Immunogold labeling of keratin filaments in normal human epidermal cells with two anti-keratin monoclonal antibodies

We report on application of the highly sensitive and specific immunogold labeling method for ultrastructural investigation of keratin intermediate filament antigens in human epidermal cell suspensions. Triton X-100 pretreated cells proved accessible to the colloidal gold conjugate, thus enabling keratin filament bundles to be labeled. Anti-keratin KL1 and KL2 monoclonal antibodies were raised in mice after immunization with either human stratum corneum-isolated keratins or keratins extracted from human epidermal cells suspensions, respectively. Immunoelectron microscopy confirmed immunofluorescence and immunoperoxidase results of epidermal keratinocyte staining, and revealed two different antibody reactivity patterns: KL2 reacted with keratin filaments in keratinocytes of all epidermal layers, whereas antigen to KL1 was detected only on keratin of the suprabasal layers, not on the basal keratinocyte tonofilaments. The monoclonal antibody-recognized epitopes were specific for the keratin filaments. Vimentin-rich cells (melanocytes) were not stained in the same epidermal cell suspensions. Additionally, two distinct ultrastructural patterns of keratin filament epitope labeling were observed. KL1 and KL2 monoclonal antibodies react with two different antigenic determinants, depending on the stage of keratinocyte differentiation, and may therefore be used for immunohistochemical studies of various keratin-containing cells in normal and pathologic conditions.     

Expression of keratins during experimentally induced carcinogenesis in hamster cheek pouch visualized polyclonal and monoclonal antibodies

Summary We obtained immnohistochemical profiles of several keratin proteins during experimentally induced carcinogenesis in hamster cheek-pouch mucosa using a polyclonal antibody (TK; detecting keratins with molecular masses of 41 65 kilodalton) and two monoclonal antibodies (KL1, 55- to 57-kilodalton keratins; PKK1; 40-, 45- and 52.5-kilodalton keratins). The squamous epithelium of normal pouch mucosa exhibited positive TK staining in all layers. KL1 staining in the spinous layer and PKK1 staining in the basal layer, thus indicating a regional or zonal distribution pattern. Epithelia undergoing basal hyperplasia showed irregular localization of PKK1 binding, while hyperkeratinized lesions exhibited the binding pattern found in normal epithelium. In case of epithelial dysplasia, there was reduced KL1 staining in spinous cells and decreased PKK1 staining in the basal and parabasal layers. Papillomas exhibited a rather zonal distribution of keratin staining. All squamous-cell carcinomas, irrespective of their degree of keratinization and infiltration pattern, showed slight or no PKK1 staining. Such lesions were only positive for KL1-detectable keratins in keratinizing tumour cells and exhibited an irregular distribution of TK binding. The expression of keratin proteins during carcinogenesis in hamster cheekpouch mucosa may parallel that of keratins in human squamous-cell carcinomas originating in the oral mucosa.     

Expression of keratins during experimentally induced carcinogenesis in hamster cheek pouch visualized polyclonal and monoclonal antibodies

We obtained immunohistochemical profiles of several keratin proteins during experimentally induced carcinogenesis in hamster cheek-pouch mucosa using a polyclonal antibody (TK; detecting keratins with molecular masses of 41-65 kilodalton) and two monoclonal antibodies (KL1, 55- to 57-kilodalton keratins; PKK1; 40-, 45- and 52.5-kilodalton keratins). The squamous epithelium of normal pouch mucosa exhibited positive TK staining in all layers, KL1 staining in the spinous layer and PKK1 staining in the basal layer, thus indicating a regional or zonal distribution pattern. Epithelia undergoing basal hyperplasia showed irregular localization of PKK1 binding, while hyperkeratinized lesions exhibited the binding pattern found in normal epithelium. In case of epithelial dysplasia, there was reduced KL1 staining in spinous cells and decreased PKK1 staining in the basal and parabasal layers. Papillomas exhibited a rather zonal distribution of keratin staining. All squamous-cell carcinomas, irrespective of their degree of keratinization and infiltration pattern, showed slight or no PKK1 staining. Such lesions were only positive for KL1-detectable keratins in keratinizing tumour cells and exhibited an irregular distribution of TK binding. The expression of keratin proteins during carcinogenesis in hamster cheek-pouch mucosa may parallel that of keratins in human squamous-cell carcinomas originating in the oral mucosa.     

Alpha-enolase is restricted to basal cells of stratified squamous epithelium.

We have developed a monoclonal antibody against a 50-kDa protein that binds preferentially to basal cells in the limbus of rat, rabbit, and human corneas (J. D. Zieske, G. Bukusoglu, and M. A. Yankauckas, Invest. Ophthalmol. Visual Sci. 33, 143-152, 1992). Here we report on the purification and identification of the antigen. The 50-kDa antigen was purified from rabbit limbal and corneal epithelium using HPLC methodology including anion exchange (DEAE) followed by reverse-phase (C18) chromatography. The purified 50-kDa protein was then digested with endoproteinase Lys-C, and a reproducible profile comprising approximately 20 peptides was observed by reverse-phase HPLC of the digest. Sequence analysis of five peptides ranging in length from 4 to 20 residues revealed that the 50-kDa protein was alpha-enolase, a glycolytic enzyme. Overall, 57 amino acids were identified with a 95% sequence homology. Localization of alpha-enolase in rat epithelium by immunofluorescence microscopy demonstrated that simple epithelium contained low or undetectable levels of the enzyme. Stratified squamous epithelium, however, showed high levels of alpha-enolase, which was localized specifically to cells of the basal layer. Epidermal, corneal limbal, oral mucosal, vaginal, and laryngeal epithelium all showed cytoplasmic binding specific to the basal cells. These data indicate that the glycolytic enzyme alpha-enolase is preferentially localized in the basal cell layer of stratified squamous epithelium and suggest that glycolytic activity is concentrated in these cells. The localization pattern suggests that a major change in metabolism occurs as cells leave the mitotically active basal cell layer and migrate toward terminal differentiation in the suprabasal cell layers.     

Quantitative studies of keratin expression with the post-embedding immunogold labeling method

Immunoreactivity of the 56.5 KD acidic (type I) keratin was localized ultrastructurally and quantified in normal human epidermis using the specific monoclonal antibody KL1 and post-embedding immunogold labeling. The protein was detected in keratin intermediate filament bundles of all suprabasal keratinocytes. Keratohyalin granules and desmosomal plaques were labeled only on the periphery, in regions where keratin filaments penetrate these structures. The 56.5 KD keratin immunoreactivity increased from the first suprabasal layer onwards and reached its maximum in the outmost spinous layer. A subsequent abrupt decrease of the specific immunogold labeling was observed in the granular layer. This low reactivity, which persisted also in the horny layer, may be partially explained by either protein degradation or masking of the antigenic sites by a filament-aggregating material occurring at these stages of keratinocyte terminal differentiation. Statistical comparison of the quantitative results obtained in various cell and tissue compartments revealed no significant differences between the background labeling levels observed in the basal layer of epidermis with KL1, a control monoclonal antibody, or the immunogold conjugate alone. Our results confirm the specificity of 56.5 KD keratin for terminally differentiating suprabasal keratinocytes and demonstrate the importance of appropriate control studies when a post-embedding immunogold labeling method is employed.     

Spreading of embryologically distinct urothelial cells is inhibited by SPARC

The AON epitope of secreted protein acidic and rich in cysteine (SPARC) is a conserved motif expressed by human SPARC in a variety of human cell types. Through the use of a monoclonal antibody that recognizes this epitope, transitional epithelium was found to restrict expression of SPARC to the suprabasal and intermediate layer. Such intracellular expression was defined by immunoreactive signals that localized to the apical plasma membranes of suprabasal and intermediate cells. Polarization of SPARC to apical plasma membranes of suprabasal cells was retained in vitro by a subpopulation of cells that exhibited characteristics of suprabasal cells--cell-cycle quiescence, large cell volumes, and multiple nuclei. In contrast, the basal layer of transitional epithelium in vivo and cycling cells in vitro did not exhibit this apical staining pattern, but instead sequestered the SPARC polypeptide within urothelial cytoplasm and/or nuclei, as revealed by immunohistochemical analysis. Elution of soluble proteins and DNA from urothelial cells revealed the presence of SPARC within the nuclear matrix--and that SPARC colocalized with the nuclear matrix Ki-67 antigen. rSPARC activity was demonstrated and quantified with a rounding assay whereby the spreading of freshly plated cells was inhibited by recombinant SPARC in a concentration- and time-dependent manner. Inhibition of spreading was observed in urothelial cells derived from endoderm (bladder) and mesoderm (ureter) germ layers. Statistically significant differences were seen between urothelial cells from these two layers. Mesodermal cells recovered more slowly from the inhibitory effects of rSPARC, such that at hour 6 endodermal cells underwent significantly more spreading, as shown by a rounding index (RI). These experiments provide new insights about the matricellular trafficking of SPARC and suggest that intra- and extra-cellular localization patterns influence the development, homeostasis, and differentiation of transitional epithelium.     

Phenotypical characterization of lymphocytes infiltrating regressing papillomas.

Papillomavirus-induced lesions often regress spontaneously in both humans and animals. Papilloma regression is deemed to be due to a cell-mediated immune response, the nature of which is still ill defined, and is accompanied by immune cell infiltrates. To gain further information on the nature and role of the immune cells present in regressing papillomas, we have analyzed biopsies of papillomas induced in the soft palate of cattle by bovine papillomavirus type 4 (BPV-4) and have phenotypically characterized and quantified the lymphocytes present in these lesions. Eleven papilloma biopsies and seven biopsies of noninfected palate were analyzed for the presence of activated CD4+, CD8+, and gamma delta(WC1+) lymphocytes. We found large numbers of lymphocytes in the subepithelial derma of papillomas but not in normal palate tissue; these cellular masses consisted predominantly of CD4+ lymphocytes, with only a few CD8+ and gamma delta(WC1+) lymphocytes, generally positioned at the periphery of these masses. All three subtypes of lymphocytes were found interdigitated with the cells of the basal layer both in papillomas and in normal palate tissue, but while basal layer CD8+ and gamma delta(WC1+) T cells were detected with similar frequencies in papillomas and uninfected palate, basal layer CD4+ T cells were much more frequent in papillomas. CD4+, CD8+, and gamma delta(WC1+) lymphocytes were found in the suprabasal layers of papillomas, but the CD8+ and gamma delta(WC1+) T cells were more numerous and had migrated further into the differentiating keratinocytes of the papilloma fronds than the CD4+ T cells. We conclude that T-cell infiltration is characteristic of regressing BPV-4 papillomas, that CD4+ lymphocytes are specifically and massively recruited into the regressing papillomas, and that although all three lymphocyte subsets can penetrate the papilloma, only the CD8+ and gamma delta(WC1+) lymphocytes are able to migrate into the fronds. These results suggest that all three lymphocyte subsets have an important role to fulfill during natural regression of papillomas.     

Expression of retinoblastoma gene product in respiratory epithelium and sinonasal neoplasms: relationship with p16 and cyclin D1 expression

Transition from G1 to S phase of the cell cycle is mediated by interactions between the Retinoblastoma gene product (pRb), p16, and cyclin D1. To determine the expression of these proteins in the sinonasal mucosa immunohistochemistry was carried out on archived tissue sections from 46 patients (37 men, 9 women, age range 17 to 82 years, median 55 years). Nuclear immunostaining for these proteins was assessed and the expression rates (percentages of immunoreactive nuclei) in normal respiratory epithelium, inverted sinonasal papillomas, cylindrical (oncocytic) sinonasal papillomas, and squamous cell carcinomas were compared. Normal respiratory epithelium showed significantly higher pRb expression in surface cells compared to basal cells (p < 0.05). In contrast, abundant pRb expression in surface and basal cells was detected in columnar differentiation in sinonasal papillomas and adjacent mucosa. Cuboidal and squamous metaplasia in inverted papillomas showed significantly reduced pRb expression in surface cells compared to columnar epithelium in inverted papillomas (p < 0.05, respectively). Expression of p16 was detected in all epithelial cell layers of normal respiratory epithelium, sinonasal papillomas, and adjacent mucosa. Cuboidal and squamous metaplasia in inverted papillomas showed increased p16 expression in surface cells compared to columnar epithelium in inverted papillomas (p < 0.05 between squamous metaplasia and columnar epithelium). Sinonasal squamous cell carcinomas showed the coexpression of pRb and p16. Expression rates of cyclin D1 higher than 10% were detected only in invasive carcinomas but not in carcinoma in situ, sinonasal papillomas or respiratory epithelium. Conclusively, pRb expression accompanies terminal differentiation in columnar surface cells. Expression of pRb in proliferating basal cells is present in sinonasal papillomas and adjacent mucosa but not in normal respiratory epithelium. Cuboidal and squamous metaplasia in inverted papillomas involves downregulation of pRb expression along with increased p16 expression in surface cells. Sinonasal squamous cell carcinomas coexpress pRb and p16. Overexpression of cyclin D1 in sinonasal lesions is confined to invasive squamous cell carcinomas.     

Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion

The alpha 6/beta 4 complex is a member of the integrin family of adhesion receptors. It is found on a variety of epithelial cell types, but is most strongly expressed on stratified squamous epithelia. Fluorescent antibody staining of human epidermis suggests that the beta 4 subunit is strongly localized to the basal region showing a similar distribution to that of the 230-kD bullous pemphigoid antigen. The alpha 6 subunit is also strongly localized to the basal region but in addition is present over the entire surfaces of basal cells and some cells in the immediate suprabasal region. By contrast staining for beta 1, alpha 2, and alpha 3 subunits was very weak basally, but strong on all other surfaces of basal epidermal cells. These results suggest that different integrin complexes play differing roles in cell-cell and cell-matrix adhesion in the epidermis. Immunoelectron microscopy showed that the alpha 6/beta 4 complex at the basal epidermal surface is strongly localized to hemidesmosomes. This result provides the first well-characterized monoclonal antibody markers for hemidesmosomes and suggests that the alpha 6/beta 4 complex plays a major role in epidermal cell-basement membrane adhesion. We suggest that the cytoplasmic domains of these transmembrane glycoproteins may contribute to the structure of hemidesmosomal plaques. Immunoultrastructural localization of the BP antigen suggests that it may be involved in bridging between hemidesmosomal plaques and keratin intermediate filaments of the cytoskeleton.     

EEDA: a protein associated with an early stage of stratified epithelial differentiation

Using suppressive subtractive hybridization, we have identified a novel gene, which we named early epithelial differentiation associated (EEDA), which is uniquely associated with an early stage of stratified epithelial differentiation. In epidermis, esophageal epithelium, and tongue epithelium, EEDA mRNA, and antigen was abundant in suprabasal cells, but was barely detectable in more differentiated cells. Consistent with the limbal location of corneal epithelial stem cells, EEDA was expressed in basal corneal epithelial cells that are out of the stem cell compartment, as well as the suprabasal corneal epithelial cells. The strongest EEDA expression occurred in suprabasal precortical cells of mouse, bovine, and human anagen follicles. Developmental studies showed that the appearance of EEDA in embryonic mouse epidermis (E 15.5) coincided with morphological keratinization. Interestingly, EEDA expression is turned off when epithelia were perturbed by wounding and by cultivation under both low and high Ca2+ conditions. Our results indicate that EEDA is involved in the early stages of normal epithelial differentiation, and that EEDA is important for the "normal" differentiation pathway in a wide range of stratified epithelia.     

Characterization of a monoclonal antibody, OVB1, which binds to a unique determinant in human ovarian carcinomas and myeloid cells

A monoclonal antibody, OVB1, was generated against a human ovarian carcinoma cell line, OVCAR-3. The antigen reacting with this antibody was strongly expressed on the external surface of the plasma membrane of OVCAR-3 cells and cells of 4/4 other ovarian carcinoma lines. Variable density and homogeneity of expression was found on cells from 5/5 breast carcinoma lines. Various ovarian tumor specimens and normal human tissues were frozen, cryostat-sectioned, and examined for OVB1 reactivity using immunoperoxidase methods. A strong, uniform, homogeneous reaction on 10/10 ovarian carcinoma specimens and variable, non-homogeneous reactions on breast tumors were seen. Normal tissues reacting with the antibody include thyroid, pituitary pars intermedia, breast ductal epithelium, Auerbach's plexus and neuronal processes in the GI tract, colonic mucosal epithelium, and salivary gland ductal epithelium. Polymorphonuclear leukocytes, eosinophils, and approximately 13% of peripheral lymphocytes, as well as cells around germinal centers in lymph nodes and spleen, showed strong reactivity by immunofluorescence and/or immunoperoxidase. Expression of the OVB1 antigen in the myeloid cells of normal human bone marrow occurred from the promyelocyte stage through to more mature cells in a subpopulation of myeloblasts. Indirect immunofluorescence of live peripheral blood cells showed localization to the surface of PMNs, eosinophils, and certain lymphocytes. Double-immunofluorescence studies (with a direct fluorescein-anti-lactoferrin antibody conjugate) showed co-localization of OVB1 and OKM1 (anti-C3bi receptor) antibodies to specific granules of PMNs. Localization of OVB1 and OKM1 antibodies to granular structures in the PMN was confirmed by electron microscopy using the ferritin bridge technique. The antigen reacting with the OVB1 antibody was shown to be neuraminidase sensitive, but protease insensitive. The OVB1 monoclonal antibody may be useful in identification of ovarian tumors and subclassification of myeloid leukemias.     

Macrophage-specific RAM11 monoclonal antibody cross-reacts with basal cells of stratified squamous epithelia

RAM11 is a mouse monoclonal anti-rabbit macrophage antibody recognizing connective tissue and vascular (atheromatous tissue) macrophages. This study demonstrates a cross-reaction of RAM11 with an unknown antigen in rabbit normal epithelial cells. Formalin-fixed, paraffin sections of the New Zealand White rabbit normal skin, oral mucosa, esophagus, small intestine and lung were immunostained with RAM11 antibody followed by goat anti-mouse Cy-3-conjugated antiglobulin. RAM11-positive immunofluorescence was observed in basal layer cells of stratified squamous epithelia (skin, oral mucosa, esophagus). No RAM11 immunostaining was found in any cells of simple (intestinal, bronchial) epithelia. These findings show that basal cells of stratified squamous keratinized and non-keratinized epithelia of the rabbit express an antigenic epitope which is common with that of macrophage antigen recognized by RAM11 monoclonal antibody.     

Concurrent expression of basal and luminal epithelial markers in cultures of normal human breast analyzed using monoclonal antibodies

The aim of this study was to investigate the retention in culture of the antigens characteristic of the two mammary epithelial subclasses, basal and luminal epithelium. Primary and secondary cultures of normal human mammary-gland cells were used for immunolocalization experiments with monoclonal antibodies to luminal and basal epithelium. In contrast to the in vivo situation, in which reactivity was only seen in basal cells that were negative for the luminal antigen, we found the homogeneous expression of the basal marker by all of the cultured cells at second passage, and the simultaneous expression of the luminal marker by some of these cells. Characterization of the basal antigen expressed in culture using sodium-dodecyl-sulfate/polyacrylamide gel electrophoresis and immunoblotting techniques showed it to be a 51-kilodalton keratin peptide with an isoelectric pH of 5.4, and confirmed its similarity to the antigen expressed in vivo. Our findings thus demonstrated the coordinate expression of the basal and luminal antigens in cells cultured on solid substrates. The availability of monoclonal antibodies to epithelial-subclass-specific markers of the human mammary gland now makes it feasible to search for culture conditions that would allow the maintenance and manipulation of cell differentiation in vitro.     

Suprabasal expression of a 64-kilodalton keratin (no. 3) in developing human corneal epithelium

We have previously shown that a basic 64-kilodalton (no. 3 in the catalog of Moll et al.) and an acidic 55-kilodalton (no. 12) keratin are characteristic of suprabasal cell layers in cultured rabbit corneal epithelial colonies, and therefore may be regarded as markers for an advanced stage of corneal epithelial differentiation. Moreover, using an AE5 mouse monoclonal antibody, we showed that the 64-kilodalton keratin marker is expressed suprabasally in limbal epithelium but uniformly (basal layer included) in central corneal epithelium, suggesting that corneal basal cells are in a more differentiated state than limbal basal cells. In conjunction with previous data implicating the centripetal migration of corneal epithelial cells, our data support a model of corneal epithelial maturation in which corneal epithelial stem cells are located in the limbus, the transitional zone between the cornea and conjunctiva. In the present study, we analyzed the expression of the 64-kilodalton keratin in developing human corneal epithelium by immunohistochemical staining. At 8 weeks of gestation, the presumptive corneal epithelium is composed of a single layer of cuboidal cells with an overlying periderm; neither of these cell layers is AE5 positive. At 12-13 weeks of gestation, some superficial cells of the three- to four-layered epithelium become AE5 positive, providing the earliest sign of overt corneal epithelial differentiation. At 36 weeks, although the epithelium is morphologically mature (four to six layers), AE5 produces a suprabasal staining pattern, this being in contrast to the adult epithelium which exhibits uniform staining.(ABSTRACT TRUNCATED AT 250 WORDS)     

The intermediate filament system of the keratinizing mouse forestomach epithelium: Coexpression of keratins of internal squamous epithelia and of epidermal keratins in differentiating cells

Summary The internal epithelium of mouse forestomach represents a fully keratinized tissue that has many morphological aspects in common with the integumental epidermis. In the present study we have, therefore, analyzed keratin expression in the total epithelium, in subfractions of basal cells and in living and dead suprabasal cells that were obtained by Percoll density gradient centrifugation of trypsin-dissociated forestomach keratinocytes. The keratin analysis revealed that basal forestomach keratinocytes synthesize the same keratin types as basal epidermal cells (60 000, 52 000 and 47 000 daltons), whereas differentiating cells contain both the epidermal suprabasal keratin pair (67 000 and 59 000 daltons) and the suprabasal keratin pair characteristic for other internal squamous epithelia (57 000 and 47 000 daltons). Indirect immunofluorescence using an antibody recognizing the members of the epidermal-type suprabasal keratin pair and in-situ-hybridization experiments using specific cDNA probes for the members of the internal-type keratin pair showed that the two keratin pairs are uniformly coexpressed in living suprabasal forestomach keratinocytes. Furthermore, it could be shown that distinct cells in the basal cell layer acquire the ability to express both the 67 000/59 000 dalton and the 57 000/47 000 dalton keratin pair and that some basal cells apparently lose the ability to synthesize mRNAs for basal keratins.     

A human monoclonal antibody directed to blood group i antigen: heterohybridoma between human lymphocytes from regional lymph nodes of a lung cancer patient and mouse myeloma

A fusion of human lymphocytes released from regional lymph nodes of papillary adenocarcinoma of lung cancer with mouse myeloma P3-X63-Ag8-U1 cells resulted in a stable hybridoma-secreting human IgM antibody (NCC-1004) that reacts with a large proportion of squamous cell carcinomas of lung and esophagus as well as carcinoma of thyroid glands. However, the antibody also reacts with normal red blood cells, B lymphocytes, and a few other limited loci in normal tissues such as the basal cells of bronchial epithelium and the basal cell layer of stratified squamous epithelium, as well as endothelium and alveolar lining epithelium. The antigen defined by NCC-1004 has been characterized as blood group i antigen on the basis of the following results. The antibody preferentially agglutinates cord erythrocytes in contrast to adult erythrocytes. The agglutination was obvious at 4 degrees C, but diminished greatly at 37 degrees C, and was enhanced after sialidase treatment. The antibody specifically reacts with lacto-norhexaosylceramide (nLc6) and sialosyllacto-norhexaosylceramide (IV3NeuAcnLc6), but does not react with lacto-neotetraosylceramide (nLc4), sialosyllacto-neotetraosylceramide (IV3NeuAcnLc4), lacto-isooctaosylceramide (IV6Gal beta 1----4GlcNAcnLc6; I antigen), and other standard glycolipids so far tested. The properties of the antibody and its antigen are identical to those previously described for the i blood group system. Inasmuch as the hybridoma was established by hybridization of lymphocytes derived from regional lymph nodes of lung cancer, and the antigen was found in the patient's lung cancer tissue, the i antigen in lung cancer is probably recognized as a tumor-associated antigen by the host's immune cell system.     

Immunological and histochemical analysis of regional variations of epidermal Langerhans cells in normal human skin

Summary Epidermal Langerhans' cells (LC) were enumerated in normal human skin from various anatomical sites using a monoclonal antibody (NA1/34) to human thymocyte antigen (HTA-1) and the standard ATPase reaction on frozen sections. The same population of cells was identified with each technique. LC densities were found to be significantly higher in hair bearing skin than in skin from the palm and sole. LC were also identified in hair follicles (where the numbers decreased from the superficial to the deep portions) and sebaceous glands but in no other adnexal structure. Normal numbers were encountered in patients who had received radiotherapy or systemic chemotherapy for malignant disease for periods of greater than two months before death. As LC are important antigen presenting cells, the variation in their density suggests that the immunological properties of normal skin may not be uniform throughout the body. This may be related to the varying anatomical distribution of some skin disorders with an immunological basis.