Clin. exp. Immunol. (1980) 39, 750-755.

Large mononuclear (veiled) cells with 'Ia-like'membrane antigens in human afferent lymph

C. J. F. SPRY, A. J. PFLUG, G. JAN0SSY* & J. H. HUMPHREY Departments of Immunology and Surgery,Royal Postgraduate Medical School, Ducane Road, and * Department of Immunology, The Royal Free Hospital Medical School,

Pond Street, London

(Accepted for publication 10 September 1979)

SUMMARY

Studies were done to see whether large mononuclear cells in human afferent lymph possesscytoplasmic veils or Ia-like antigens on their surface. Small numbers of veiled cells were seen inlymph from five subjects: one with post-phlebitis oedema, one with trophic ulcers of the legs,one with common variable immunoglobulin deficiency, and two control subjects. They were notseen in afferent lymph from two other patients with oedema of the legs, and one control subject.Only occasional large mononuclear (veiled) cells formed rosettes with IgG-coated erythrocytes,and they did not attach to glass after overnight culture. They had a distinctive nuclear structureand fluorescent antisera showed that their membranes and veils possessed large amounts of 'Ia-like' antigens. They did not have surface immunoglobulin or a monocyte membrane antigen. Itwas concluded that the membrane and nuclear structure of human large mononuclear (veiled)cells is in keeping with the possibility that they are derived from Langerhans' cells in the skin,and that they can become interdigitating cells in lymphoid tissues. It is suggested that these cellshave an important role in the transport of antigens from the skin and the stimulation of Tlymphocytes in lymphoid tissues.

INTRODUCTION

Afferent lymph contains small numbers of lymphocytes, mononuclear cells, granulocytes and erythro-cytes. This has been shown both in sheep (Morris & Courtice, 1977), rabbits (Kelly et al., 1978), pigs(Drexhage et al., 1979), and man (Engeset, Sokolowski & Olszewski, 1977). Although the output ofnucleated cells in human afferent lymph from the foot is variable (Engeset et al., 1977) most of the cellsare small lymphocytes which lack surface immunoglobulin (Godal & Engeset, 1978), and are thereforeprobably T lymphocytes (Engeset, Bremer & Froland, 1977). Recently a large cell type in human afferentlymph, comprising about 1-5% of the mononuclear cells has been shown to have a distinctive nuclearstructure and to contain Birbeck granules which are found also in Langerhans' cells in the skin and inter-digitating cells in the paracortex of lymph nodes (Sokolowsky, Jakobsen & Johannessen, 1978). AsLangerhans' cells in the skin and interdigitating cells in lymph nodes have large amounts of Ia-likeantigen on their surface (Klareskog et al., 1977; Rowden, Lewis & Sullivan, 1977; Stingl et al., 1978;Lampert et al., 1979), the possibility arose that if these three cells belonged to the same cell lineage,veiled cells in afferent lymph might also contain equally large amounts of Ia-like antigens on theirsurface.

Large mononuclear cells in afferent lymph have also been studied in animals (Morris & Courtice, 1977;Kelly et al., 1978; Drexhage et al., 1979). Cultures of these cells have shown that they often extend large

Correspondence: Dr C. J. F. Spry, Department of Immunology, Royal Postgraduate Medical School, 150 Ducane Road,London W12 OHS.

0099-9104/80/0300-0750$02.00 (0 1980 Blackwell Scientific Publications

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Ia-like antigens on human veiled cells 751cytoplasmic veils which are retracted periodically in culture. Similar studies on human lymph have notbeen reported.For these reasons experiments were done on afferent lymph obtained from patients who were under-

going lymphography to examine the cause for their swollen legs. Experiments were done to see whetherlarge mononuclear cells in the lymph also had veils and whether their plasma membranes expressedlarge amounts of Ia-like antigens. It was hoped that this would show how the cells might be related toLangerhans' cells in the skin and interdigitating cells in lymph nodes.

MATERIALS AND METHODS

Patients. Lymph was collected from five patients and two normal subjects. One patient (K.S.) with swelling of one legdue to phlebitis, also provided lymph from his normal leg. Eight samples oflymph, therefore, were examined. The collectionswere done after informed consent had been given by these subjects.Lymph collections. Canulae were placed in the dorsal lymph vessels of the feet after they had been outlined with Patent Blue

V (May and Baker, Dagenham) injected into the web between the toes (Johnson & Pflug, 1975). One canula was put into thedistal part of the vessel collecting lymph from the feet while the other was inserted proximally and was used later for lympho-graphy in the patients with swollen legs. The subjects were encouraged to move their feet and lymph was collected forj-1 hr while they were walking. The lymph drained into glass vials which had been wetted with two drops of heparin, 1000units/ml.

Cell counts. After about 0-2 ml of lymph had been collected, 10 A1 was mixed with 10 p1 of white blood cell counting fluidand a nucleated cell count was done in an improved Neubauer counting chamber. When cell counts were below 104/ml,lymph was examined directly by phase contrast microscopy without dilution.

Veiled cells. Ten microlitres of lymph were placed in waxed coverslip/microscope slide preparations and examined at370C or room temperature. Veiled cells were found to be large rounded cells with undulating cytoplasmic projections.Cytocentrifuge preparations were made of lymph cells after they had been concentrated by centrifugation, and were stainedwith Leishman's stain. Receptors for rabbit IgG-coated ox erythrocytes (EA) were done by the method of Tai & Spry(1976).

Fluorescent antisera andIF staining. Antisera to purified human 'Ia-like' (p28,33) antigens were made in rabbits and chickensas described previously (Janossy et al., 1977a). These antisera reacted strongly with B lymphocytes (but not with thymocytesand most T lymphocytes), and a subset of blood monocytes. In tissue sections these anti-Ia-like reagents reacted particularlystrongly with Langerhans' cells in the skin, interdigitating cells in the lymph nodes (Lampert et al., 1979) and macrophages/epithelial cells in the thymic medulla (Janossy, 1979). An antiserum specifically detecting human thymocyte/T cell antigens(huTLA) was raised in rabbits by immunizing with monkey thymocytes (Janossy et al., 1977b). Two rabbit antisera weremade against peritoneal macrophages and chronic granulocytic leukaemia cells respectively. After absorptions, both antiserareacted with human monocytes and polymorphonuclear cells but were unreactive with lymphocytes (Greaves, Falk & Falk,1978). These reagents were used in indirect IF test using goat anti-rabbit Ig-FITC, and sheep anti-chicken Ig-TRITC asdescribed previously (Janossy et al., 1977a, b). Both first and second layers were used in 1: 20-1:40 dilutions (except anti-huTLA serum which was used at 1: 10). A goat anti-human Ig-FITC (Behringwerke) detected surface Ig in direct IF test.The staining of cells was carried out in live cell suspensions. Each layer was incubated for 15 min at 20'C and washed threetimes. Cytocentrifuge preparations of the stained cells were made, fixed in cold ethanol (10 sec) and covered with glycerol andglass cover slips. These preparations were then examined under a Zeiss microscope with x 63 phase oil objective and IV/Fepifluorescence condenser containing interchangeable selective filter sets for FITC and TRITC. Some preparations werecounterstained for nuclear DNA with acridine orange.

RESULTS

The number of nucleated cells/ml in the afferent lymph from the eight subjects was between 5 x 102and 8f5 x 104/ml, and the majority were small lymphocytes. Veiled cells were seen in five of the eightsamples oflymph and made up to 15% of the nucleated cells (Table 1). The veiled cells were not attachedto other cells in the living state, although they appeared to be juxtaposed to T lymphocytes in fixedpreparations. They did not adhere to glass when left overnight at 370C in waxed coverslip/microscopeslide preparations. They continued to put out undulating cytoplasmic projections at this period but didnot move across the surface of the slide (Fig. 1).

Cytocentrifuge preparations of the cells showed that large mononuclear (veiled) cells had a distinctivenuclear structure and often had prominent cytoplasmic processes which confirmed that they were

C. ]. F. Spry et al.TABLE 1. Large mononuclear (veiled) cells in afferent lymph from patients and

control subjects

Afferent lymph

Age Nucleated 'Veiled'Subjects Sex (years) Diagnosis cells/ml cells (%)

P.J. F 56 Trophic leg ulcers - OccasionalR.H. M 54 Post-phlebitis oedema 1 8 x 104 3J.H. M 62 Varicose veins 5 x 102 None seenK.S. M 29 Post-phlebitis oedema (1. leg) 5 x 102 None seenC.F. M 21 Hypogammaglobulinaemia 8-5 x 10i 15

A.L. M 29 Normal 1P7x 103 None seenK.S. M 29 Normal (r. leg) 2'8x 104 8J.H. M 63 Normal - Occasional

FIG. 1. A large mononuclear cell from human afferent lymph in culture. The cell possesses long cytoplasmicextensions which were seen to undulate and to be extended and withdrawn once or twice each minute. (Phasecontrast x 650.)

identical to the cells seen in the living state. Less than 10% of the large mononuclear (veiled) cells formedrosettes with EA (Fig. 2).

Immunofluorescent studies revealed spectacularly strong staining of the large mononuclear (veiled)cells with both rabbit and chicken anti-Ia-like antisera. The membrane staining of the projections andveils was smooth with no evidence of patch- or cap-formation during the short staining period (Fig. 3).The staining intensity of large mononuclear (veiled) cells with anti-Ia-like antisera was considerablystronger than that of the (scanty) Ig-positive B lymphocytes in the same lymph sample, or in the controltonsil cell suspension stained for Ia-like antigens under identical conditions. Small lymphocytes and thefew polymorphs present showed no staining with the anti-Ia-like reagents. Large mononuclear (veiled)cells failed to show membrane fluorescence with anti-human T lymphocyte antiserum, anti-human Igor the two different batches of anti-human monocyte/granulocyte sera.

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Ia-like antigens on human veiled cells

FIG. 2. Cytocentrifuge preparation of large mononuclear (veiled) cells in human afferent lymph. (a) A largemononuclear (veiled) cell and a small lymphocyte showing their relevant sizes. (b) A large mononuclear cellshowing long cytoplasmic veils (arrows). (c) Some large mononuclear cells had nuclei with a convulutedappearance and cytoplasmic vacuoles. (d) A large mononuclear (veiled) cell and three attached lymphocytesafter the cells had been concentrated by centrifugation. (e) A large mononuclear (veiled) cell with attachedlymphocytes, forming a rosette with EA. (Leishman's stain x 450.)

FIG. 3. Detection of Ia-like antigens on large mononuclear (veiled) cells using fluorescent antisera. (a) A largemononuclear (veiled) cell in human afferent lymph showing strong membrane fluorescence when stained withchicken anti-human Ia-like antibody, and sheep anti-chicken Ig-TRITC. Two small T lymphocytes counter-stained with acridine orange are attached to the cell surface. (b) A large mononuclear (veiled) cell stained as in (a),showing large amounts of Ia-like antigens on the long cytoplasmic processes. (x 80.)

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DISCUSSION

This study has shown that human afferent lymph contains large mononuclear cells which have a charac-teristic nuclear structure and possess veils which are extended and retracted in culture. The numberof these cells was always small, and occasionally samples contained no veiled cells. This contrasts withafferent lymph from rabbits where up to 5300 of the nucleated cells may have veils (Kelly et al., 1978).The entry of large mononuclear (veiled) cells into afferent lymph may be intermittent as injections ofantigens into the skin (Hoffsmit et al., 1979) and application of contact sensitizing agents to the skin ofguinea-pigs (Drexhage et al., 1979) increased their output. The absence of cutaneous lesions in the areadrained by the dorsal foot lymphatics may account for the low output of veiled cells found in thesepatients. Large numbers of veiled cells may enter afferent lymph draining skin which is involved incertain diseases, especially those which lead to dermatopathic lymphadenopathy (see below).

Large mononuclear (veiled) cells in human afferent lymph were found to contain large amounts ofIa-like antigen on their surface. Similar findings have been made in pigs (R. M. Binns & B. M. Balfour,personal communication). As they did not possess other membrane markers which are characteristic oflymphocytes or monocytes, they are probably a distinct cell type. They are probably identical to thelarge mononuclear cells in human afferent lymph which have been shown recently by electronmicroscopyto contain Birbeck granules (Sokolowsky et al., 1978).The finding that few large mononuclear (veiled) cells in afferent lymph formed rosettes with EA

should be compared with the report that most epidermal Langerhans' cells which are isolated usingtrypsin treatment can bind EA (Stingl et al., 1977). However, as pointed out by Rowden et al. (1977),trypsin may increase the binding capacity of epidermal Langerhans' cells for EA, and recent work hasconfirmed that trypsin increases EA binding by several cell types (Tai & Spry, 1979, unpublished data).It is therefore possible that epidermal Langerhans' cells in situ also have a low binding capacity for EA.As Langerhans' cells in human skin also contain Birbeck granules (Shelly & Juhlin, 1978), and have

large amounts of Ia-like antigens on their surface (Klareskog et al., 1977; Rowden et al., 1977; Stinglet al., 1978), it is likely that Langerhans' cells leave the skin to become large mononuclear (veiled) cellsin afferent lymph. This possibility is strengthened by related studies in animals (Kelly et al., 1978,Drexhage et al., 1979). Recently it has been suggested that large mononuclear (veiled) cells enter lymphnodes to become interdigitating cells in the paracortex (Humphrey, 1979). The finding of large amountsofIa-like antigens on epidermal Langerhans' cells and interdigitating cells in lymph nodes draining theskin of patients with dermatopathic lymphadenopathy supports this hypothesis (Lampert et al., 1979),and as the same reagents were used in that study and the present one, a common lineage for Langerhans'cells, large mononuclear (veiled) cells and interdigitating cells seems very probable.The presence of large amounts of Ia-like antigens on the surface of large mononuclear (veiled) cells

may have functional importance. It has been proposed that contact sensitizing agents could bind to Iaantigens on Langerhans' cells in the skin and induce local T lymphocyte cytotoxic reaction (Forsum et al.,1978), but there is no direct evidence to show how Ia antigens on Langerhans' cells are involved inallergic reactions. It has also been suggested that large mononuclear (veiled) cells transport antigensfrom the skin to lymph nodes (Drexhage et al., 1979), and stimulate T lymphocytes in the paracortex(Humphrey, 1979). The finding of T lymphocytes attached to laige mononuclear (veiled) cells in someexperiments strengthens the concept that these two cell types may interact with each other in vivo.

It is concluded that large mononuclear cells with veils are present in small numbers in normal afferentlymph, and that these cells possess large amounts ofIa-like antigens on their surface. Further studies onthese cells may be of value in determining the nature of sensitizing antigens in the skin of patients withchronic skin diseases, and they could be used to study the mechanisms which lead to the induction of Tlymphocyte-dependent processes in draining lymph nodes.

We should like to thank Mrs Eira Rawlings for technical help and Miss Ruth Frearson for typing the manuscript.

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Ia-like antigens on human veiled cells 755

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