralizing anti-TNF mAb.
Maturation of BM-DCs in the presence of GM-CSF [7
] depends on intact TNF signaling. The increase of TNF in the SN at day 6 correlates with the simultaneous maturation of DC precursor cells. Therefore, TNF-dependent cell maturation must be mediated in a paracrine or autocrine way. It is tempting to speculate that the adherent macrophage-like cells in the tissue culture are responsible for the production of TNF and thus, support the DC maturation in a paracrine way. RT-PCR analysis of adherent and nonadherent cells demonstrates that TNF is expressed in all cells (U. Ritter and H. Körner, unpublished results). However, mixing TNF-positive and TNF-negative BM cells results in a comparable maturation and points to a paracrine effect of TNF. In the absence of TNF, the differentiation step from immature to mature BM-DCs is defective. In our tissue-culture system, earlier steps of murine DC development are not affected by the absence of TNF or LT. In contrast, the neutralization of TNF in a culture of early human CD34+ precursor cells results in a block of DC development and a shift to myelogranulocytic hematopoiesis [18
]. This finding could indicate differences in the cytokine requirements of human and murine precursor DCs. The residual, spontaneous maturation of murine DCs in the absence of TNF also does not depend on LT. Therefore, the remaining maturation must be caused by TNF-independent mechanisms and has yet to be explained. One candidate cytokine could be the TNF-superfamily member TNF-related activation-induced cytokine. The receptor of this cytokine is expressed on mature DCs, and it serves primarily as a survival factor but could also have functions in DC maturation [19
].
Our results demonstrate a direct role for TNF but neither for soluble nor membrane LT in DC differentiation. This is in contrast with the analysis of the in vivo situation in gene-deficient models. In LT-negative mice, splenic DCs are strongly reduced [12
], whereas in TNF and TNF-receptor-negative mice, DCs are distributed normally in lymphatic tissues [12
]. Efficient homing of leukocytes needs the presence of tissue-associated chemokine gradients and the expression of CCRs on migratory cells [20
, 21
]. The CC-chemokines 19 and 21 are strongly expressed in the T cell area of the spleen [22
] and have been shown to be a DC chemoattractant [23
]. These chemokines are regulated by membrane LT and are therefore absent in LT-negative mice [24
], but their expression and therefore the splenic phenotype can be reconstituted with transgenes expressing TNF [25
] or LIGHT [26
]. This indicates that in LT-negative mice, a chemokine-dependent migration defect is the basis of the reduced number of splenic DCs in vivo. We show that CCR7 is expressed by mature BM-DCs (Fig. 2)
, irrespective of the gene deficiency. Furthermore, we demonstrate that LT is not involved directly in DC maturation. The data argue therefore that the described LT-dependent chemokine defect is indeed responsible for the reduced splenic DC number in LT-negative mice.
TNF-negative DCs are able to replenish tissue compartments in an unchallenged, steady-state situation, and the cell number reaches normal density in the epidermis. Furthermore, in TNF-negative mice, the DCs of the epidermis show the normal, ramified, in situ phenotype (U. Ritter and H. Körner, unpublished results). This indicates that under noninflammatory conditions, TNF-deficient DCs are able to home to peripheral tissues normally [27
]. As shown in Figure 3
, matured, TNF-deficient BM-DCs are indeed able to home to the LN. The defective DC maturation of TNF-deficient BM precursor cells in vitro raises the question of the performance of the cells in the absence of TNF under challenge. In the infection model of murine, experimental, cutaneous leishmaniasis, it could be demonstrated that the cellular-immune response in TNF-negative mice is delayed by approximately 1 week [28
]. This delay is a result of the diminished, local, inflammatory response and to a defect in antigen transport to the draining LN by CD8-negative, inflammatory DCs (U. Ritter and H. Körner, unpublished results). It is tempting to speculate that in the absence of TNF, a deficiency in maturation of DCs results in a diminished number of cells migrating to draining lymphoid organs.
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ACKNOWLEDGEMENTS |
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Received December 2, 2002; revised February 19, 2003; accepted March 24, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and tumour necrosis factor in lymphoid tissue organogenesis and spatial organisation defined in gene targeted C57BL/6 mice Eur. J. Immunol. 27,2600-2609[Medline]
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