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IMMUNOBIOLOGY
From the Center for Vascular Medicine,
Friedrich Schiller University of Jena, Erfurt, Germany; the Department
of Pharmaceutical Chemistry, University of Frankfurt, Frankfurt,
Germany; the Division of Carcinogenesis and Differentiation, German
Cancer Research Center, Heidelberg, Germany; the Department of
Dermatology, Faculty of Medicine, Kyoto University, Kyoto, Japan; and
the Department of Medical Biochemistry and Biophysics, Division of
Chemistry II, Karolinska Institutet, Stockholm, Sweden.
The 5-lipoxygenase (5-LO) pathway in human
CD34+ hematopoietic progenitor cells, which were induced to
differentiate into dendritic cells (DCs) by cytokines in vitro and in
DCs of lymphoid tissues in situ, was examined. Extracts prepared from
HPCs contained low levels of 5-LO or 5-LO-activating protein.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) plus tumor
necrosis factor- Leukocyte lineages of the innate immune system,
including neutrophils, monocyte-macrophages, mast cells, and
eosinophils, express the 5-lipoxygenase (5-LO;
arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) pathway and
produce leukotrienes (LTs) in response to inflammatory agonists in
vitro.1 Investigators have therefore considered LTs as
inflammation- and/or allergy-associated molecules.2,3 Several lines of evidence, however, support an expanded role of the
5-LO pathway in the regulation of antigen-specific adaptive immunity.
Epidermal Langerhans cells (LCs), ie, immature members of the dendritic
cell (DC) family capable of initiating antigen-specific immune
responses in naive lymphocytes, markedly express the 5-LO pathway.4 Among human tissues studied by Northern blot
analysis, LTB4 receptor messenger RNA (mRNA) is
strongly expressed in both thymus5 and lymph nodes (LNs)
(R.S., unpublished data, October 1998), while cysteinyl
LT1 receptor mRNA is expressed in spleen.6 Probably most significantly, 5-LO-deficient mice show altered ovalbumin-dependent cellular and humoral immune
responses.2,3,7,8
Although these studies have provided substantial support for an
indispensable role of the 5-LO pathway in the regulation of adaptive
immunity in general, important questions remain. The identities of the
5-LO pathway- and LT receptor-expressing cells in lymphoid tissues
are not known. Moreover, the significance of 5-LO expression by the 2 other professional antigen-presenting cells (APCs), B
lymphocytes9,10 and monocyte-macrophages,11 for the regulation of immune responses is far from clear. Accordingly, the impact of LTs within the adaptive immune system is presently difficult to conceive at cellular and molecular levels.
Antigens invade the host through epithelial surfaces, yet primary
immune reactions require prolonged interaction of DCs with naive T
lymphocytes in the extrafollicular (paracortical) areas of lymphoid
organs.11 Hence, antigens must be translocated to these
areas to trigger immunity. Moreover, T-cell activation by DCs requires
the antigen to be taken up, processed, and presented at the DC surface
in an immunogenic form on major histocompatibility complex (MHC)
molecules. It has been established that these tasks are carried out by
LCs. LCs form a network of initially quiescent sentinel DCs that are
located throughout epithelial surfaces. They are endowed with high
antigen-binding capacities and thus can effectively monitor their
environment for antigen. Upon antigen capture, they become activated,
migrate via afferent lymph vessels as veiled cells, home in
paracortical areas of draining LNs, and eventually mature into
interdigitating DCs (IDCs). IDCs have largely lost antigen-binding
activity, but they have acquired the ability to attract, cluster, and
activate naive T lymphocytes. In addition to DCs and T cells, lymphoid
organs harbor other cells that participate in the regulation of immune
reactions within anatomically and functionally highly organized
compartments including B lymphocytes at various stages of
differentiation, tingible body macrophages (TBMs), germinal center (GC)
DCs, and follicular DCs.11 Nothing is known about 5-LO
expression in these cells, the anatomy of the 5-LO pathway, or 5-LO
pathway target cells in any lymphoid organ.4 We reasoned
that analyses of in vitro DC systems, together with anatomical studies,
would conceivably enhance understanding of the potential role
of 5-LO in immunity.
To establish in vitro models of 5-LO in DCs, we studied DC
differentiation systems from human CD34+ hematopoietic
progenitor cells (HPCs) isolated from cord blood (CB). These systems
have permitted us to generate DCs at different stages of
differentiation and at sufficient quantities to explore LT formation in
intact DCs. In parallel studies, we have begun to outline the anatomy
of the 5-LO pathway in human LNs, tonsil, oral mucosa, and the Waldeyer
tonsillar ring by 5-LO and 5-LO-activating protein (FLAP) in situ
hybridization (ISH) and fluorescence immunohistochemistry. We show that
(1) cytokines known to affect the immune response in vivo up-regulate
the 5-LO pathway in DCs in vitro, (2) these DCs are capable of
producing large amounts of 5-hydroxyeicosatetraenoic acid (5-HETE) and
LTB4, and (3) distinct DC phenotypes in vivo, as well as GC
TBMs, express the 5-LO gene at marked levels.
Materials
Cell culture
RT-polymerase chain reaction and ISH 5-LO and FLAP RT-polymerase chain reaction (RT-PCR) analyses were performed as described.4 For ISH, human 5-LO complementary DNA (cDNA) EcoRI fragment (nucleotides, 1-2496) and human FLAP ApaI-NotI fragment (nucleotides, 13-504) were subcloned into pBluescript II KS vectors (Stratagene, La Jolla, CA). Frozen sections of normal human LNs were obtained during routine surgery from axillae of patients undergoing neck dissection and were free of metastases. Mucosal tissue and tonsil were obtained during routine surgery from patients undergoing tonsillectomy. Details of tissue preparation, hybridization conditions, probe specificity, and PCR parameters are available upon request.Fluorescence immunohistochemistry Frozen tissue sections or cytocentrifuged cells were fixed in 80% ice-cold methanol followed by 1 minute in 100% acetone at 20°C. Before use, anti-5-LO antiserum 1550 was affinity-purified on a 5-LO Sepharose column. For 2-color immunofluorescence, slides were
incubated with donkey antirabbit IgG F(ab')2 conjugated
with Cy3 and goat antimouse IgG F(ab')2 conjugated with Cy2
for one hour. Additional details are available upon request.
Fluorescence-activated cell sorter analyses Fluorescence-activated cell sorter (FACS) analyses were performed using anti-5-LO antiserum 1550 as described.4 Unconjugated antibodies (5-LO, Lag, mannose receptor, and eosinophil peroxidase) were added for 2 hours, then washed twice in saponin buffer. Fluorochrome-conjugated monoclonal antibodies (mAbs) (CD1a-PE, CD80-PE, CD83-PE, CD86-PE, CD40-PE, CD14-FITC, and HLA-DR-FITC) and fluorochrome-conjugated secondary antibodies (goat antirabbit IgG-FITC, donkey antirabbit IgG-PE, and donkey antimouse IgG-PE) were added in saponin buffer for one hour. Negative controls were performed with unrelated mouse mAbs and rabbit anticollagen IV antiserum. Fluorescence analyses were performed with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Data were analyzed with the CellQuest program (Becton Dickinson). 5-LO mean fluorescence intensity (MFI) was calculated by 5-LO MFI minus collagen MFI.Assays Immunoblots were performed on 10 µg total cell protein as described using anti-5-LO antiserum or a polyclonal anti-FLAP antiserum (gift from J. Evans, Merck Frosst, Canada).4 5-HETE and LTB4 formation in intact cells were determined upon stimulation with 10 µmol/L Ca++ ionophore A23187 and 40 µmol/L arachidonic acid or in cell-free systems in the presence of 40 µmol/L arachidonic acid as described.15
Freshly isolated highly purified CD34+ HPCs contain a small number of immature 5-LO+/HLA-DR+ cells Transcript and protein levels of 5-LO pathway constituents in CD34+ HPCs were determined by RT-PCR, immunoblot, and immunohistochemical analyses. When compared with buffy-coat cells or CD14+ blood monocytes, HPCs showed low but detectable levels of 5-LO and FLAP transcripts (Figure 1; additional data not shown). Consistent with low transcript levels, 5-LO and FLAP proteins were below the detection limit in immunoblots of 10 µg protein (Figure 2). These data indicated the presence of small numbers of cells with significant expression of 5-LO mRNA and protein or low-level expression in larger numbers of cells. To detect single rare 5-LO protein-expressing cells, we applied double-fluorescence immunohistochemical analyses. CD14+ monocytes and CD66b+ myeloid precursor/granulocytes, ie, leukocytes that could possibly have contaminated the HPC preparations, were used to evaluate the assay. We found that the great majority of cells expressed CD34 and HLA-DR but were 5-LO (Figure
3; see below). However, rare cells (less
than 2%) did show significant nuclear 5-LO fluorescence (Figure 3C,
arrow). These 5-LO+ cells resembled HPCs by several
criteria including CD14 and CD66b negativity, small size, scarcity of
cytoplasm, and round or moderately lobulated nuclei (Figure 3D).
However, while CD34 antigen was detectable, the immature
5-LObright cells displayed a CD34dim phenotype
(Figure 3C, arrow at patches of CD34 signals). It is noteworthy that
all 5-LObright HPCs were also HLA-DR+, whereas
we failed to detect 5-LObright cells that were
HLA-DR Induction of the 5-LO pathway during generation of DCs from HPCs in
response to SCF, GM-CSF, and TNF-  , known to promote DC
differentiation from CB-derived CD34+
HPCs,12-14 was chosen.18 Upon addition of
cytokines, 5-LO and FLAP transcripts rose within 3 days (Figure 1). The
increase of 5-LO and FLAP transcripts was largely caused by the
combination of GM-CSF and TNF- because either cytokine alone was
much less effective, and SCF alone had only small effects. The other
major human hematopoietic SCG factor, the fms-like tyrosine protein kinase ligand 3,19 yielded similar results. This indicates
that SCF and fms-like tyrosine protein kinase ligand 3 act primarily to
promote proliferation of HPCs rather than to influence the expression
of the 5-LO pathway. The rise in 5-LO and FLAP transcripts continued
for up to 22 days and was accompanied by a large increase in 5-LO and
FLAP proteins (Figure 2; additional data not shown).
TGF-  -1 has been demonstrated
to promote LC maturation in vivo, as shown by the absence of epidermal
LCs in TGF--1 knockout mice.20-22 When we added
TGF--1 to SCF/GM-CSF/TNF-, we observed a further strong increase
in 5-LO and FLAP transcript and protein levels in the total cell
population (Figures 1 and 2). The effect on transcripts became
significant within 3 days and continued for up to 22 days. Using RT-PCR
analysis, we determined whether TGF--1 alters transcript levels of
leukocyte and DC markers and those of other constituents of the
arachidonic acid cascade. TGF--1 decreased transcript levels of
LTA4 hydrolase and CD14 while it increased transcript
levels of LTC4 synthase, 15-LO, cyclo-oxygenase 2, CD1a,
and CD83. No change was observed in transcript levels of
cyclo-oxygenase 1. Transcript levels of 12-LO were low at 35 cycles at
all time points, but we cannot rule out 12-LO regulation in
subpopulations of cells. Again, in view of the heterogeneity of
leukocyte differentiation in SCF/GM-CSF/TNF--containing medium (see
below), further work is needed to determine the leukocyte lineage in
which each arachidonic acid cascade constituent is expressed
and/or regulated.
Double-fluorescence immunohistochemistry of
SCF/GM-CSF/TNF- specifically promotes the DC differentiation
pathway,12 other leukocyte lineage differentiation
programs may not be entirely suppressed. To identify 5-LO+
cell lineages and DC phenotypes, we applied double-fluorescence immunohistochemistry using a combination of 5-LO antisera, the nuclear
proliferation-associated antigen Ki67, antisera of leukocyte lineages,
and DC maturation markers. In contrast to freshly isolated HPCs, the
majority of their cytokine-derived progeny was strongly nuclear 5-LO+ (compare Figure
4A-B). The majority of Ki67+
cells were 5-LO, indicating that 5-LO up-regulation
during leukopoiesis occurs in cells that have left the cell cycle (data
not shown). Of 5-LObright cells, many (but not all) were
HLA-DR+, indicating that they are capable of antigen
presentation; other cells were 5-LO/HLA-DR
(Figure 4A-B, arrows at lower left) or
5-LO/HLA-DR+ (Figure 4A-B, arrows in upper
center). A small but significant number of 5-LO+ cells were
HLA-DR/CD66b+, revealing 5-LO expression in
myeloid precursor/granulocytes (data not shown). In addition, a minor
subpopulation of cells (less than 3%) was identified as eosinophil
peroxidase-positive/5-LO+ eosinophil precursors (Figure
4H). 5-LO+ eosinophils also showed strong cytoplasmic 15-LO
positivity, revealing 5-LO/15-LO double-positive cells, yet no other
cell subpopulation, including all DC phenotypes, expressed 15-LO
protein (data not shown).
We next focused on the 5-LO+/HLA-DR+ DC
population after extended periods of cytokine exposure. A
large number of the HLA-DR+ progeny expressed the monocytic
markers, CD14 (Figure 4I) and mannose receptor (Figure 4J), and the
monocytic lysosome marker, CD68 (data not shown). A lesser number
expressed the LC markers CD1a (compare Figure 4C-D) and Lag (Figure
4G); the costimulatory molecule, CD40 (Figure 4E); and the marker for
mature DCs, CD83 (Figure 4F). Caux et al13,14 described
several DC populations in SCF/GM-CSF/TNF- 5-LO FACS analyses reveal profound TGF- -derived HPC progeny (Figure 4). In addition, TGF--1 was found to strongly affect leukocyte lineage and DC maturation markers as well as 5-LO transcript and protein levels (Figures 1 and 2; additional data not shown). To characterize and
quantitate 5-LO protein expression in the entire cell population at the
single cell level, in each leukocyte lineage, and at distinct DC
differentiation stages, we employed FACS analyses. A sensitive 5-LO
FACS assay was established using the affinity-purified anti-5-LO antiserum 1550.4 In mixing experiments (confirmed by
immunohistochemical analyses), we used CHO cells as negative controls
and added RBL1 cells as positive controls. We noted that this antiserum
detected less than 2% RBL1 cells in a suspension of greater than 98%
CHO cells. When we applied the assay to the cytokine-derived HPC
progeny, we observed that approximately 50% of all cells were
5-LO+ after 15 days of exposure to SCF/GM-CSF/TNF-
(Figure 5). At this time a significant
number of cells were still proliferating due to the action of SCF.
These cells were Ki67+ and largely lacked 5-LO, as shown by
immunohistochemical analyses (data not shown). At 15 days
of exposure to SCF/GM-CSF/TNF- in the presence of TGF--1, 85%
of all cells had been recruited into the 5-LO+ cell
population (Figure 5).
This effect of TGF- The effects of TGF- HPC progeny produce significant amounts of 5-HETE and LTB4 in response to Ca++ ionophore A23187 and arachidonic acid To assay the ability of HPC-derived progeny to produce LTs, we added Ca++ ionophore A23187 in the presence of arachidonic acid to intact cells that had differentiated in the presence of SCF/GM-CSF/TNF- or SCF/GM-CSF/TNF-/TGF--1. We
observed that both populations of cells produced significant amounts of
5-HETE and LTB4 and that TGF--1-derived cells produced
amounts of 5-LO pathway products that exceeded those of control cells
by a factor of 3-4 (Figure 6). Similar
amounts of 5-HETE and LTB4 were produced by cell-free systems (data not shown). In a single preliminary experiment we found
that several immune agonists, CD40 ligand trimer (Immunex, Seattle,
WA), 2 members of the chemokine family,23-25 macrophage inflammatory protein (MIP)-3- and MIP-3-, and prostaglandin E2 stimulated LTB4 formation in intact cells
(data not shown). These data reveal that intact DCs can produce
significant amounts of 5-HETE and LTB4.
In situ hybridization and double-fluorescence immunohistochemistry identify scattered 5-LO+ cells in the paracortex of LNs or tonsil, in DCs of the Waldeyer tonsillar ring, and in GC TBMs We studied 5-LO and FLAP mRNA expression by in situ hybridization (ISH) in LNs, tonsil, oral mucosa, and mucosa overlying the Waldeyer tonsillar ring. Specific scattered 5-LO and FLAP ISH signals were recorded in paracortical areas (Figure 7A,C-D) and tonsil (data not shown). Signals were not found when the sense RNAs were used as probes (Figure 7B; data not shown). The distribution patterns of 5-LO and FLAP signals in these lymphoid organs were similar and consistent with the localization of LCs (peripheral paracortex) and IDCs (peripheral and central paracortex).11 There was no apparent difference in either the signal density/surface area or signal intensity/cell when peripheral and central paracortical areas were compared, which indicates that there was no major regulation of 5-LO during differentiation of immature (CD1a+) to mature DCs (CD83+). Scattered 5-LO and FLAP ISH signals were also found in cells of the mucosa of the Waldeyer tonsillar ring in proximity to the tonsil (Figure 7G-H). It is noteworthy that we had failed earlier to identify FLAP ISH signals in the normal human epidermis.4
Immunohistochemical analyses of T-cell-rich paracortical areas of LNs
and tonsil localized nuclear 5-LO to cells that were dendritic in
morphology and expressed CD1a (Figure
8A-B) and/or CD40 (data not shown) or
CD83 (Figure 8C) and HLA-DR (Figure 8D). To show the T-lymphocyte
cellularity of this area and the scattered location of the
5-LO+ DCs, we stained identical specimens with the
DNA-binding dye, Hoechst 33258 (Figure 8B). Similar findings were
obtained in human tonsil and the lymphoid tissue of the Waldeyer
tonsillar ring. These data reveal that LCs of the oral mucosa and 2 phenotypes of DCs in T areas in normal human LNs, tonsil, and Waldeyer
tonsillar ring express the 5-LO gene at significant levels.
Most of the 5-LO protein-expressing cells in the oral mucosa were
CD1a+, which indicates that the majority of ISH signals did
not stem from cells of innate immunity such as mast cells, neutrophils, or macrophages (data not shown). These data extend the fact
that 5-LO is expressed in epidermal LCs of epithelia other than the epidermis.4 Although we were unable to identify the
FLAP-expressing cell lineage by immunohistochemistry, the distribution
of FLAP and 5-LO ISH signals were similar, indicating that the majority of 5-LO-expressing cells in the normal oral mucosa were LCs. It is
noteworthy in this connection that the LTB4
receptor5 and LTC4
receptor6 RT-PCR analyses revealed significant mRNA
levels of both receptors in LNs and tonsil, thereby indicating
the presence of LTB4 and LTC4
receptor-expressing cells in secondary lymphoid organs. Further work
is required to determine the identity of these cells.
In addition to T-lymphocyte-rich areas, we observed scattered 5-LO and FLAP ISH signals in GCs of both tonsil and LNs (Figure 7E-F). However, while these 5-LO- expressing cells also displayed a dendritic morphology, they stained for CD68 (lysosomal marker of monocytes) and CD71 (transferrin receptor), ie, markers previously associated with follicular TBMs (Figure 8E). In some GC TBMs we found 5-LO protein fluorescence in patches within the cytoplasm (Figure 8E). In view of the known ability of TBMs to phagocytose apoptotic B cells, the origin of this 5-LO remains to be demonstrated. Because GCs largely consist of rapidly proliferating centroblasts and because B lymphocytes have been reported to express 5-LO,9,10 we double-stained GCs with Ki67 and 5-LO antisera. It became evident, however, that centroblasts did not express significant 5-LO protein, as Ki67 signals of centroblasts were clearly dissociated from 5-LO signals (Figure 8F). Moreover, using the affinity-purified antiserum 1550, we did observe cytoplasmic fluorescence in significant numbers of cells in the B-lymphocyte-rich GC mantle zones. These presumptive 5-LO+ cells were small when compared to DCs and TBMs, did not have a dendritic morphology, and most likely represented differentiated B lymphocytes26,27 (data not shown). These results raise the interesting possibility that the 5-LO gene is up-regulated during differentiation of GC centroblasts into B lymphocytes. However, more direct evidence is needed to demonstrate that this assumption is legitimate. We are presently attempting to clarify these issues by using confocal laser scan microscopy and isolation of B lymphocytes from human lymph nodes at different stages of differentiation. Of note also, as single 5-LO+ cells in GCs expressed CD11c+, a marker previously found in GCDCs,14 it is possible that GCDCs express 5-LO. However, because fluorescence intensity of CD11c signals was found to be relatively weak in our preliminary studies, GCDC purification is required to substantiate this notion.
Most investigators have favored the view that 5-LO pathway products act distally of a T-lymphocyte helper 2 type-dependent immune response that is governed by interleukin-4 (IL-4), IL-5, IL-6, and IL-13 involving eosinophils, mast cells, and macrophages, as typified by extrinsic asthma.3,28 This notion implies that the major function of LTs in diseased states is to contribute to the organization of inflammatory infiltrates or to trigger other inflammatory tissue reactions, such as contraction of bronchial smooth muscle, and to enhance mucus production by epithelial cells. Studies by other investigators29 have provided evidence that the 5-LO pathway plays a role in both cellular and humoral immunity, thereby implying its action at one or more proximal steps within the immune response cascade.2,7,8 However, the identity of immune cells that produce LTs and their target cells, as well as their functional impact, all remain to be defined. Primary immune reactions are initiated by LC-type sentinel DCs within epithelial surfaces.11 These DCs first monitor and sample antigen, then are triggered to emigrate from the sites of antigen capture, pass through lymph vessels as "veiled" cells, home in T-lymphocyte-rich areas of draining lymphoid tissues, and finally present antigen to instruct naive lymphocytes during prolonged multiple-cell interactions. The complexity of these events and the lack of information regarding the anatomy of the 5-LO pathway in lymphoid tissues have precluded predictions of specific roles of LTs in the immune response. Two lines of evidence detailed above are consistent with a proximal action of the 5-LO pathway within the immune-response cascade. DC differentiation and maturation from CD34+ HPCs is associated with strong up-regulation of the 5-LO pathway in vitro, and CD1a+ DCs of epithelia and the peripheral paracortex (immature) and central paracortical CD83+ IDCs of T-lymphocyte-rich areas of LNs and tonsil (mature) express both 5-LO and FLAP genes in vivo. While 5-LO expression in freshly prepared embryonic CB-derived
CD34+ HPCs is restricted to a scarce immature leukocyte
HLA-DR+ (Figure 3) subpopulation (indicating early
up-regulation of 5-LO in APCs during hematopoiesis),
SCF/GM-CSF/TNF- The HPC progeny produce significant amounts of 5-HETE and
LTB4 in response to arachidonic acid and Ca++
ionophore A23187, although mature DCs generated in the presence of
TGF- Our ISH and immunohistochemical data provide new information on the organization of the 5-LO pathway in epithelia and secondary lymphoid organs. Both immature CD1a+ DCs and mature CD83+ IDCs in paracortexes of normal LNs express the 5-LO and FLAP genes (Figures 7 and 8). Similar results have been obtained in inflamed tonsil (data not shown) and the epithelium of the Waldeyer tonsillar ring (Figure 7). Other DCs, including those located in buccal mucosa, CD1a+ LCs in the dermis, and those in nonasthmatic bronchial epithelium of the lung (obtained from lung cancer patients), express 5-LO (data not shown). Moreover, scattered cells in LN GCs that are strongly 5-LO+ and FLAP+, display a dendritic morphology, and stain for CD68 (Figure 8) and CD71 (the transferrin receptor; data not shown), probably represent GC TBMs. Together with previous studies of naive epidermal LCs,4 these data reveal that the 5-LO gene is expressed in both immature and mature DCs and possibly in GC TBMs in vivo. They raise the possibility that the entire myeloid-derived human DC system11 expresses the 5-LO pathway. There is no information presently available on 5-LO expression in lymphoid-derived DCs.30 Paracortical CD1a+ cells of LNs are believed to represent recently arrived LCs that are in the process of down-regulating CD1a and maturing into CD83+ IDCs.11,18 What is the function of this prominent 5-LO pathway expression in several DC phenotypes located in the epidermis, epithelia, and lymphoid organs? Major roles of immature DCs are antigen sampling, processing, and antigen transport to sites of lymphocyte activation in LNs, whereas major functions of mature DCs are antigen presentation and initiation of primary immune reactions. Thus, the immune response involves multiple cell-cell interactions that are separated both in time and space and range from LC/keratinocyte interaction in the epidermis, DC/endothelial cell interactions during retroendothelial migration of DCs into dermal lymph vessels, homing events at specific sites within lymphoid organs, and DC/lymphocyte interactions in the paracortex and GCs. We hypothesize that LTs play a role in one or more of these events. Moreover, as the paracortex of LNs does not contain inflammatory cells of the innate immune system, such as neutrophils, mast cells, or eosinophils, it is unlikely that 5-LO products produced by LCs,4 IDCs, GC TBMs, or GCDCs31 participate in any bona fide inflammatory/allergic tissue reaction previously associated with the action of 5-LO pathway products.1-3,7,8,28,29 Instead, it is more likely that these products affect one or more proximal steps of the immune response cascade either by acting on the DCs in an autocrine way or by acting on neighboring cells, possibly lymphocytes. The fact that LTB4 can affect B-lymphocyte functions has already been shown in vitro.32 To delineate molecular mechanisms within immune responses that are affected by 5-LO pathway products, future studies should be directed toward DC/lymphocyte cocultures, the identification of LT receptor-expressing cells in lymphoid organs, and immune-response studies of 5-LO knockout mice.
We are grateful to Dr F.-X. Bosch, Ear Nose and Neck Clinic, University of Heidelberg, Heidelberg, Germany, for gifts of normal human LNs and tonsil, and to the nurses of the Department of Gynecology of the University of Heidelberg for providing CB. Antiserum 1550 was prepared by Drs Y.-Y. Zhang, T. Hammarberg, and H. Okamoto.
Submitted May 10, 2000; accepted August 3, 2000.
Supported by grant Ha 1083/12-1 from the German Research Council, Germany; grant 01GB 9401/6 from the German Bundesministerium für Forschung und Technologie, Germany; grants BMH4-CT96-0229 and BMH4-CT98-3191 from the EU concerted actions; grant TP5.11 from the Verbund für Klinische Forschung of the University of Jena, Erfurt, Germany; grant 03X-217 from the Swedish Medical Research Council, Stockholm, Sweden; grant A95067 from the Vårdal Foundation, Stockholm, Sweden; and the Stiftung VERUM, Munich, Germany.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Rainer Spanbroek, Center for Vascular Medicine, Friedrich Schiller University of Jena, Nordhäuserstr. 78, 99089 Erfurt, Germany; e-mail: spanbroek{at}zmkh.ef.uni-jena.de.
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R. van de Ven, M. C. de Jong, A. W. Reurs, A. J. N. Schoonderwoerd, G. Jansen, J. H. Hooijberg, G. L. Scheffer, T. D. de Gruijl, and R. J. Scheper Dendritic Cells Require Multidrug Resistance Protein 1 (ABCC1) Transporter Activity for Differentiation J. Immunol., May 1, 2006; 176(9): 5191 - 5198. [Abstract] [Full Text] [PDF]
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R. Spanbroek, M. Hildner, A. Kohler, A. Muller, F. Zintl, H. Kuhn, O. Radmark, B. Samuelsson, and A. J. R. Habenicht IL-4 determines eicosanoid formation in dendritic cells by down-regulation of 5-lipoxygenase and up-regulation of 15-lipoxygenase 1 expression PNAS, April 24, 2001; 98(9): 5152 - 5157. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
