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Free Hapten Molecules are Dispersed by Way of the Bloodstream During Contact Sensitization to Fluorescein Isothiocyanate

1999; Elsevier BV; Volume: 113; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.1999.00770.x

1523-1747

Jolanthe Pior, Thomas Vogl, Clemens Sorg, E. Macher,

Contact Dermatitis and Allergies

The fate of the contact sensitizer fluorescein isothiocyanate was traced by means of fluorescence spectrophotometry and flow cytometry. The hapten applied to one ear rapidly entered the circulation by way of local lymphatics and blood vessels. It was dispersed for several hours essentially as free hapten, released from a reservoir left behind at the site. Hapten molecules coupled to plasma proteins while circulating and reacted with white blood cells. Total cells of regional lymph nodes, spleen, and distant lymph nodes became fluorescent in successive order. Fluorescence of CD11c-positive dendritic cells exceeded significantly that of lymphoid cells. Total spleen cells and total nonregional lymph node cells were shown in vitro to drive committed lymph node cells to proliferation. The mechanism disclosed is proposed to counterbalance the action of epidermal Langerhans cells for regulation of contact hypersensitivity. The fate of the contact sensitizer fluorescein isothiocyanate was traced by means of fluorescence spectrophotometry and flow cytometry. The hapten applied to one ear rapidly entered the circulation by way of local lymphatics and blood vessels. It was dispersed for several hours essentially as free hapten, released from a reservoir left behind at the site. Hapten molecules coupled to plasma proteins while circulating and reacted with white blood cells. Total cells of regional lymph nodes, spleen, and distant lymph nodes became fluorescent in successive order. Fluorescence of CD11c-positive dendritic cells exceeded significantly that of lymphoid cells. Total spleen cells and total nonregional lymph node cells were shown in vitro to drive committed lymph node cells to proliferation. The mechanism disclosed is proposed to counterbalance the action of epidermal Langerhans cells for regulation of contact hypersensitivity. dendritic cells fluorescence-activated cell sorter Contact allergens are believed to act chiefly within the skin at the site of application. It is generally agreed that epidermal Langerhans cells pick up hapten molecules there and subsequently migrate to the regional lymph nodes for antigen presentation (Kripke et al., 1990Kripke M.L. Munn C.G. Jeevan A. Tang J.M. Bucana C. Evidence that cutaneous antigen-presenting cells migrate to regional lymph nodes during contact sensitization.J Immunol. 1990; 145: 2833-2838PubMed Google Scholar). The fate of the sensitizer on the whole, however, is but vaguely known. Experimental contact sensitization is performed, for the most part, by painting lipid-soluble sensitizers onto the skin. Small hapten molecules penetrate the skin barrier and bind to endogenous proteins to form an antigen which can then elicit an immunologic response. But antigen formation need not only occur in the skin and the draining lymph nodes. Both chemical and permeation data indicate that lipid soluble molecules penetrate into and diffuse through lipid regions in the tissue until they reach the capillary plexus and are transferred to the circulating blood (Scheuplein, 1978aScheuplein R. The skin as a barrier.in: Jarrett A. The Physiology and Pathophysiology of the Skin. Vol. 5. Academic Press, London1978: 1669-1692Google Scholar). It is therefore reasonable to question how fast penetration proceeds of a topically applied hapten, how far the chemical travels, and how long it takes for those hapten molecules to combine. Connecting these quantities could help to better understand the scope for action of lipophilic haptens in general. We addressed these questions by using the lipophilic compound fluorescein isothiocyanate (FITC) shown to be a strong contact sensitizer (Thomas et al., 1980Thomas W.R. Edwards A.J. Watkins M.C. Asherson G.L. Distribution of immunogenic cells after painting the contact sensitizers fluorescein isothiocyanate and oxazolone. Different sensitizers form immunogenic complexes with different cell populations.Immunology. 1980; 39: 21-27PubMed Google Scholar). Its attached fluorochrome permits detection in liquids by means of fluorescence spectrophotometry and in cells by flow cytometry. Following skin painting the fluorescent chemical escaped from the site, rapidly at first, and then more slowly, was dispersed essentially as free hapten and combined both with soluble proteins and cells. These results suggest that lipophilic haptens such as FITC not only act locally but, from the beginning, do so systemically. Female BALB/C and C57Bl/6 mice, 8–12 wk of age, were purchased from Harlan-Winkelmann GmbH (Borchen, Germany) and maintained in a light- and temperature-controlled environment with free access to food and water. Experimental groups of animals were painted once on the right ear with 500 μg of FITC dissolved in 15 μl of acetone/dibutyl phthalate 50:50 (vol/vol) with 5% dimethyl sulfoxide. Challenge on the left ear with 40 μg FITC 5 or 6 d later proved sufficient sensitization. FITC, dibutyl phthalate, dimethylsulfoxide, and xylazine were purchased from Sigma (Deisenhofen, Germany). Ketamine (Ketanest) was obtained from Parke-Davis (Berlin, Germany), heparin Na from B. Braun (Melsungen, Germany), and [methyl-3H]thymidine (spec. act. 20 Ci per mmol) from Hartmann Analytic GmbH (Braunschweig, Germany). For collecting venous blood the right axillary vein was dissected out under Ketanest anesthesia and slit by scissors. The blood flowing out was taken up into a heparin-wetted Pasteur pipette. Blood flow was sustained for 1–2 min; volumes of 0.7–1.0 ml of blood could be collected. Samples were added to 100 μl of heparin-Na each to avoid clotting, and pooled if appropriate. Blood plasma was separated by centrifugation and red and white blood cells were isolated by Ficoll density gradient centrifugation. Fluorescence measurements were carried out in a SPEX FluoroMax-II fluorescence spectrophotometer (ISA Instruments, München, Germany). This instrument was equipped with a water-jacketed cell holder and a NESLAB RTE 111 circulation bath for temperature control. The actual temperature inside the cell was measured using a Pt 100 resistance thermometer. All experiments were done at 20°C. Wavelength scans were performed with samples at appropriate dilutions and data were collected at varying times. Uptake of FITC was followed by measuring the change in fluorescence emission at 517 nm. The excitation wavelength was 488 nm (bandpass 2 nm). All spectra were corrected for blood plasma, which was prepared as described above without application of FITC but with solvent. The pathlength of the cuvette was 0.4 × 1 cm. Separation was achieved by Sephadex G-25 column chromatography (Pharmacia, Freiburg, Germany). Columns were equilibrated to phosphate-buffered saline, the flow rate was kept at 0.6 ml per min. Plasma proteins both with and without bound FITC were collected in a first fraction comprising 4.8 ml. The second fraction comprising 36 ml contained the low molecular unbound FITC. Accurate separation was secured by recording simultaneously the absorption of proteins at 280 nm and of FITC at 488 nm. Fluorescence of both fractions was measured immediately after collecting by fluorescence spectrophotometry. Animals were killed at varying times following painting with FITC, and lymph nodes (retroauricular, axillary, inguinal, mesenteric) and spleens were taken. Single cell suspensions were prepared under aseptic conditions by mechanical disaggregation and passed through a sterile nylon gauze. Cells were washed three times and resuspended in RPMI-1640 complete. Viability was noted to be > 95% as determined by Trypan blue exclusion. Before depleting CD11c+ cells, aliquots of total lymph node cells or spleen cells were set aside for final fluorescence-activated cell sorter (FACS) analysis. The remaining cells were suspended in ice-cold phosphate-buffered saline containing 2 mM ethylenediamine tetraacetic acid and 0.5% bovine serum albumin with 100 ml of monoclonal hamster–anti-mouse CD11c microbeads, and were incubated at 6°C for 20 min. Mini-MACS separation columns type MS+ (Miltenyi-Biotec, Bergisch-Gladbach, Germany) were used according to the manufacturer's instructions. "Negatively depleted" lymph node cells or spleen cells (total cells except CD11c+ cells), were eluted first and subsequently, after removal of the column from the magnet, positively depleted CD11c+ cells were eluted. Both fractions and the original suspension were washed twice in phosphate-buffered saline for FACS analysis. The intensity of FITC fluorescence of red and white blood cells, total lymph node cells, total spleen cells, positively depleted CD11c+ cells, and negatively depleted cells was determined using a FACS Scan Flow Cytometer (Becton Dickinson, Heidelberg, Germany). Data acquisition and analysis was obtained by using Lysis-II Software (Becton Dickinson). Dead cells were gated out after addition of propidium iodide (100 mM) to the cell suspension. Cells were considered to exhibit FITC fluorescence when the median of fluorescence intensity was above that of the control population. Spleens of untreated mice were disaggregated into single cell suspensions. Erythrocytes were lysed and the remaining cells washed three times in RPMI-1640 complete. Cells (1 × 108) were resuspended in 100 ml RPMI-1640 supplemented with 5% fetal bovine serum, added to 100 ml phosphate-buffered saline containing 1.25 mg FITC, and incubated for 20 min at 37°C, pH 7.4. To remove free hapten, cells were washed at least three times in ice-cold RPMI-1640 with 10% fetal bovine serum. Spleen cells or lymph node cells (1 × 105 per well) taken 3, 12, and 24 h after epicutaneous FITC application and subsequently irradiated with 30 Gy (60Co source) were cocultured with lymph node cells (3 × 105 per well) from FITC-sensitized donors. In vitro haptenized and irradiated normal spleen cells served as a positive control. On day 5 of culture in a humidified atmosphere of 5% CO2 at 37°C cells were pulsed with 20 μl per well of 3H thymidine (1 μCi per well). Sixteen hours later cells were harvested by blotting onto filter discs and dried at 80°C for 2 h. The acid-insoluble material was counted using a liquid scintillation counter. Differences in log cpm significantly greater than replication variability were calculated using analysis of variance or Student's t test. Statistical significance of differences in the medians of experimental groups was evaluated using the Student's t test. Differences of p < 0.05 were considered significant. FITC was applied in a sensitizing dosage (500 μg per 15 μl solvent = 1.28 μM FITC) onto one ear. Blood samples drawn from the axillary vein at varying time intervals and examined by fluorescence spectrophotometry were found to exhibit significant fluorescence emission at 517 nm. The excitation wavelength was 488 nm, the excitation maximum of FITC. In the absence of FITC, as with blood samples drawn from animals treated with the solvent only, the basic fluorescence intensity was about 1 × 105, largely owing to the autofluorescence and light scattering properties of the blood plasma (Figure 1). Data presented in Figure 2 show that FITC emission was recorded in the blood plasma from 1 min up to 72 h following epicutaneous application of the fluorescent contact sensitizer. The curve depicts that FITC was traceable in the blood already by 1 min after painting. FITC fluorescence rose further by 1.5 log, peaked at 24 h, and continued to be on rather high levels for 2 more days. Also single doses of FITC lower than the regular one (50 μg, 5 μg) could definitely be traced (Table 1).Figure 2Time dependence of FITC fluorescence in the blood. Fluorescence emission at 517 nm (FITC) was measured by means of fluorescence spectrophotometry in blood plasma samples drawn after epicutaneous application of 1 × 500 μg FITC. The emission spectra were recorded from 500 to 600 nm with an integration time of 1 s. Error bars are median ± SD.View Large Image Figure ViewerDownload (PPT)Table 1Single paintings with 50 μg or 5 μg FITC are traceable in blood plasmaaFluorescence intensities/cps. Subsensitizing but tolerizing doses of 50 μg or 5 μg of FITC dissolved in 15 μl of solvent were painted once onto one ear. After time intervals indicated blood was drawn from the axillary vein and plasma separated by centrifugation. Plasma samples were investigated by fluorescence spectrophoto- metry, and emission at 517 nm (FITC) was recorded.Time interval1 × 50 μg FITC1 × 5 μg FITC3min2.59 × 105bValues presented are single measurements (n =1).Not donebValues presented are single measurements (n =1).15minNot done0.48 ×1051hNot done1.99 ×10524 h8.40 ×1065.11 ×10548h5.14 ×1063.56 ×10572h2.19 ×1062.32 ×105a Fluorescence intensities/cps. Subsensitizing but tolerizing doses of 50 μg or 5 μg of FITC dissolved in 15 μl of solvent were painted once onto one ear. After time intervals indicated blood was drawn from the axillary vein and plasma separated by centrifugation. Plasma samples were investigated by fluorescence spectrophoto- metry, and emission at 517 nm (FITC) was recorded.b Values presented are single measurements (n =1). Open table in a new tab Blood samples shown to emit FITC fluorescence were subjected to Sephadex G-25 column chromatography. By recording the absorption of proteins (280 nm) and of FITC (488 nm) simultaneously it was secured that high molecular conjugation products and low molecular FITC molecules were separated nicely. Both elutions were immediately measured by fluorescence spectrophotometry. As illustrated in Figure 3 the readings of both protein-bound hapten and free chemical increased strikingly during the first 3 h, suggesting that free chemical continuously seeped in from the skin, whereas at the same time coupling to plasma proteins occurred. At 6 h, more so at 24 h, the inflow of uncoupled FITC slowed down, suggesting that the reservoir at the site gradually descended. Conversely, the rate of coupling ascended. While circulating in the bloodstream, FITC was found to attach also to blood cells as determined by FACS analysis (Figure 4). Polymorphonuclear granulocytes, enriched by Ficoll density gradient centrifugation, exhibited FITC fluorescence significantly higher than did mononuclear cells or erythrocytes.Figure 4Circulating polymorphonuclear leukocytes (PMN) attain more FITC fluorescence than do other blood cells. After paintings with 500 μg FITC, white and red blood cells were isolated by Ficoll density gradient centrifugation, and FITC fluorescence was determined by flow cytometry. Measured were 104 cells throughout, plotted are medians of fluorescence intensities ± SD. MNC, mononuclear cells; RBC, red blood cells.View Large Image Figure ViewerDownload (PPT) An estimate of FITC being drained through lymphatic vessels was essayed. Single cell suspensions derived from draining lymph nodes were subjected to FACS analysis at varying times after epicutaneous application of FITC and compared with those of nonregional lymph nodes, such as axillary, inguinal, and mesenteric nodes. Figure 5 shows that lymph nodes from different regions acquired FITC fluorescence not all alike. Cells from retroauricular nodes exhibited fluorescence far earlier and at higher degrees than did those from nonregional nodes. This suggests that free hapten escaped at least partially through local lymphatics and was directly drained into retroauricular lymph nodes. Spleen and distant, nonregional lymph nodes, in particular mesenteric nodes, are independent of lymphatic drainage from the site. Yet single cell suspensions obtained from those organs were observed to exhibit FITC fluorescence too, although at lower degrees than did retroauricular lymph nodes (Figure 5). As compared with regional nodes, which seemingly obtained FITC at first hand through lymphatic vessels, FITC fluorescence in spleen (Figure 6b) and nonregional nodes rose not quite as fast; however, within 3 h after painting the increase of fluorescence was statistically significant. CD11c-positive dendritic cells (CD) separated from total lymph node cells or spleen cells by depletion were found to exceed by far the residual cells with regard to their fluorescence intensity. This phenomenon was detected in all of the lymphoid organs examined (Figure 6a,b). Moreover, evident time differences were observed in reaching the various peaks of fluorescence intensity. Again, regional lymph nodes peaked already at 1 h, CD derived from spleen cells peaked at 3 h, and CD from inguinal nodes did so not until 24 h. Spleen cell suspensions obtained from animals painted with 500 μg of FITC 3 or 12 h previously were irradiated (30 Gy) and used as antigen-presenting cells. Responder cells were syngeneic lymph node cells from animals sensitized to FITC. Cells were cocultured for 5 d, pulsed with 3H-thymidine, and harvested 16 h later. The proliferation responses (Figure 7a,b,) were similar to those obtained with normal spleen cells exposed to FITC in vitro and used as a positive control, but significantly different from that obtained with spleen cells exposed to the solvent only. Spleen cells taken later, however, i.e., at 24 h after painting, were no longer capable of triggering proliferation (Figure 7b). In contrast, cells derived from nonregional, inguinal lymph nodes taken from the same donors at the same time and cocultured in the same experiment induced significant proliferation responses. The goal of this study was to trace the fate of the lipophilic contact sensitizer FITC after application onto the skin by means of fluorescence spectrophotometry and flow cytometry. A single dose of 500 μg was painted onto one ear, a site of which direction of venous flow and lymphatic drainage are clearly defined. The dosage used was adequate to induce quite satisfactory sensitization (data not shown). Following application, FITC was observed to disseminate from the site rapidly. Already 1 min after painting FITC could be traced in the blood plasma by fluorescence spectrophotometry. The allergen escaped from the site in decreasing quantity, but only after 24 h the inflow into the blood had ceased, indicating that the reservoir had finally disappeared. The lipophilic allergen was found to enter the blood as free hapten in chemically active form because it was shown to couple increasingly to soluble plasma proteins and to attach also to white blood cells. Blood-borne CD were not measured separately. The coupling reaction occurring in the blood was observed to start instantaneously but to proceed rather slowly according to the reaction constant to proteins, as illustrated in Figure 3. Thus, within a few hours after painting a considerable amount of hapten–protein conjugate had formed, which entered the spleen directly and should be expected to cause production of anti-hapten antibody. This is a well-known phenomenon in contact hypersensitivity (Asherson et al., 1983Asherson G.L. Colizzi V. Watkins M.C. Immunogenic cells in the regional lymph nodes after painting with the contact sensitizers picryl chloride and oxazolone: evidence for the presence of IgM antibody on their surface.Immunology. 1983; 48: 561-569PubMed Google Scholar;Macatonia et al., 1987Macatonia S.E. Knight S.C. Edwards A.J. Griffiths S. Fryer P. Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate.J Exp Med. 1987; 166: 1654-1667Crossref PubMed Scopus (520) Google Scholar), presumed to limit the persistence of antigen in its immunogenic form by modulating the removal of the CD carrying antigen. The continual inflow of chemically active hapten into the circulation implies that free FITC molecules were dispersed throughout the body by the pumping action of the heart. They can be expected to move to all tissues and every organ, of which the peripheral lymphoid organs are of particular interest. FACS analysis of spleen and distant lymph nodes revealed that the cells had acquired FITC fluorescence according to their blood supply. Spleen and mesenteric nodes gained FITC fluorescence earlier and at higher degrees than did axillary and inguinal nodes. The even stronger fluorescence of the regional nodes must be attributed to the additional supply by lymphatic vessels draining the ear site. CD11c+ CD depleted from all of the lymphoid organs examined, including the spleen, were found to attain more FITC fluorescence than the negatively depleted lymphoid cells. Seemingly, FITC molecules were particularly attracted by CD, irrespective of their autofluorescence being basically stronger than that of lymphoid cells. It cannot be determined by flow cytometry whether lipid-soluble FITC molecules penetrate cell membranes directly or bind to membrane-bound proteins or do both. In general, nonpolar lipid-soluble compounds as well as polar water-soluble substances that retain sufficient lipid solubility can cross the lipid portion of the cell membranes by passive diffusion (Fingl and Woodbury, 1975Fingl E. Woodbury D.M. General principles.in: Goodman L.S. Gilman A. The Pharmacological Basis of Therapeutics. 5th edn. Macmillan Publishers Co, New York1975: 1-45Google Scholar). It has been shown earlier (Macatonia et al., 1986Macatonia S.E. Edwards A.J. Knight S.C. Dendritic cells and the initiation of contact sensitivity to fluorescein isothiocyanate.Immunology. 1986; 59: 509-514PubMed Google Scholar,Macatonia et al., 1987Macatonia S.E. Knight S.C. Edwards A.J. Griffiths S. Fryer P. Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate.J Exp Med. 1987; 166: 1654-1667Crossref PubMed Scopus (520) Google Scholar) that FITC was preferentially located on CD but only draining lymph nodes had been examined. These authors painted FITC on the shaved thorax and abdomen and investigated axillary and inguinal lymph nodes. We intentionally selected the ear site for application because lymph draining from this site is supposed to enter solely the retroauricular lymph node before subsequently passing through the cervical duct into the circulating blood. This technique should make it possible to distinguish draining lymph nodes from distant ones. CD11c+ cells from spleen and mesenteric lymph nodes reached maximal FITC fluorescence at 3 h (Figure 5), whereas those from inguinal lymph nodes did so not until 24 h. Accounting for the fact that the applied FITC solution is considerably diluted upon entering the blood, and also an indefinable number from the circulating pool of FITC molecules is constantly withdrawn through coupling to plasma proteins and blood cells, it is surprising that enough free hapten is left for reacting at the periphery with billions of cells. It is noteworthy that cells need to bind at least 5000 FITC molecules to be detectable by a fluorescence-activated cell sorter (Loken and Herzenberg, 1975Loken M.R. Herzenberg L.A. Analysis of cell population with fluorescence-activated cell sorter.Ann N Y Acad Sci. 1975; 245: 163-171Crossref Scopus (287) Google Scholar). When spleen cells or lymph node cells, haptenated in this fashion in vivo, were used as antigen-presenting cells in vitro, they were found to drive syngeneic committed lymph node cells to proliferation (Figure 7a,b,b), indicating that antigen was presented and recognized. Healthy intact skin is a good barrier to the mass transport of topically applied substances, but it allows some permeation of almost every substance (Scheuplein, 1978aScheuplein R. The skin as a barrier.in: Jarrett A. The Physiology and Pathophysiology of the Skin. Vol. 5. Academic Press, London1978: 1669-1692Google Scholar). Percutaneous absorption refers to the entire process of mass transport of substances and includes absorption by the stratum corneum, diffusion through each layer of the skin, and finally uptake by the microcirculation. Diffusion through the horny layer is usually the controlling or rate-limiting process. The nature of passive skin permeation allows the usual physical laws regarding passive diffusion processes to be applied to skin permeability. Diffusion through intact skin is in no way dependent on metabolic assistance (Allenby et al., 1969Allenby A.C. Creasey N.H. Edginton J.A.G. Fletcher J.A. Schock C. Mechanisms of action of accelerants on skin penetration.Br J Dermatol. 1969; 81: 47-55Crossref PubMed Scopus (52) Google Scholar). As we intentionally used the auricle as the site of application, the sensitizing dose of 500 μg of FITC was dissolved in just 15 μl of solvent. This was only achieved with the addition of 5% dimethyl sulfoxide. We tested whether percutaneous absorption of FITC is thereby accelerated and found that it is not (data not shown). This result is in perfect agreement with experimental data (Scheuplein and Ross, 1970Scheuplein R.J. Ross L. Effects of surfactants and solvents on the permeability of epidermis.J Soc Cosmet Chem. 1970; 21: 853-873Google Scholar;Scheuplein, 1978bScheuplein R. Site variation in diffusion and permeability.in: Jarrett A. The Physiology and Pathophysiology of the Skin. Vol. 5. Academic Press, London1978: 1731-1752Google Scholar), indicating that solvent proportions of dimethyl sulfoxide in excess of 50–70% are necessary before permeability is appreciably enhanced. A matter for consideration is unintentional oral ingestion of hapten. This can definitely be excluded concerning the earliest measurements, i.e., at 1 and 3 min after painting. The animals were already deeply anesthetized for the subsequent collection of blood before FITC was painted on their ears. For measurements at 15 min animals were anesthetized soon after application. Thus, early systemic distribution of hapten can be assigned entirely to diffusion through the skin. At later times, the treated animals may have ingested some hapten by wiping their ears and licking their feet, although this was not observed. In light of the findings at early points, however, it seems unlikely that hapten ingested later would add significantly to the amount resulting from diffusion and would thus have to be considered to act as oral tolerogen. The fluorescent sensitizer FITC has been used to track migration of epidermal Langerhans cells or dermal CD from the skin to draining lymph nodes.Macatonia et al., 1987Macatonia S.E. Knight S.C. Edwards A.J. Griffiths S. Fryer P. Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate.J Exp Med. 1987; 166: 1654-1667Crossref PubMed Scopus (520) Google Scholar have reported that after skin painting with FITC on the shaved thorax and abdomen fluorescent CD appeared in the regional lymph nodes between 4 and 8 h and reached a peak at 24 h. Morphologically, these cells were classified as functionally activated, and in some of them Birbeck granules were identified. The timing of the appearance of these cells seems to support the view that they are derived from migratory Langerhans cells. We agree with their results and largely with their conclusions but in light of the findings reported here one should be cautious of relating the ''appearance'' of fluorescent cells in regional nodes to their migratory activity. Free hapten molecules ready to bind get into close contact with lymph node cells long before Langerhans cells migrate. Just after the painting of a sensitizer, Langerhans cells are immersed in a solution of dissolved hapten molecules. Their high level of macropinocytosis allows them to take up large amounts of fluid and then to concentrate the solutes (Sallusto et al., 1995Sallusto F. Cella M. Danieli C. Lanzavecchia A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products.J Exp Med. 1995; 182: 389-400Crossref PubMed Scopus (2178) Google Scholar;Cella et al., 1997Cella M. Sallusto F. Lanzavecchia A. Origin, maturation and antigen presenting function of dendritic cells.Curr Opin Immun. 1997; 9: 10-16Crossref PubMed Scopus (1157) Google Scholar). This is known to promote maturation and migration to the draining lymph nodes and homing to T cell areas. These are the crucial events in contact sensitization. But at the same time, and independently, free FITC molecules escape from the skin through lymph and blood vessels and enter distant lymphoid organs. Whether their accumulation on lymphoid CD proceeds in accordance with the mechanism described for immature CD such as Langerhans cells is not known. The data presented suggest that FITC, although applied in limited dosage onto a confined area of the skin, rapidly enters the skin by diffusion and is swept into the circulation essentially as free hapten. Reactive hapten molecules disseminate throughout the body by way of the bloodstream accumulating preferentially on CD11c+ CD in virtually all of the peripheral lymphoid organs. Antigen presentation and recognition is, in principle, shown to occur. It seems reasonable to assume that this chain of events is of biologic significance. Systemic haptenization compares with local haptenization at the site, where epidermal Langerhans cells and perhaps dermal CD become engaged in eliciting an immunologic response of the delayed type. Simultaneous induction of feedback suppressor cells is a regular part thereof (Claman et al., 1980Claman N.H. Miller S.D. Conlon P.J. Moorhead J.W. Control of experimental contact sensitivity.Adv Immunol. 1980; 30: 121-157Crossref PubMed Scopus (81) Google Scholar); however, its mode of induction is still poorly understood. Hapten molecules bypassing the draining lymph nodes and reacting with antigen-presenting cells far distant from the site could well be the eliciting elements. Earlier sensitization experiments in guinea-pigs (Macher and Chase, 1969Macher E. Chase M.W. Studies on the sensitization of animals with simple chemical compounds. XII. The influence of excision of allergenic depots on onset of delayed hypersensitivity and tolerance.J Exp Med. 1969; 129: 103-121Crossref PubMed Scopus (91) Google Scholar) used spaced separation of the site for differentiating the roles of dispersed and residual allergen. By interrupting abruptly, at varying times, both further drainage and effectiveness of the reservoir deposited at the site, it was found that the fraction of allergen which escaped had a definite tolerogenic effect, whereas the remaining fraction effected sensitization. They concluded that during the induction period of contact hypersensitivity, two immunologic processes occur simultaneously and independently, which in principle act antagonistically. The resultant sensitization would then be a measure of the balance attained between the two processes. If the conclusions cited above can be transferred to the findings presented here, systemically haptenated CD are interpreted as representing a counterbalance to epidermal Langerhans cells. We thus tentatively propose systemic haptenization to be a means required to achieve systemic regulation of locally induced contact hypersensitivity. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (grant no. 87/11–3). We thank Drs Volker Gerke and Cord Sunderkötter for helpful discussions. We also thank Ms Claudia Solé-Amell and Ms Ruth Goez for expert technical assistance, and Ms Brunhilde Scheibel for secretarial help. This study is dedicated to Dr. Merrill W. Chase on the occasion of his 94th birthday. Parts of this work were presented at the annual meeting of the ''Arbeitsgemeinschaft für Dermatologische Forschung'', Bonn, Germany, February 18–20 1999 (Arch Dermatol Res 291:163 1999).