| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Departments of Pathology,*
Urology,
Nephrology and
Rheumatology,
Gastroenterology,
and
Dermatology,¶
Georg-August University of
Göttingen, Göttingen, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The traffic of inflammatory cells at sites of inflammation includes four distinct phases: circulation, adhesion, diapedesis, and migration. Chemokines are known to play a crucial role in these steps.3 Chemokines are small secreted proteins stimulating chemotaxis and leading to selective attraction of inflammatory cells. The chemokine superfamily can be subdivided according to the organization of the N-terminal conserved cysteine (C) motif into four groups, which are designated as C-, C-C-, C-X-C-, and C-X3-C-chemokines.4 The minor differences in the N-terminal structure result in actions on different cell types.5 C-X-C-chemokines generally attract neutrophils and T cells, C-C-chemokines are chemoattractive for monocytes, eosinophils, basophils, and T cells.6,7 Whereas the C-X-C- and C-C-subfamilies constitute several members, the C- and C-X3-C-chemokine subfamilies are represented by only one member so far, designated lymphotactin (Lptn)8 also known as activation-induced T cell-derived and chemokine-related molecule (ATAC)9 or single C motif-1 (SCM-1)10 and fractalkine/neurotactin,11 respectively. Preliminary characterization revealed that in contrast to the members of the C-C- and C-X-C subfamilies, Lptn and fractalkine are powerful attractants for lymphocytes, in particular T cells, but are inactive in inducing the migration of neutrophils and monocytes.8
In vitro and in vivo Lptn was found to be more
chemotactic for CD8+ than CD4+ T cells and, in addition, modestly
chemoattractive for natural killer cells.12-16
In
contrast to other chemokine subfamilies Lptn lacks two of the four
characteristic cysteine residues, but shows strong amino acid
similarities with the C-C-chemokines.17
Expression of Lptn
mRNA was found in certain subsets of T cells, including activated CD8+
and CD4+ T cells,9
intraepithelial 
-type T
cells,17
ß-type thymocytes,8
mast
cells,18
and natural killer cells.15
More
recently, a unique G-protein-coupled receptor (GPR-5) has been found to
function as the natural Lptn ligand.19,20
Other biological
functions of Lptn are still unknown.
So far the expression of Lptn at sites of inflammation has rarely been investigated. The in vitro observed functional properties of Lptn suggest an important function in the recruitment of T cells in the pathogenesis of chronic inflammatory disorders. Because focal lymphocytic infiltrates represent a predominant feature of the chronic inflammatory infiltrate observed in CD, we characterized and determined the phenotype of inflammatory cells expressing this potent chemokine. Our results demonstrate an increased local expression of Lptn in CD, which could be attributed not only to activated T cells, but also to mast cells. In addition, we will show for the first time expression of Lptn mRNA by dendritic cells (DCs) in CD in vivo and by monocyte derived DCs generated in vitro.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Surgical specimens derived from involved areas of large intestine of 15 patients with active CD were studied. CD was active in all of the patients as confirmed by clinical features, laboratory parameters, as well as macroscopic and histopathological alterations. As controls surgical specimens from six patients with noninflammatory gut disorder were analyzed (four colon carcinomas and two diverticular diseases). For routine morphological analysis, immunohistochemistry, and nonradioactive in situ hybridization appropriate tissue specimens were fixed in formalin and embedded in paraffin. Additional representative samples were snap-frozen in liquid nitrogen, stored at -80°C, and used for RNA isolation.
RNA Extraction and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
RNA was isolated from cryopreserved tissue samples and isolated cells using TriReagent according to the manufacturer’s instructions (Sigma, Deisenhofen, Germany) and finally obtained in 30 µl of RNase-free water. RNA content was determined spectrophotometrically. One µg of RNA was used for reverse transcription after a DNase (fast protein liquid chromatography-pure DNaseI; Pharmacia, Freiburg, Germany) digestion step. cDNA synthesis was performed in a total volume of 20 µl of RNase-free water including 1.5 µmol/L of oligo-dT primer, 0.4 mmol/L of each deoxynucleotidetriphosphate (both from MBI Fermentas, St. Leon-Rot, Germany), and 200 U of reverse transcriptase (SuperScriptII; Gibco, Karlsruhe, Germany).
The oligonucleotide primer sequences were determined manually according to the published sequences in GenBank for ß-actin (X00351), Lptn (U23772), and GPR-5 (NM005283). PCR conditions were as follows: ß-actin (ß-actin forward primer: 5'-CTACAATGAGCTGCGTGTGGC-3'; ß-actin reverse primer: 5'-CAGGTCCAGACGCAGGATGGC-3'; product size: 270 bp) 3 minutes at 95°C, 35 cycles of 40 seconds at 95°C, 45 seconds at 57°C, 1.5 minutes at 72°C, followed by a final extension step of 7 minutes at 72°C; Lptn (Lptn forward primer: 5'-GTGGAAGGTGTAGGGAGTGAAGTC-3'; Lptn reverse primer: 5'-GTCCATGAGGGTGTAAAGTGAAAT-3'; product size: 354 bp) 3 minutes at 95°C, 35 cycles of 40 seconds at 95°C, 40 seconds at 55°C, 1 minute at 72°C, and final elongation of 7 minutes at 72°C; Lptn receptor GPR-5 (forward primer: 5'-GCTTTCTTCGGGCTGTGATTATTC-3'; reverse primer: 5'-GCGGCAGATGAGCAGGGCGTATT-3'; product size: 310 bp) 3 minutes at 95°C, 35 cycles of 40 seconds at 95°C, 45 seconds at 58°C, 1.30 minutes at 72°C, and final elongation of 10 minutes at 72°C. The PCR products were visualized on 1% agarose gels by staining with ethidium bromide. To exclude amplification of genomic sequences PCR primers were designed to amplify cDNA fragments that span at least one intron of the corresponding gene. cDNA from a human lymph node was used as a positive control template for each primer pair. Negative controls with water instead of cDNA were always included. A PCR reaction was considered to be positive when a single band of correct product length could be detected. Amplification of correct PCR products was controlled by asymmetric restriction enzyme digestion and sequencing with an ABI310 automatic sequencer using the rhodamine-dye terminator kit according to the manufacturer’s instructions (Perkin Elmer, Darmstadt-Weiterstadt, Germany).
Cloning of PCR Products
For generation of the Lptn and GPR-5 riboprobes for quantitative
real-time RT-PCR or in situ hybridization PCR products were
cloned into the blunt-end SmaI restriction site of the
transcription vector pBluescript II KS plasmid. Briefly, blunt-end PCR
products were generated by using Vent polymerase (Pharmacia,
Heidelberg, Germany) according to the PCR-cycling conditions as
mentioned above. After microspin column purification (Pharmacia) the
blunt-end PCR products were ligated into a SmaI restricted
pBluescript II KS plasmid using T4-DNA ligase at 16°C overnight and
transformed into the CaCl2-competent
Escherichia coli strain DH5. After propagation under
standard culture conditions the plasmids were isolated using the
alkaline lysis procedure. Cloning of Lptn- and GPR-5-specific PCR
fragments was confirmed by cycle-sequencing with the Rhodamine-dye
terminator kit using an ABI 310 Sequencer according to the
manufacturer’s instructions (Perkin-Elmer). Riboprobes were generated
by in vitro transcription using T3- or T7-polymerase
(Boehringer, Mannheim, Germany). The amount of cRNA transcripts was
monitored by 1% agarose gel electrophoresis and determined
spectrophotometrically.
Real-Time Quantitative RT-PCR Analysis
Quantification of Lptn and GPR-5 mRNA expression in a RT-PCR set up was performed on an iCycler iQ real-time detection system (Bio-Rad, Hercules, CA). For RT-PCR a one-step RT-PCR kit (Qiagen, Hilden, Germany) was used. The 15-µl reaction from this kit was supplemented with 150 ng of total cellular RNA, 0.6 µmol/L each of the gene-specific primers, 4 U of RNase inhibitor (Promega, Mannheim, Germany) and 0.5 µl of SYBRgreen I dye (Molecular Probes, MoBiTec, Göttingen, Germany) and diluted in 1x RT-PCR buffer (Qiagen) to 12.5x. The dilution was checked spectrophotometrically at 260 nm. The final concentration of SYBRgreen I dye was 0.4x. The reaction mix was loaded into rows of eight 200-µl PCR reaction tubes (Sarstedt, Nümbrecht, Germany) and covered with optical sealing tape (Bio-Rad). The thermal cycling proceeded as follows: 50°C for 30 minutes for reverse transcription, followed by 15 minutes 95°C for denaturation of reverse transcriptase, and activation of HotStar Taq polymerase. A maximum of 50 PCR cycles was run in the following profile: initial denaturation at 95°C, annealing at 55°C, and extension at 72°C for 30 seconds, each. Acquisition of fluorescence signals was monitored on the iCycler and terminated when all reactions reached an amplification plateau and template-free control stayed at basement levels. A PCR standard curve was generated using the same reaction mix including in vitro generated Lptn and GPR-5 cRNA (dilution range from 1 to 1000 attomoles) instead of total cellular RNA. Data analysis was done with iCycler iQ real-time detection system software (Bio-Rad). Results derived from the PCR standard curve for Lptn and GPR-5 mRNA expression are given in attomoles per µg total cellular RNA.
To verify that only specific PCR products evoked fluorescence signals a melting curve of PCR products was obtained in a cycler program of denaturation (95°C), renaturation (50°C), and a temperature ramp from 60°C to 95°C with 0.5°C increments for 10 seconds. Melting peaks were related to PCR product size on 1% agarose gels.
Nonradioactive in Situ Hybridization
For in situ hybridization of Lptn- and GPR-5-specific mRNA transcripts paraffin sections were mounted on slides coated with 3-aminopropyl-triethoxysilane (Sigma) under RNase-free conditions. The slides were allowed to dry at 37°C overnight. Digoxigenin-11-dUTP-labeled antisense and sense probes were generated by in vitro transcription using T3 or T7 polymerase (Boehringer). The amount of transcripts was monitored by 1% agarose gel electrophoresis and labeling efficiency was controlled by dot-blot analysis of serial probe dilutions.
In situ hybridization was performed modified according to the method described by Breitschopf and colleagues.21 In brief, tissue sections were deparaffinized, rehydrated in serial dilutions of ethanol, and postfixed in 4% Tris-buffered saline (TBS)- buffered paraformaldehyde. Samples were permeabilized using proteinase K (10 µg/ml) for 30 minutes at 37°C. Digestion was stopped by washing the samples in a phosphate-buffered saline solution (pH 7.4) and the slides were dehydrated in serial dilutions of ethanol. Digoxigenin-labeled riboprobes were diluted in hybridization buffer (Amersham, Braunschweig, Germany). After application of sense or antisense cRNA probes the samples were covered with sterile coverslips and placed on a hot plate at 85°C for 5 minutes for probe and target denaturation. Hybridization was performed overnight at 58°C in a sealed humid chamber containing 50% formamide. Nonspecific binding or unbound probes were removed by the following posthybridization washes: 1x standard saline citrate/0.1% sodium dodecyl sulfate at room temperature (2 x 5 minutes) and 0.2x standard saline citrate/0.1% sodium dodecyl sulfate at 58°C (2 x 10 minutes) followed by RNase digestion (20 µg/ml, Gibco) for 30 minutes at 37°C. Finally, sections were washed in TBS containing 0.1% Tween-20 (TBST, Boehringer). Hybridization signals were detected using a sheep polyclonal antibody F(ab)2-fragment against digoxigenin conjugated with alkaline phosphatase (1:500, Boehringer). Alkaline phosphatase activity was visualized using 5-bromo-4-chloro-3-indolyl phosphate as substrate and nitro blue tetrazolium as coupler (Boehringer). For each tissue section sense riboprobes served as controls.
Detection of GPR-5-specific mRNA required signal amplification. Briefly, hybridization signals were detected using a sheep polyclonal antibody F(ab)2-fragment against digoxigenin conjugated with horseradish peroxidase (1:300, Boehringer) and applying the tyramide signal amplification method as described below in the next section using diaminobenzidine as chromogen for visualization.
To determine the specified cell types expressing Lptn mRNA hybridized specimens were incubated with the monoclonal antibodies (mAbs) Ki-M1P recognizing monocytes/macrophages22 (1:1000; kindly provided by Prof. Parwaresch, Kiel, Germany) or CD3 for T cells (undiluted; Beckmann Coulter, Heidelberg, Germany) followed by a rabbit anti-mouse Ig fluorescein isothiocyanate (1:50; DAKO, Hamburg, Germany) for evaluation by immunofluorescence. In case of the antigens CD4 (1:50) and CD8 (1:50) for T-cell subsets (both Novocastra, Dossenheim, Germany), CD20 for B-cells (1:50), CD57 for natural killer cells (1:50), tryptase for mast cells (1:300; all DAKO), and S100 protein recognizing DCs (undiluted; Beckman Coulter) immunohistochemistry was performed on serial sections. To visualize bound primary antibodies, the following detector components were applied as recommended by the manufacturer: a biotin-streptavidin-amplified system containing biotinylated secondary antibody (multilinker 1:20), alkaline-phosphatase-conjugated streptavidin (label 1:20), fast red (chromogen), and naphthol phosphate (substrate; Biogenex, Hamburg, Germany).
Immunohistochemistry and Double Immunofluorescence for Lptn
Paraffin-embedded sections were immunostained applying the tyramide signal amplification system method according to the manufacturer’s instructions (Dupont-NEN, Boston, MA). For the detection of Lptn (1:600, rabbit anti-human Lptn; TEBU, Frankfurt, Germany) sections were pretreated by repeated boiling in citrate buffer (pH 6.1) in a microwave (700 W, 5 x 3 minutes). After cooling to room temperature nonspecific binding was blocked with 20% bovine serum albumin (Sigma) in 0.05 mol/L Tris/HCl-buffered NaCl solution (TBS, pH 7.4) followed by incubation with the primary antibody overnight at 4°C in a humidified chamber. After rinsing in TBS the slides were incubated for 30 minutes with a biotinylated goat anti-rabbit Ig polyclonal antibody (1:500, DAKO) diluted in Tris-HCl buffered NaCl blocking buffer (TNB, pH 7.5) (Dupont-NEN). After washing three times in TBS slides were incubated with streptavidin-horseradish peroxidase complex (diluted 1:100 in TNB) for an additional 30 minutes. Three washes with TBS were followed by application of biotin-tyramide diluted 1:100 in Amplification Diluent (Dupont-NEN) for 10 minutes. Reactive sites were detected with streptavidin-horseradish peroxidase (1:100 in TBS, 30 minutes at room temperature). Peroxidase activity was visualized by using aminoethyl carbazole as chromogen. In control reactions irrelevant primary antibody was applied or primary antibodies were omitted.
To characterize in situ the co-expression of Lptn and other antigens such as CD3 (DAKO), CD4, CD8 (both Novocastra), CD31, CD45RA (both DAKO), CD103 (HML-1; Santa Cruz, Heidelberg, Germany), tryptase, and the proliferation-associated antigen Ki67 (Dianova, Hamburg, Germany) dual-color digital confocal laser microscopy on paraffin-embedded sections was performed. Pretreatment of the deparaffinized slides was performed as mentioned above. The Lptn mAb was applied first, incubated overnight in a humidified chamber at 4°C, and detected by using the biotin-tyramide signal amplification system (Dupont-NEN): biotinylated secondary antibodies (goat anti-rabbit antibody conjugated with biotin, DAKO) were diluted in TNB (1:500) and applied for 30 minutes at room temperature. After washing three times in TBS slides were incubated in streptavidin-horseradish peroxidase complex (diluted 1:100 in TNB) for an additional 30 minutes. Three washes with TBS were followed by application of biotin-tyramide diluted 1:100 in amplification diluent (Dupont-NEN) for 5 minutes. Reactive sites were detected with streptavidin-fluorescein isothiocyanate (1:50 in TBS, 30 minutes at room temperature). Subsequently, the polyclonal antibody CD3 and the mAbs CD4, CD8, CD31, CD45RA, CD103, Ki67, and tryptase were applied overnight at 4°C. The mAbs were detected by indirect immunofluorescence using rhodamine-Red-X-conjugated F(ab)-fragments of rabbit anti-mouse Ig (1:50, Dianova). CD3 was detected by application of rhodamine-Red-X-conjugated goat anti-rabbit Ig (1:50, Dianova). After each incubation the sections were thoroughly washed in TBS and embedded in fluoromount fluorescent mounting media before examination to prevent fading (DAKO). Irrelevant antibodies or omission of first and second step antibodies served as negative controls. Analysis of fluorescence signals was performed on a Leica TCS NT laser scanning microscope (Heidelberg, Germany).
Separation of Monocytes and T Cells from Blood
Monocytes and T lymphocytes were obtained by Ficoll (Biochrom, Berlin, Germany) gradient centrifugation of peripheral mononuclear cells separated from the buffy coats of blood samples from healthy volunteers. For positive selection of interphase cells the mAbs anti-CD14 and anti-CD3 directly coupled to magnetic beads were used for enrichment of monocytes and T lymphocytes, respectively (all Miltenyi Biotec, Bergisch-Gladbach, Germany). All selection steps were repeated once. Separated cells were >95% pure as determined by flow cytometry (data not shown).
T-Cell Stimulation Experiments
For cell stimulation experiments T cells were cultured at a density of 106 T cells/ml RPMI 1640 culture medium supplemented with 10% fetal calf serum, 2 µmol/L L-glutamine, and gentamicin (50 µg/ml), further referred as complete culture medium, in the presence of 5% CO2 at 37°C. Cells were stimulated for 12 hours with phorbol-12-myristate-13-acetate (PMA, 50 ng/ml) or concanavalin A (ConA, 200 µg/ml). Dexamethasone (1 µmol/L), cyclosporine A (500 ng/ml), and FK506 (100 ng/ml) were added immediately before stimulation. To investigate a possible regulatory effect of Lptn itself on its expression by activated T cells, a blocking anti-human Lptn antibody (TEBU, Frankfurt, Germany) was supplemented to the cultures at a neutralizing concentration of 10 µg/ml of complete culture medium. The expression of Lptn and GPR-5 was determined by real-time quantitative RT-PCR analysis as described above.
Dendritic Cell Differentiation
For the generation of mature DCs, CD14-positive selected monocytes were cultured as described previously.23 Briefly, the monocytes were plated in 6-well tissue-culture plates at a density of 2 x 106 cells/well in 4 ml of complete culture medium. Granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4 were added at final concentrations of 800 and 1000 U/ml, respectively. Cultures were fed on days 2 and 4 by removing 1 ml of the medium and adding back 1.5 ml of fresh medium with cytokines (1600 U/ml granulocyte-macrophage colony-stimulating factor and 1000 U/ml IL-4). Cells were harvested on day 6 and transferred into new wells and cultured for 1 more day with fresh granulocyte-macrophage colony-stimulating factor and IL-4 supplemented with 25% monocyte-conditioned media, which was prepared by culturing 107 peripheral blood mononuclear cells on immunoglobulin-coated bacteriological plates for 1 hour at 37°C in 8 ml of complete culture medium. The DCs harvested at day 7 proved to be fully mature as determined by their strong expression of the CD83 antigen, the expression of which ranged from 55 to 85% of the population as shown by fluorescence-activated cell sorting analysis (data not shown). Total mRNA of DCs at different stages of maturation was analyzed for expression of Lptn by RT-PCR as described above.
Statistical Analysis
Statistical data analysis was performed using the InStat statistical program (GraphPad-Software, San Diego, CA). Statistical differences were evaluated by unpaired Student’s t-test. P values were determined by a two-sided calculation. Results were considered to be statistically significant if P values were <0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Lptn mRNA expression was analyzed in tissue samples affected by active CD (n = 6) and from normal controls without signs of inflammation (n = 6) by RT-PCR with oligonucleotide primers specific for the human Lptn cDNA. As internal control RT-PCR for the housekeeping gene ß-actin was performed. Lptn-specific products could be amplified from mRNA probes isolated from all tissue samples investigated.
For quantification of Lptn mRNA expression in tissue samples a method
of real-time quantitative RT-PCR was established using iCycler
technology. Lptn mRNA levels of equal amounts of total RNA from the
tissue samples were calculated by reference to a standard curve, which
was obtained by one-step RT-PCR containing different amounts of
in vitro generated Lptn cRNA (Lptn standards), showing a
dilution range from 1 to 1000 attomoles (Figure 1, A and B)
. Quantification results were
given as attomoles per µg total cellular RNA of each investigated
tissue sample. In the total RNA isolated from tissue with active CD a
significant increased expression of Lptn mRNA could be determined in
comparison to RNA samples obtained from bowel tissue without signs of
inflammation (P < 0.001). In RNA samples from
CD Lptn mRNA was found to be approximately eightfold more expressed
than in controls (Figure 1C)
.
|
As Lptn mRNA expression was shown to be increased in active CD the results of quantitative RT-PCR were confirmed by nonradioactive in situ hybridization using digoxigenin-labeled cRNA probes and by immunohistochemistry with a Lptn-specific antibody.
In control tissue specimens only few Lptn-positive cells could be
observed. Cells expressing Lptn mRNA and protein were almost restricted
to the lamina propria (Figure 2, a and b)
. In situ hybridization experiments and immunostainings on
tissue specimens from CD revealed numerous Lptn-positive cells within
the inflammatory infiltrate of the entire bowel wall. In the mucosa
numerous positive cells could be observed within the lymphocytic
infiltrates of the lamina propria and additionally within the
epithelium (Figure 2, c and d)
. Furthermore, numerous Lptn-positive
cells were observed adjacent to blood vessels. The perivascular pattern
was predominantly found in the deeper layers of the bowel wall
comprising the submucosa, the muscularis propria, and the subserosal
tissue (Figure 2e)
. Granulomas, occasionally observed in CD, did not
show any expression of Lptn mRNA or protein by epithelioid cells or
giant cells, which was in contrast to the strong intracytoplasmic Lptn
expression by surrounding lymphoid cells (Figure 2f)
. Furthermore, a
clear relationship was observed between the increased numbers of
Lptn-expressing cells and the extent of lymphocytic infiltration in CD
versus controls without significant signs of inflammation.
|
) showed a significant increased number of Lptn-positive cells
within the lamina propria, which were found to be significantly reduced
in remission on therapy with steroids 8 months later (severity activity
index, 35; Figure 2h
). However, these data have to be confirmed by
further cases.
To characterize the phenotype of inflammatory cells expressing Lptn
mRNA or protein combined nonradioactive in situ
hybridization combined with immunofluorescence or
double-immunofluorescence investigations were performed. The great
majority of Lptn-positive cells could be identified as CD3+ T cells
(Figure 3a
and Figure 4a
) comprising both the CD4+ and CD8+
subtype (Figure 3, c and d)
. Other cells expressing Lptn were
identified as mostly perivascular located CD117/c-Kit-positive mast
cells (Figure 3e)
and in addition occasionally S100 protein-positive
DCs (Figure 3f)
. The latter were mostly observed within the upper part
of the lamina propria or in the vicinity of subserosal vessels. By
extensive further investigations no expression of Lptn mRNA or protein
was found by other cell types such as natural killer cells (detected by
mAb CD57), monocytes/macrophages, or granuloma histiocytes (detected by
mAb Ki-M1P; Figure 3b
), and B-lymphocytes (detected by mAb CD20).
Furthermore, no expression was found by epithelial cells of the
mucosa or granulocytes as determined by morphology.
|
|
. Furthermore, numerous predominantly
perivascular-located cells expressed the antigens CD31 (Figure 4c)
and
CD45RA (Figure 4d)
thus representing immigrating naïve
inflammatory cells. Additionally, Lptn-positive cells proved to express
the proliferation-associated antigen Ki67 (Figure 4e)
thus comprising
predominantly proliferating CD4+ or CD8+ T cells within the
inflammatory infiltrate (Figure 4, f and g)
. In parallel, performed
negative controls did not show any specific staining pattern (Figure 4h)
. Expression of GPR-5 mRNA in CD
The expression of GPR-5 mRNA was investigated by nonradioactive
in situ hybridization. In controls GPR-5 mRNA expression was
found to be restricted to cells of the lamina propria (Figure 2i)
. In
CD numerous GPR-5 mRNA-positive cells could be observed within the
inflammatory infiltrate of the entire bowel wall. Within the mucosa
increased numbers of positive cells were found within the lymphocytic
infiltrates of the lamina propria (Figure 2j)
. Occasionally, expression
of GPR-5 mRNA could be observed by intraepithelial lymphocytes.
Furthermore, abundant GPR-5 mRNA could be observed in cells adjacent to
blood vessels (Figure 2k)
and in lymphocytes surrounding granulomas
within the deeper layers of the inflammatory affected bowel. No
expression of GPR-5 mRNA could be observed by epithelioid cells, giant
cells (Figure 2l)
, and epithelial cells.
The comparison of biopsy specimens before and after conservative therapy with steroids demonstrated comparable results to those obtained for Lptn: in nonactive CD a significant reduced number of GPR-5 mRNA-positive cells could be found compared to the increased number of GPR-5 mRNA-positive cells observed within the inflammatory affected mucosa from active CD (data not shown).
Regulation of Lptn mRNA Expression by T Cells in Vitro
Quantitative real-time RT-PCR was performed on unstimulated or Con A-stimulated or PMA-stimulated CD3-positive selected T cells from peripheral human blood. Freshly isolated T cells demonstrated a very weak expression of Lptn mRNA expression, which was strongly enhanced by addition of Con A or PMA, whereby PMA was more powerful by inducing Lptn mRNA expression than ConA. The addition of a neutralizing Lptn antibody at saturating concentrations during the culture period did not show any regulatory effect on the expression of Lptn mRNA by T cells either without or with stimulation by ConA or PMA (data not shown).
Dexamethasone, FK506, and cyclosporine A are powerful immunosuppressive
drugs, which are successfully used for therapy in CD. Their therapeutic
effect is partly mediated by inhibiting transcriptional activation of
different cytokine genes such as of the IL-2 gene.24
Addition of FK506 and cyclosporine A prevented the observed
up-regulation of Lptn mRNA by PMA, whereas dexamethasone led to a
decrease of Lptn mRNA expression below the amount of Lptn mRNA
expression of untreated T cells (Figure 5A)
.
|
. Lptn mRNA Expression by Monocyte-Derived Dendritic Cells in Vitro
Because of the unexpected expression of Lptn mRNA by small numbers
of S100-positive DCs, monocyte-derived DCs were generated in
vitro.23
Lptn mRNA expression was investigated at
different stages of DC differentiation and maturation. RT-PCR analysis
of freshly isolated monocytes and day 3-harvested immature DCs showed
no expression of Lptn mRNA. First Lptn-specific amplification products
became detectable on day 5 of in vitro differentiation, the
expression of which increased until day 7, when full maturation with
conditioned media was induced (Figure 6)
.
The results demonstrate a maturation-dependent expression of Lptn mRNA
in DCs.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our results show a statistically significant increase of Lptn mRNA
expression in CD versus normal controls
(P < 0.001) as determined by quantitative
real-time RT-PCR analysis (Figure 1)
. The amount of Lptn mRNA per µg
total RNA was found to be approximately eight times higher in CD than
in controls. These results could be confirmed by in situ
hybridization experiments for Lptn mRNA. In CD increased numbers of
Lptn mRNA and protein-expressing cells were found within the entire
inflammatory affected bowel wall (Figure 2)
. No differences could be
observed by comparing the staining pattern of Lptn mRNA by in
situ hybridization or protein by immunohistochemistry. A
comparable distribution could be observed for the Lptn receptor GPR-5
in CD and controls as determined by in situ hybridization
(Figure 2)
. Cells demonstrating the most abundant GPR-5 mRNA expression
were predominantly located adjacent to blood vessels, a finding that is
in accordance to the in vitro observed chemoattractive
function of Lptn.
By applying simultaneous immunostaining of serial sections we found
most of these Lptn-positive cells to express the T-cell antigen CD3,
which comprised by subset analysis both CD4- and CD8-positive T cells
(Figures 3 and 4)
. Furthermore, we found the Lptn-positive cells to
express the integrin CD103, which is known to play a key role for
homing of inflammatory cells such as T cells into the gut. In addition,
numerous Lptn-positive cells demonstrated expression of the antigens
CD31 and CD45RA (Figure 4)
, which have been shown to be expressed by
naive T cells in CD.2
Because CD31/CD45RA and Lptn
co-expressing T cells were found predominantly in a perivascular
pattern, these cells represent newly immigrated inflammatory cells.
By additional investigations we found a substantial number of
Lptn-positive cells to express the proliferation-associated antigen
Ki67 in CD. The proliferating T cells were found to represent both the
CD4+ and CD8+ subset (Figure 4)
. Thus, active proliferation of
Lptn-positive cells is at least in part responsible for the observed
increase of Lptn-expressing cells in CD.
More recently, different abilities of Lptn have been demonstrated on
proliferation and stimulation of CD4+ and CD8+ T cells. Lptn could be
shown to inhibit CD4+ T-cell proliferation in vitro through
a decreased production of Th1 (IL-2, interferon-) but not Th2 (IL-4,
IL-13) cytokines. In contrast, Lptn exerted a potent stimulatory effect
on CD8+ T-cell proliferation and IL-2 secretion. CD4+ T-cell
proliferation was completely restored by addition of
IL-2.25
Because the inflammatory process in CD represents
a Th1-mediated immune response reflected by an increased expression of
interferon-, tumor necrosis factor-, and IL-2, increased
expression of IL-2 might compensate the inhibitory effect of Lptn on
CD4+ T-cell proliferation in CD as observed by us.
The observed expression of Lptn mRNA or protein by CD4- and
CD8-positive T cells is in accordance with previous reports
demonstrating that both activated T-cell subsets represent the most
important source of Lptn.14
In addition, Lptn production
has been detected in ß-type thymocytes8,9
and murine
intraepithelial -type T cells.17
The latter finding
seems to be of special importance, because Lptn was found to be the
most abundant chemokine produced by intraepithelial T cells,
peripheral blood T cells did not express Lptn at all in
mice.17
This latter finding is in accordance with our
finding of intraepithelial lymphocytes demonstrating expression of Lptn
in CD but not in controls.
The control of Lptn gene expression is not well understood. Although it
is clear that expression is essentially activation-dependent, there are
only a few studies that have examined this in detail. For murine T
cells it could be shown that Th1 clones and CD8+ T cells express Lptn
maximally on engagement of the T cell receptor and that co-stimulation
through CD28 inhibited expression.26
In humans this effect
seems to be restricted to the CD4+ subpopulation, whereas CD8+ T cells
are resistant. In our study Con A and PMA induced up-regulation of Lptn
mRNA expression in T cells isolated from peripheral blood, showing that
signals either provided by T-cell receptor engagement (Con A) or
directly by activation of protein kinase C (PMA) induce the expression
of Lptn as observed in CD (Figure 5)
. Unexpectedly, we found an
increased expression of Lptn in unstimulated T cells after 24 hours in
culture, which may be because of the separation method used in this
study by applying CD3-positive selection. CD3 itself has been shown to
activate T cells by T-cell receptor ligation.25
Furthermore it has been shown that T-cell stimulation by CD3 caused a
significant up-regulation of Lptn.7
Thus, the T cells
investigated by us were at least partly activated by CD3 stimulation
leading to an increased basal Lptn mRNA expression. To investigate
possible effects of Lptn by regulating its expression via an autocrine
feedback loop, additional experiments applying a neutralizing
polyclonal antibody at saturating concentrations during the culture
period were performed. In these specific blocking experiments no
significant modulation on the expression of Lptn mRNA in T cells could
be observed. Therefore, an autocrine regulation mechanism controlling
the expression of Lptn seems not to be likely. This conclusion is
further supported by our results obtained in tissue specimens in CD
demonstrating that almost the entire T-cell population expressed Lptn.
Beside glucocorticosteroids the immunosuppressive drugs FK 506 and
cyclosporine A are used for the therapy of CD. We found all these three
agents to prevent completely the PMA mediated up-regulation of Lptn
mRNA by T cells (Figure 5)
. This finding is in agreement with the
observation by Wang and colleagues,3
who demonstrated Lptn
expression by activated lymphoid cells in the acute phase of rejection
in a rat model of renal graft rejection, whereas no expression of Lptn
mRNA was observed in chronic rejection; expression of Lptn was found to
depend on the activation of lymphocytes and could be down-regulated by
cyclosporine A and FK506. A similar regulation mechanism on stimulation
and inhibition by immunosuppressive drugs as for Lptn could be shown in
peripheral T cells for its receptor GPR-5 (Figure 5)
. Thus, the
expression of Lptn and its receptor seems to be tightly linked to
activation of T cells, which points to a possible autocrine or
paracrine regulation mechanism of the recruitment of activated T cells
in autoimmune disease such as CD. This finding is further supported by
the observed decrease in the number of cells expressing Lptn and its
receptor GPR-5 in biopsy specimens of CD after sufficient therapy by
systemic steroids as shown in this study (Figure 2)
.
Other cells expressing Lptn mRNA were identified as mast cells or DCs
(Figure 3)
. Expression of Lptn mRNA could not be observed by other cell
types including granulocytes, natural killer cells, B-lymphocytes,
monocytes/macrophages, or fibroblasts as also shown by other
authors.27
The capacity of mast cells to produce Lptn provides a possible link
between mast cell activation and recruitment of T cells. Particularly,
because mast cells are predominantly found in the vicinity of blood
vessels, Lptn expressed by these cells may function in the recruitment
of T cells from the blood stream. Furthermore, mast cells have been
shown to express a wide range of chemokines, some of which possess
chemotactic activities for T cells such as RANTES, IL-8, IL-16, MCP-1,
macrophage inflammatory protein-1, and macrophage inflammatory
protein-1ß.28
Recently, the expression of Lptn by mast
cells as well as by the mast cell line HMC-1 and the basophil cell line
kU812 has been examined in vitro.18
It could be
demonstrated that expression of Lptn was dependent on aggregation of
the Fc
RI and that its expression was inhibited by cyclosporine A and
dexamethasone as described for T cells in this study.
Interestingly, we observed by careful examination expression of Lptn
mRNA by DCs in CD (Figure 3)
. To confirm this result further
experiments were performed with in vitro-generated
monocyte-derived DCs. No amplification products for Lptn mRNA could be
found in CD14+ monocytes and immature DCs on day 3. Lptn mRNA
expression appeared after 5 to 7 days of culturing, when DC maturation
has occurred (Figure 6)
. Thus, Lptn mRNA expression seems to be
restricted to the mature stages of DCs. This finding is in accordance
with our observations that functionally immature DCs such as the
Langerhans cells of the epidermis do not show any Lptn mRNA expression,
whereas interdigitating DCs within the lymph node paracortex, which
represent fully mature T cell accessory DCs, demonstrate expression of
Lptn mRNA (data not shown). Furthermore, we found expression of Lptn
within some germinal centers probably representing germinal center DCs,
an observation that has also been suggested previously.27
More recently, the obvious capability of Lptn to induce recruitment of T cells in immune response29 has successfully been used in a model of tumor immune therapy in mice. Mouse bone marrow-derived DCs were genetically modified with the Lptn gene by adenovirus vector-mediated transfection. Immunization with tumor RNA-pulsed Lptn-modified DCs improved the therapeutic efficacy of tumor rejection by enhanced chemotaxis of DCs to CD4+ and CD8+ T cells resulting in a higher efficiency of antigen peptide delivery for induction of T-cell-mediated tumor rejection.30,31 These results provide evidence that induction of expression and release of Lptn by mature DCs would not only represent a powerful tool for novel therapy approaches using DCs for tumor vaccination, but also point to the possible Lptn-mediated chemotactic properties of mature DCs in chronic inflammatory diseases such as CD.
The results indicate that increased expression of Lptn by activated T cells, mast cells, and DCs in CD seems to provide a powerful chemotactic signal for circulating T lymphocytes; persistent expression of Lptn at sites of inflammation seems to be involved in the continuous recruitment of peripheral T cells from the blood stream expressing the Lptn receptor. Down-regulation mRNA expression of Lptn and its receptor in T cells by dexamethasone, FK506, and cyclosporine A represents a possible mechanism preventing activation of T cells and being responsible for the beneficial effects of these immunomodulatory-acting drugs in CD. It seems worthwhile to look for Lptn expression in other autoimmune diseases that are characterized by increased recruitment of activated T cells such as sarcoidosis, rheumatoid arthritis, or atopic dermatitis.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Accepted for publication July 27, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chemokine, SCM-1. FEBS Lett 1995, 360:155-159[Medline]
T cells. Implications for the recruitment of inflammatory cells to damaged epithelia. J Immunol 1996, 157:985-992[Abstract]
) receptor I aggregation: modulation by TGF-b, IL-4, dexamethasone, and cyclosporine A. J Immunol 1997, 158:1353-1360[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  M. M. Zanone, E. Favaro, E. Ferioli, G. C. Huang, N. J. Klein, P. C. Perin, M. Peakman, P. G. Conaldi, and G. Camussi Human pancreatic islet endothelial cells express coxsackievirus and adenovirus receptor and are activated by coxsackie B virus infection FASEB J, October 1, 2007; 21(12): 3308 - 3317. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P Middel, D Raddatz, B Gunawan, F Haller, and H-J Radzun Increased number of mature dendritic cells in Crohn's disease: evidence for a chemokine mediated retention mechanism Gut, February 1, 2006; 55(2): 220 - 227. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. H. Schimmelpfennig, S. Schulz, C. Arber, J. Baker, I. Tarner, J. McBride, C. H. Contag, and R. S. Negrin Ex Vivo Expanded Dendritic Cells Home to T-Cell Zones of Lymphoid Organs and Survive in Vivo after Allogeneic Bone Marrow Transplantation Am. J. Pathol., November 1, 2005; 167(5): 1321 - 1331. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. F. Hedges, K. J. Lubick, and M. A. Jutila {gamma}{delta} T Cells Respond Directly to Pathogen-Associated Molecular Patterns J. Immunol., May 15, 2005; 174(10): 6045 - 6053. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Toth, M. Alexander, T. Daniel, I. H. Chaudry, W. J. Hubbard, and M. G. Schwacha The role of {gamma}{delta} T cells in the regulation of neutrophil-mediated tissue damage after thermal injury J. Leukoc. Biol., September 1, 2004; 76(3): 545 - 552. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. C. Peterson, E. S. Elgin, T. J. Nelson, F. Zhang, T. J. Hoeger, R. J. Linhardt, and B. F. Volkman Identification and Characterization of a Glycosaminoglycan Recognition Element of the C Chemokine Lymphotactin J. Biol. Chem., March 26, 2004; 279(13): 12598 - 12604. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. O. Kim, Y. Liu, B. Y. Zhou, and J. J. He Induction of C Chemokine XCL1 (Lymphotactin/Single C Motif-1{alpha}/Activation-Induced, T Cell-Derived and Chemokine-Related Cytokine) Expression by HIV-1 Tat Protein J. Immunol., February 1, 2004; 172(3): 1888 - 1895. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Sato, T. Higuchi, S. Yoshioka, K. Tatsumi, H. Fujiwara, and S. Fujii Trophoblasts acquire a chemokine receptor, CCR1, as they differentiate towards invasive phenotype Development, November 15, 2003; 130(22): 5519 - 5532. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Stievano, V. Tosello, N. Marcato, A. Rosato, A. Sebelin, L. Chieco-Bianchi, and A. Amadori CD8+{alpha}{beta}+ T Cells That Lack Surface CD5 Antigen Expression Are a Major Lymphotactin (XCL1) Source in Peripheral Blood Lymphocytes J. Immunol., November 1, 2003; 171(9): 4528 - 4538. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Palena, J. Schlom, and K.-Y. Tsang Differential Gene Expression Profiles in a Human T-cell Line Stimulated with a Tumor-associated Self-peptide versus an Enhancer Agonist Peptide Clin. Cancer Res., May 1, 2003; 9(5): 1616 - 1627. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. S. Kuloglu, D. R. McCaslin, J. L. Markley, and B. F. Volkman Structural Rearrangement of Human Lymphotactin, a C Chemokine, under Physiological Solution Conditions J. Biol. Chem., May 10, 2002; 277(20): 17863 - 17870. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||
