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Department of Dermatology, University of Mainz, Mainz, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In former work, we performed in vitro studies on the endocytotic activation of murine LC by haptens (3, 4, 15, 16, 17). Because usage of human LC for an in vitro model is limited by the inconsistent availability of human skin, we established an in vitro system using human blood-derived DC to study the endocytotic activation of human cells by contact sensitizers (18).
We were interested in the underlying signal-transduction mechanism of this hapten-mediated activation of DC. As a first approach, the induction of tyrosine phosphorylation by contact sensitizers was studied. We concentrated on the function of protein tyrosine kinases (PTKs) because of their pivotal role for the transduction of signals received by cell membrane receptors in immune cells. The role of a large number of PTKs of the src, janus, syk, and csk families has been studied in immune cells, especially in lymphocytes (19, 20, 21, 22). Compared with T and B cells, less is known about the PTK-mediated signal transduction in monocytes (23, 24, 25, 26), and only single reports addressed elements or mechanisms of signal transduction in LC and DC (27, 28, 29, 30). Although a possible role for the protein kinase C-ß in contact hypersensitivity has been suggested (31, 32), data on any signal-transduction mechanism involved in the hapten-mediated activation of LC or DC are lacking.
In a preliminary communication, we described the selective induction of phosphotyrosine (p-tyr) in human blood-derived DC by haptens (33). In this work, the capacity of haptens to induce tyrosine phosphorylation in different human MHC class II-positive cell populations will be further characterized, and biochemical data on proteins involved in this early event of cellular activation by contact sensitizers are presented.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-CD3, CD14, CD16, and CD19, minimally cross-reacting
phycoerythrin- and biotin-conjugated mouse IgG- and rat IgG-specific
second step reagents as well as phycoerythrin-coupled CD14- and
CD19-specific Abs were obtained from Dianova (Hamburg, Germany).
Streptavidin-Quantum Red was purchased from Sigma Chemical Co. (St.
Louis, MO); anti-HLA-DR (clone Ye2/36 HLK, rat IgG 2a) was supplied
by Serotec (Oxford, U.K.). The p-tyr-specific Ab PY20 FITC (mouse
IgG2b) was supplied by Leinco Technologies (St. Louis, MO). mAb 4G10
(anti-p-tyr, mouse IgG2b) and anti-p85
subunit of human PI3
kinase (mouse IgG1) were obtained from Upstate Biotechnology (Lake
Placid, NY). Mouse IgG2b FITC (Southern Biotechnology, Birmingham, AL)
and mouse IgG2b (PharMingen, San Diego, CA) were used as isotype
control. Polyclonal rabbit IgG Abs specific for lyn and
MAP-kinase p38 were supplied by Santa Cruz Biotechnology (Santa Cruz,
CA). The secondary reagents for Western blotting were purchased from
Dianova.
Cells
Preparation of human PBMC was performed using standard procedures, as outlined recently (18). Different cell types in these mixed preparations, such as immature fresh DC (f-DC), monocytes, B cells, and HLA-DRnegative cells, were studied by flow cytometry without further enrichment using phenotypic markers, as outlined below. LC were prepared from human skin by overnight incubation at 4°C in thermolysin (protease X, 500 µg/ml in PBS, 1 mM CaCl2; Sigma Chemical Co.). After removing the dermis, epidermal sheets were treated for 30 min at 37°C with PBS/0.25% trypsin (type III; Sigma Chemical Co.), followed by addition of 100 U/ml deoxyribonuclease I (type IV; Sigma Chemical Co.) and 0.02% CaCl2 for 30 min. Single cell suspensions were harvested by filtering through a stainless steel mesh. Short-term culture for 24 h was performed in Iscove’s modified Dulbecco’s medium supplemented with 10% FCS.
Cultured DC (c-DC) were generated from mononuclear progenitors using a published modification (34) of the protocol described by Romani et al. (35).
Briefly, heparinized whole blood was separated by Ficoll gradient. T cells and B cells were removed from the PBMC fraction using immunomagnetic beads coated with anti-CD2 and anti-CD19 mAb (Dynal, Oslo, Norway). The remaining cells were cultured in six-well plates (Costar, Cambridge, MA) using the culture medium ex vivo (BioWhittaker, Gaithersburg, MD) supplemented with 1000 U/ml human IL-4 (Pharma Biotechnologie Hannover, Hannover, Germany), 800 U/ml human granulocyte-macrophage CSF (Leukomax, Sandoz, Nürnberg, Germany), as well as 1% autologous plasma. Cells were fed with fresh medium every 2 days.
On day 5, nonadherent cells were harvested, because at that time point optimal response to stimulation with haptens with respect to induction of IL-1ß was found. Nearly 70 to 75% of these cells were found to be HLA-DRpositive, but CD2/14/16/19negative; no other MHC class IIpositive populations were detectable, and viability routinely exceeded 90%. Further characterization of these early DC revealed an immature phenotype (CD80+/-CD86low, CD83negative). As outlined previously (34), further culture and addition of 10 ng/ml TNF (R&D Systems, Wiesbaden, Germany) and 10 ng/ml IL-1 (R&D Systems) at day 7 induced their differentiation to mature DC expressing high levels of MHC class II, CD80, CD86, and CD83 as early as 9 days after onset of culture.
Monocytes were enriched by short-term incubation of PBMC in culture medium (Iscove’s modified Dulbecco’s medium, 3% FCS) on cell culture dishes for 45 min at 37°C and removal of nonadherent cells by washing carefully with warm PBS. The adherent cells were cultured overnight in autologous serum before their detachment by vigorous rinsing. Contaminating B cells were depleted using CD19-coupled immunomagnetic beads (Dynal) following a standard protocol (34). At the end of this procedure, approximately 10% of the PBMC could be harvested with a content of 80 to 90% CD14+ monocytes, less than 3% CD19+ B cells, and a viability routinely exceeding 95%.
Stimulation with chemicals
Cells (106/ml) were stimulated with strong haptens (2, 4-dinitrofluorobenzene (DNFB), 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone (MCI/MI), thimerosal, and formaldehyde), weak haptens (methyl-4-hydroxy-benzoate (methyl paraben), 2-phenoxyethanol), irritants (SDS, benzalkonium chloride), and activators of distinct signal-transduction mechanisms (PMA, calcium ionophore A23187, and sodium orthovanadate) in subtoxic concentrations for 15 min at 37°C in Iscove’s modified Dulbecco’s medium. Methyl paraben and phenoxyethanol were obtained from Fluka (Neu-Ulm, Germany); MCI/MI was obtained from Hermal (Reinbeck, Germany). DNFB, sodium orthovanadate, benzalkonium chloride, thimerosal, formaldehyde, PMA, and calcium ionophore A23187 were supplied by Sigma Chemical Co. Stock solutions of DNFB, and the PTK inhibitors herbimycin A, tyrphostin B46 and B56 (Calbiochem, San Diego, CA), and genistein (Upstate Biotechnology) were prepared in DMSO; the other chemicals were dissolved immediately before use in culture medium.
Quantification of p-tyr by flow cytometry
For selective analysis of f-DC, PBMC were stained with mAb specific for CD3, CD14, CD16, CD19, and R-phycoerythrin-conjugated donkey anti-mouse IgG Abs before stimulation. Reactivity of CD14+ or CD19+ cells was studied by labeling of PBMC with the corresponding phycoerythrin-coupled primary Abs after stimulation with the hapten. Afterward, cells were treated with HLA-DR-specific Ab, biotin-SP-conjugated goat anti-rat IgG, and streptavidin-Quantum Red. Finally, cell suspensions were incubated for 20 min with the p-tyr-specific FITC-labeled mAb PY20 in PBS supplemented with 2% FCS and 0.25% saponin. Selective analysis of c-DC was achieved by subjecting the cultured populations to the staining procedure, as outlined for f-DC. For analysis of MHC class IInegative cells, PBMC were stimulation and stained for HLA-DR and p-tyr. Incubations were performed on ice; between each step, probes were washed twice with cold washing buffer (PBS, 2% FCS, and 0.25% saponin). Any cross-reactivity between the different components was excluded using isotype control Abs.
Flow-cytometric determination of p-tyr in f-DC and c-DC was performed according to principles published recently (18) using software gates for collection of the HLA-DRpositive, but CD3/14/16/19negative DC. Selective quantification of p-tyr-specific mAb bound to B cells, monocytes, and MHC class IInegative populations was performed by gating based on their marker expression. Measurement of p-tyr in LC was achieved after staining for MHC class II molecules and p-tyr using software gates for expression of HLA-DR.
Analysis was performed using a FACScan (Becton Dickinson, Heidelberg, Germany) equipped with Lysis II software and a standardized instrument setup.
Phosphorylation of cellular proteins in vitro
PBMC prestained for phenotypic markers and stimulated with MCI/MI as outlined above were subjected to permeabilization with saponin and further cultured for 8 min in the presence or absence of 5 µM ATP dissolved in tyrosine protein kinase buffer (20 mM 3-(N-morpholino) propanesulfonic acid, 5 mM MgCl2, pH 7) at 37°C, according to a protocol described for an in vitro PTK assay (36). Flow-cytometric analysis for p-tyr in f-DC was performed as described. The effects of PTK inhibitors were studied by addition of 100 µM tyrphostin B46 or 100 µg/ml genistein. To exclude an inhibition of protein tyrosine phosphatases (PTPs), unstimulated cells were permeabilized and subjected to this in vitro phosphorylation procedure in the presence or absence of 0.4 µg/ml MCI/MI and 8 mM sodium orthovanadate, followed by analysis for p-tyr.
Biochemical analysis
Each 107 cell was stimulated in an appropriate volume of culture medium with MCI/MI (0.4 µg/ml) or DNFB (1 µg/ml). Cells were lysed in 50 µl lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 2 mM PMSF, 10 µg/ml aprotinin, and 1 mM sodium orthovanadate) by vigorous resuspension and incubation on ice for 20 min, followed by centrifugation at 14,000 x g. The supernatant was stored at -70°C until protein determination using the BCA protein assay (Pierce, Rockford, IL) and electrophoresis on a 10% polyacrylamide gel with 0.5% SDS using standard procedures (45 mA, maximum 500 V). A biotin-labeled m.w. marker (Boehringer Mannheim Corp., Mannheim, Germany) was run in parallel for quantification of the m.w. after Western transfer on a polyvinylidene fluoride membrane (Immobilon-P; Millipore, Bedford, MA). Transfer was performed at constant current (1.5 mA/cm2 membrane) and maximum 25 V for 30 to 50 min using a blotting buffer containing methanol (96 mM glycine, 12 mM Tris, and 10% methanol) and a semidry transfer unit (SemiDry Transfer Cell; Bio-Rad, Hercules, CA).
Phosphotyrosine, lyn, and the MAP-kinase p38 and p85 of human PI3 kinase were detected after blocking the membrane in blocking buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, 0.25% gelatin, and 0.1% Triton X-100, pH 7.4) by incubation with 1 µg/ml of the specific Abs for 2 h. Following washing the membrane in blocking buffer, incubation with secondary Abs (peroxidase-coupled goat anti-rabbit IgG or goat anti-mouse IgG; 1:5000) was performed for 30 min. After thoroughly washing, enhanced chemiluminescence reagent (Amersham, Buckinghamshire, U.K.) was added for 60 s before exposure to an x-ray film (Hyperfilm-MP; Amersham) for 60 min. Sequential immunodetection of different epitopes was performed after prior incubation of the membrane in stripping buffer (62.5 mM Tris/HCl, 2% SDS, and 100 mM 2-ME, pH 6.7) for 15 min at 50°C and washing with blocking buffer.
RT-PCR analysis
Semiquantitative RT-PCR analysis for IL-1ß-specific mRNA in c-DC was performed as described previously (1, 37). Briefly, c-DC (106/ml) were treated with 1 µg/ml DNFB or 0.1% DMSO as control for 15 min at 37°C. Alternatively, DC were preincubated with genistein (100 µg/ml) for 30 min before addition of DNFB. Total RNA was extracted by RNAzol B (Wak Chemie, Bad Homburg, Germany) and quantified by spectrophotometric analysis. For each probe, 100 ng of RNA was subjected to an RT-PCR following standard protocols with conditions of 60 s at 94°C, 90 s at 55°C, and 90 s at 72°C. Primer sequences were as follows: IL-1ß 5' primer, GAC ACA TGG GAT AAC GAG GC; IL-1ß 3' primer, ACG CAG GAC AGG TAC AGA TT; ß-actin 5' primer, GAG CGG GAA ATC GTG CGT GAC ATT; and ß-actin 3' primer, GAA GGT AGT TTC GTG GAT GCC. The sequence of the internal oligonucleotide probe for IL-1ß was CAG GGA CAG GAT ATG GAG CAA GTG GTG, and for ß-actin, CTG GAC TTC GAG CAA GAG ATG GCC ACG GC. PCR products were separated by agarose gel electrophoresis at 60 V for 2 h in 1.5% agarose in tris/acetic acid/EDTA buffer containing 0.4 µg/ml of ethidium bromide. To validate the predicted size of the PCR amplificates, a pBR322/AluI digest was used as a DNA m.w. marker. To confirm the origin of the amplified PCR products, a nonisotopic Southern blot hybridization with internal digoxigenin (DIG)-tailed oligonucleotide probes was performed. By a neutral Southern transfer technique, PCR products were blotted onto a positively charged nylon membrane (Boehringer Mannheim Corp.). The DNA was immobilized by baking the membrane (120°C, 30 min). The oligonucleotide probes were labeled using the DIG oligonucleotide tailing kit (Boehringer Mannheim Corp.). After Southern blot hybridization, the DIG-labeled hybridization products were detected by using an anti-DIG alkaline phosphatase conjugate and a luminescence substrate. Visualization was achieved by exposure of the membrane to an x-ray film.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stimulation of unseparated fresh epidermal cells with the strong
contact sensitizer MCI/MI before selective analysis for p-tyr in
HLA-DRpositive LC failed to modulate the amount of
p-tyr (Fig. 1
). In contrast to this
unresponsiveness of fresh LC (f-LC), LC cultured for 24 h showed a
significant increase in tyrosine phosphorylation following stimulation
with this hapten (Fig. 1), whereas in keratinocytes the amount of p-tyr
was not affected (data not shown). Because of the hyporesponsiveness of
f-LC and the inconsistent availability of human skin, the effect of
contact sensitizers on different mononuclear cell populations as well
as in vitro generated c-DC was explored. Quantification of p-tyr in
HLA-DRnegative cells, B cells, monocytes, f-DC, as well as
in vitro generated c-DC after stimulation with MCI/MI revealed no
reliable response for HLA-DRnegative cells, a significant
reactivity of B cells and c-DC, and best results for f-DC and monocytes
with respect to the relative increase in p-tyr (Fig. 2). Because of their relationship to LC,
f-DC were used as a model for the response of the DC family to
stimulation with haptens in all further flow-cytometric
experiments.
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A distinct increase in formation of p-tyr became apparent as early
as 4 min during stimulation with MCI/MI. Maximal content of p-tyr was
detectable after 15 min, followed by a slow decrease upon further
culture in the presence of the hapten (Fig. 3). In addition to the p-tyr-specific mAb
PY20, quantification of p-tyr in DC was also possible using the
specific mAb 4G10 (data not shown). Permeabilization of cell membranes
was a prerequisite for binding of these Abs to their targets (Fig. 4). Increased tyrosine phosphorylation
was detected only following stimulation of viable cells under
physiologic cell culture conditions (Fig. 4). The capacity of the PTK
inhibitors tyrphostin B56 (38) and herbimycin A (39) to block augmented
tyrosine phosphorylation pointed to the central role of these kinases
for this mechanism (Fig. 4).
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). The
concentrations used in Figure 5 represent strictly subtoxic amounts of
each compound and were determined in dose-response experiments
including propidium iodide for monitoring of toxic effects. The
nonreactive compounds were also ineffective when used at higher toxic
concentrations (data not shown).
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Further phosphorylation of tyrosine residues was demonstrated in
vitro for f-DC after prior stimulation with MCI/MI and subsequent
permeabilization with saponin before incubation at 37°C in the
presence of ATP. The potent PTK inhibitors genistein (40) and
tyrphostin B46 (38) were able to block this process (Fig. 6). To study any inhibition of PTPs by
haptens, cells were first permeabilized, followed by incubation with
ATP in the presence of MCI/MI. Whereas the PTP inhibitor sodium
orthovanadate induced an excessive formation of p-tyr by inhibition of
its disintegration, MCI/MI was without effect. These data point to an
activation of PTKs or enhanced binding to their substrates during
stimulation of viable cells with haptens rather than to an inhibition
of PTPs by simple chemical interaction.
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Because sufficient numbers of f-DC or LC were not available for
systematic biochemical analysis, studies were performed with
monocyte-enriched populations to obtain data on a defined cell
population, which also responds very well to stimulation with haptens,
as described above. In comparison with unstimulated cells, a distinct
pattern of enhanced phosphorylated proteins of approximately 47, 53,
65, 75, and 81 kDa was observed following stimulation with MCI/MI (Fig. 7, arrows). Comparable pattern of
hyperphosphorylated proteins was obtained by stimulation with DNFB or
thimerosal (not shown). The presence of the PTK inhibitor tyrphostin
B56 almost completely prevented the increase in phosphorylation of
these proteins (unpublished results). Sequential staining for proteins
involved in defined signal-transduction pathways such as the MAP-kinase
p38, the p53/p56 isoforms of the src kinase lyn,
as well as p85 of PI3 kinase (positions indicated in Fig. 7) suggested
that lyn might be part of a group of phosphorylated proteins
between 50 and 60 kDa. The hyperphosphorylated protein at 81 kDa could
be clearly distinguished from p85. A protein band below 40 kDa was
identified in the same position as p38, but was not found to be more
phosphorylated upon stimulation. Therefore, neither p38 nor p85 were
substrates for the PTKs active during stimulation with a strong contact
sensitizer. The expression of p85, p38, and p53/p56 lyn, as
determined by sequential immunodetection, was found to be identical in
all lanes (data not shown), confirming that equal amounts of proteins
were analyzed.
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for f-DC.
Biochemical analysis of monocyte-enriched populations after
cross-linking with HLA-DR-specific Ab and secondary Abs demonstrated an
increase in phosphorylation of two protein bands, with a m.w. of 65 and
75 kDa (asterisk in Fig. 7) in comparison with the control incubated
with the secondary Ab alone. With respect to their m.w., these proteins
closely resembled structures being more phosphorylated after
stimulation with haptens.
Biochemical analysis on c-DC revealed changes in the phosphorylation of
three protein bands of about 41, 65, and 75 kDa upon stimulation with
the strong sensitizer DNFB (arrows in Fig. 8). In correlation with the
flow-cytometric analysis, the increase of p-tyr detected after
stimulation was less pronounced in comparison with monocyte-enriched
populations. Although the reactivity to stimulation with DNFB and
MCI/MI was comparable, the result of stimulation with DNFB is shown to
correlate these data to the experiments on the induction of
IL-1ß-specific mRNA by stimulation with this contact sensitizer.
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To demonstrate the functional relevance of tyrosine
phosphorylation during activation of DC, the expression of
IL-1ß-specific m-RNA in c-DC after stimulation with DNFB was
determined in parallel to flow-cytometric quantification of p-tyr under
stimulation with this agent. The PTK inhibitor genistein was able to
prevent both the production of IL-1ß-specific mRNA as well as
augmented tyrosine phosphorylation under this condition (Fig. 9). In addition, Western blot analysis
demonstrated the capacity of genistein to block the increased
phosphorylation of the 65- and 75-kDa protein band seen as result of
stimulation with DNFB (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As reported earlier (33), only strong haptens induced tyrosine phosphorylation, whereas weak sensitizers and irritants failed to modulate the p-tyr content of f-DC under these conditions. This corresponds to our data concerning the modulation of endocytotic mechanisms by haptens in murine LC (17) and human f-DC (18). Activators of other important elements of intracellular signal-transduction pathways such as the PKC activator PMA or a calcium ionophore were not able to induce an increased formation of p-tyr, as detected by our flow-cytometric analysis, suggesting this early formation of p-tyr to be independent from activation of PKC or Ca2+ mobilization. Nevertheless, these structures may be involved in downstream events of the hapten-mediated activation of cells. Cross-linking of HLA-DR molecules resembled the stimulatory effect of strong haptens. This is in agreement with our finding that contact sensitizers modulate the receptor-mediated endocytosis of MHC class II molecules (17, 18), and points to a linkage between both mechanisms awaiting further characterization.
Inhibition of PTKs by tyrphostin B56 and herbimycin A almost completely blocked the hapten-mediated increase in p-tyr, suggesting an active role of PTKs sensitive to these inhibitors. Further hints for the importance of PTK activation under these conditions were obtained from experiments with hapten-stimulated f-DC subjected to an in vitro phosphorylation reaction. The enhanced tyrosine phosphorylation in hapten-stimulated cells and the capacity of potent PTK inhibitors such as genistein and tyrphostin B46 to block this process underlined the critical role of PTKs for the increase in p-tyr. Inhibition of PTPs by our model hapten MCI/MI could be excluded, making this alternative explanation for increase in p-tyr very unlikely.
It can be supposed that as result of the permeabilization of cell membranes for flow-cytometric quantification of intracellular p-tyr, most cytoplasmatic proteins are lost. This may explain the relative low level of p-tyr in the flow-cytometric analysis in contrast to the distinct protein bands found by Western blot analysis. In support for this assumption, biochemical analysis of permeabilized cells revealed a remarkable loss of phosphorylated proteins, although the increase in phosphorylation of distinct protein bands in hapten-stimulated cells was still apparent (D. Becker, P. Brand, unpublished data).
Albeit this loss of phosphorylated proteins has to be kept in mind, the detection of p-tyr by flow-cytometric analysis was advantageous for selective analysis of different cell types in mixed cell populations such as PBMC and epidermal cells. Direct analysis without prior enrichment was important, because cell collection based on the expression of membrane markers such as MHC molecules might induce tyrosine phosphorylation by cross-linking of receptors, as demonstrated in this work for f-DC.
Any influence of other cell populations (e.g., keratinocytes or T cells) on the reactivity of the cell type studied by selective analysis could not be excluded with certainty. On the other hand, data obtained for enriched populations of c-DC or monocytes suggest the capacity of APC to respond to hapten stimulation without signals delivered by contaminating populations.
Western blot analysis of enriched monocytes revealed augmented phosphorylation of distinct protein bands. Sequential immunodetection for the p53/p56 isoforms of the src kinase lyn (41), as well as the MAP-kinase p38 (42, 43) and p85 of PI3 kinase (44) revealed that a double band between 50 and 60 kDa might include p53/p56 lyn, whereas no evidence for hyperphosphorylation of p38 and p85 was found. The identity of the proteins being enhanced phosphorylated under stimulation with a hapten, as described in this work, has to be revealed. Moreover, the involvement of lyn as well as other PTKs needs to be elucidated by precipitation and in vitro kinase assays.
Concerning the functional relevance of these data for the situation in the epidermis during sensitization to contact sensitizers, it would be ideal to obtain a biochemical analysis on LC and to perform experiments demonstrating tyrosine phosphorylation as consequence of stimulation with haptens in vivo. Whereas the latter approach is presently under investigation in our lab, biochemical data on LC are hard to obtain for reasons discussed above. Because blood-derived DC are related to LC, we performed biochemical studies on immature c-DC to obtain first insights into the role of tyrosine phosphorylation for an established mechanism of activation of DC by contact sensitizers. Induction of IL-1ß following stimulation with a strong contact sensitizer has been shown for murine LC in vivo (1, 2) and previously in a preliminary report for c-DC in vitro (37). In our experiments, this molecular event was accompanied by tyrosine phosphorylation of distinct proteins and blocked in the presence of the potent PTK inhibitor genistein. We rate this finding as first hint for the importance of tyrosine phosphorylation for the activation of DC by haptens during the sensitization phase of contact sensitivity.
Irrespective of these considerations on the relation between DC and LC, our data demonstrate the capacity of contact sensitizers to induce a high degree of tyrosine phosphorylation in MHC class II molecule-positive cells. Further work should address the identity of PTKs involved, as well as their substrates and links to further downstream elements of signal-transduction pathways. Special attention has to be paid to the role of signal-transduction mechanisms induced by MHC class II molecules (26, 45, 46, 47) because in our experiments, constitutive expression of these molecules was a prerequisite for the response to haptens. Furthermore, cross-linking of class II molecules partly resembled the quantity and quality of tyrosine phosphorylation noticed after stimulation with contact sensitizers.
Another important question is how haptens induce tyrosine phosphorylation. For multiple receptors, conformational changes and dimerization, as well as stabilization of multichain complexes after binding of their ligands have been identified as the initial events leading to activation of associated tyrosine kinases or catalytic domains of the receptor itself (48, 49, 50).
Taking into account the chemical properties of haptens, monovalent binding to critical domains of membrane proteins might be a possible mechanism inducing conformational changes of the protein and subsequent activation or binding of PTKs.
Alternatively, cellular stress mechanisms leading to direct activation or translocation of PTKs might also be involved. On the other hand, this process seems not to be the result of nonspecific cellular damage, because neither subtoxic nor toxic concentrations of irritants were able to induce an increase in p-tyr in our experiments.
Unlike protein Ags, haptens are able to activate APC directly, and thereby support their presentation to T cells. Our data contribute to the understanding of the molecular details of this activation and underline the importance of further studies addressing the signal-transduction mechanisms involved.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Detlef Becker, Department of Dermatology, University of Mainz, Langenbeckstr. 1, D-55101 Mainz, Germany. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; c-DC, cultured dendritic cells; DIG, digoxigenin; DNFB, 2,4-dinitrofluorobenzene; f-DC, fresh dendritic cells; f-LC, fresh Langerhans cells; LC, Langerhans cell; MAP, mitogen-activated protein; MCI/MI, 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone; phospho inositide 3-kinase; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; p-tyr, phosphotyrosine; RT-PCR, reverse-transcriptase polymerase chain reaction.
Received for publication June 25, 1997. Accepted for publication October 1, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced transcription of the FcRI gene in human monocytes by preventing the tyrosine phosphorylation of the p91 (Stat1) transcription factor. J. Immunol. 154:318.[Abstract]
RII-mediated endocytosis in the monocytic cell line THP-1. J. Immunol. 152:30.[Abstract]
RI. J. Immunol. 155:5184.[Abstract]
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