Expression Of CD68 Antigen In Human Skin

Objective1. To observe the distribution, morphology and density of dermal mono-cytes/macrophages.2. To determine the CD68 and CDla positive cells within normal human epidermis and their relationship.3. To observe the distribution, morphology and density of dermal mono-cytes/macrophages and study the relationship between dermal monocytes/macro-phages and dermal dendritic cells4. To study the effects of UPS and some cytokine such as IFN -γ, IL - 6 and TNF - a on CD68 production and expression of human keratinocyte cell line, HaCaT cells.Methods1. Samples1.1 Normal skin from 6 locations such as the face, trunk, proximal limbs, distal limbs, and palms and soles of 8 subjects were included in the study. The horizontal and longitudinal sections of the skin were stained with an ABC immu-noperoxidase procedure with anti - CDla and CD68 monoclonal antibodies. Skin samples from 8 healthy subjects were collected. Epidermal sheets and skin sections of these samples were stained with CDla and CD68 monoclonals by an ABC immunoperoxidase procedure. The density and the location of these stained cells were determined.1.2 HaCaT cells is a gift of Free University of Berlin.2. Methods:2. 1 Immunoperoxidase2.1. 1 A three - step immunoperoxidase ( LAB - SA, Labeled[9trept] Avidin- Biotin) technique was used for staining with anti - CD68 and CD la mAb, bi-otin - conjugated goat anti mouse IgG, and horseradish - peroxidase - labeled pronase avidin in all cryostat - cut sections from normal skin.2.1. 2 Epidermal sheets and skin sections of these samples were stained with CD la and CD68 monoclonals by an ABC immunoperoxidase technique.2.2 Double - labeling immunofluorescenceCryostat sections were prepared and double staining was performed with a multistep immunofluorescence technique. Two different first monoclonal antibodies were monoclonal mouse anti - CD68 IgGl and FITC - conjugated mouse anti- human CDlaIgG2a. Rhodamine -labeled goat anti - mouse IgGl was used as secondary antibody.2. 3 Image analysisNormal skin from distal limbs of 8 subjects were included in the study. The longitudinal sections of the skin were stained with an ABC immunoperoxidase procedure with anti - CD68 monoclonal antibodies. Quantitate the dermal CD68 positive monocytes/macrophages with the technique of image analysis ( American VIC Metamorph Imaging System V4. 6 image analysis software).2.4 Flow cytometry analysisCells (106 cells/1 ml) were incubated with 20ul of R - PE labbed CD68 mAB on ice for 30m. Cells washed, twice with PBS. Thereafter, cells were ac-qucied and analyzed on a FACSCalibur. Cells were acquired and gated by FSC and SSC.2.5 Semi - quantity reverse transcriptase - polymerase chain reaction ( RT -PCR)2.5.1 Extraction of RNA from HaCaT cellsTotal tissues RNA were extracted from HaCaT cells by the Trizol kit (A-merican Invitrogen)2.5.2 Identification the concentration, purity and integrity of total RNA The optical densities (OD) of RNA was measured by violet - spectropho-tometer at 260nm and 280nm. Then the concentration and purity of total RNA were determined. 1.5% agarose formaldehyde electrophoresis was used to evaluate the integrity of total RNA.2.5.3 RT-PCREvery sample was amplified by CD68 and (3 - actin primers under certain conditions. The products were 262bp and 340bp respectively.3. Results judgement3. 1 Enumeration methodsThree sections of each skin biopsy were examined and only dendritic cells exhibiting a brightly stained cytoplasm with cell body were counted. The density of positive - staining cell in the epidermis was then calculated as follows, positive - staining cell /mm length of epidermal surface and ( or) positive - staining cell /mm of epidermis ( positive - staining cells were counted on 20 consecutive fields at 400 magnification) , measured by micrometer.3.2 Results of double - labeling immunofluoresceneeEpidermal cells with dendritic shape (red) were determinated as CD68 -positive ( CD68 + ) cells. Epidermal cells with dendritic shape (green) were confirmed as CDla positive ( CD1 a + ) Langerhans cells.3. 3 Detection of PCR productsThe amplication products were electrophoresised in 1.5% gel and scaned by ultraviolet image system. The results were analyzed by FluorChem V 2.0 microsoft (America). The gray value of every target DNA ratio to the gray value of its control |3 - actin ( e/c) were confirmed as the level of mRNA.Result1. The CD68 positive cells were detected within the superficial dermis with variable densities; distal limbs 562 ±230/mm2, trunk 517 ± 162/mm2, face 509 ±235/mm\ palms 507 ±192/mm2, proximal limbs, 472 ± 138/mm2, and soles 361 ±78/mm . Lower densities of CD68 positive dendritic cells were found in the deep( reticular) dermis, dispersed between collagen bundles. There are 3 types of CD68 positive cells within the dermis; strongly CD68 positive cells ofdendritic morphology, weakly CD68 positive cells of dendritic morphology, and CD68 positive cells of non - dendritic morphology. In the epidermal sheets, there were less regional variation for CD68 positive cells than for CDla in the 8 cases examined; palm: 310 ±97/mm2(CD68)/ 184 ±92 /mm2(CDla) , sole; 234 ± 112/mm2 ( CD68 )/252 ± 104/mm2 ( CDla) , face; 650 ± 249/mm2 (CD68) / 530 ± 216/mm2, trunk: 586 ± 21 I/mm2 ( CD68) / 809 ± 204/mm2 (CDla) , proximal limb; 637 ± 248/mm2 ( CD68)/ 574 ± 285/mm2 ( CDla) , distal limb: 524 ± 147/mm2 ( CD68) / 621 ± 235/mm2 ( CD1 a). In the skin sections , the majority of CDla positive dendritic cells were situated in the mid - epidermis, while only a proportion of CD68 positive cells were of the dendritic morphology, and were situated mainly in the mid - and lower epidermis. There were also some CD68 positive keratinocytes with nuclear and cytoplasmic staining pattern.2. 1 The majority of CDla positive dendritic cells were situated in the mid - epidermis in the skin sections, while CD68 positive cells were less dendritic in morphology, and were situated mainly in the mid - and lower epidermis. In the epidermal sheets, the densities of CDla and CD68 positive cells in the palm and sole were low; ranging from 184 -252 /mm2 and 234 -310/mm2respectively, There are more CDla and CD68 positive cells in the face, trunk and limb; the density are 530 -809/mm2 and 524 -637/mm2.2. 2. The CDla and CD68 double - labelled dendritic cells were mainly situated in the mid - epidermis. There were lowest number of CDla and CD68 double - labelled positive cells in the palm and sole, The liner density of double-labelled positive cells was less than half of those of CDla or CD68 positive cells(28.6% -49.8%).3.1 The reticulate of CD68 positive cells were detected within the superficial dermis.3.2 There are some dendritic FXIIIa positive cells surrounding the append-ant organ and blood vessel.3.3 The result of image analysis; the number of strongly CD68 positive cells of dendritic cells are higher than the number of non dendritic CD68 positive cells within the dermis; strongly CD68 positive cells of dendritic morphology,...