A method of preparing blood leucocytes for flow cytometry which prevents upregulation of leucocyte integrins.

LeuCAM (CD11/CD18) cell-surface antigens are easily upregulated on cell manipulation ex vivo. A procedure for preparing leucocytes, in which human blood is immediately treated ex vivo with buffered formaldehyde and then the erythrocytes and platelets are removed by lysis and differential centrifugation, has been successfully applied to the analysis of LeuCAM antigen expression by flow cytometry. We show that the increased expression of monocyte CD11/CD18, which occurs when mononuclear leucocytes are separated by a standard Lymphoprep density gradient separation, can be avoided if cells are fixed immediately. Following this fixation polymorphs are unable to upregulate CD11/CD18 in response to fMLP stimulation in vitro. The technique produces lymphocyte, polymorph and monocyte populations that can be clearly defined on the basis of forward scatter and side scatter, and preserves the expression of various surface antigens; the percentages of gated lymphocytes expressing CD3, CD4, and CD8 were similar to those obtained using a commercial fixing and lysis solution. The processing does not render cells permeable to antibodies, as evidenced by our failure to stain cells with antibodies to intracellular antigens. We believed the method to be useful for measuring CD11/CD18 expression on blood leucocytes from normal or pathological specimens and to have application to the measurement of other cells surface antigens which may also be upregulated by the separation procedures.

Isolation and characterisation of equine dendritic cells.

Despite their important role in initiating T-cell responses in other species, dendritic cells have not been studied in the horse. A method for isolating blood dendritic cells by adherence and metrizamide gradients was adapted to equine cells. A number of monoclonal antibodies (mAbs), including some which label dendritic cells in other species, were tested for immunochemical reactivity with the isolated blood dendritic cells, and sections of lymph node and spleen. 62 +/- 6% of the isolated blood cells were MHC Class II positive and had typical dendritic cell morphology and only 4 +/- 2% contained non-specific esterase, a marker of mature macrophages. These dendritic cells also expressed MHC Class I, LFA-1, EqWC1 and EqWC2. Amongst the potentially cross-reactive antibodies a mAb against bovine CD1b was the most interesting by staining lymph node, but not blood, dendritic cells. Monoclonal antibodies against equine CD5 (T-cells), surface immunoglobulin (B-cells) and macrophages (CZ2.2) were used to enumerate the contaminating cells in preparations from blood by flow cytometry. 39 +/- 7% of the cells did not express T and B cell markers or CZ2.2 but were large and MHC Class II positive. Comparison of immuno-chemistry and flow data, together with examination of alveolar macrophages and adhered blood cells, all support the view that CZ2.2 detects a myeloid marker not seen on mature macrophages and possibly shared with dendritic cell precursors. The functional capacity of the isolates was assessed in terms of their stimulating ability in the mixed leukocyte reaction (MLR). Dendritic cell enriched isolates were more potent stimulators of MLRs than peripheral blood mononuclear cells or adherent cells. Thus equine dendritic cells isolated from blood express high levels of MHC Class I and II and LFA-1 and stimulate a vigorous MLR. They do not express markers characterising T and B cells but, by virtue of expression of the equine macrophage marker CZ2.2, appear closely related to mononuclear phagocytes.

The anti-human CD21 antibody, BU33, identifies equine B cells.

The number of antibodies for identifying equine B cells is small and the number that react with well-defined epitopes even smaller. The monoclonal antibody, BU33, which is directed against human CD21 (Complement Receptor 2; CR2) was shown to identify (1) follicular lymphocytes in the lymph nodes and spleen of three horses, and (2) a mean of 18 +/- 6% (SEM) of peripheral blood lymphocytes from 10 horses. These findings indicate that the antibody identifies equine B cells and possibly equine CR2 or a related molecule. This study adds to the reagents available for equine research and diagnostic pathology.

Equine macrophage identification with an antibody (Ki-M6) to human CD68 and a new monoclonal antibody (JB10).

Monoclonal antibodies (mAbs) recognizing equine macrophages are scarce. The present study compared the immunocytochemical staining of various equine tissues (lymphoid tissue, lung, liver, small intestine, skin and blood leucocytes) by an antibody, Ki-M6, which detects CD68 in human macrophages and dendritic cells, and by a new anti-equine mAb, JB10, with staining produced by two previously described anti-equine macrophage mAbs, CZ2.2 and CZ3.3. Ki-M6 was shown to identify equine macrophages, which had a distribution different from those identified by CZ2.2 and CZ3.3. JB10 identified equine macrophages with a distribution similar to those identified by Ki-M6, but additionally bound to polymorphonuclear leucocytes. Flow cytometry of peripheral blood leucocyte subpopulations and tissue immunocytochemistry were used to compare staining by JB10 with that of CZ2.2 and CVS19; the latter identifies the myeloid antigen, EqCD13, found on polymorphonuclear leucocytes. The staining by JB10 differed from that of both CZ2.2 and CVS19, suggesting that JB10 detects a different molecule. These additional mAbs should prove useful for the future study of new, defined, populations of macrophages in equine immune responses and pathology, and, in the case of Ki-M6 antibody, may make possible an analysis of the structure, distribution and function of the CD68 molecule in the horse.

Antigen-presenting capacity in normal human dermis is mainly subserved by CD1a+ cells.

A proposed role for antigen-presenting dermal dendrocytes in the pathogenesis of many dermal inflammatory skin diseases remains speculative. We therefore sought to determine the phenotype and functional characteristics of antigen-presenting cells isolated from normal human dermis. Normal adult human skin was incubated overnight with dispase at 4 degrees C, the epidermis was removed, and the residual dermal preparation was then minced and digested with a mixture of hyaluronidase, collagenase, and DNAase at 37 degrees C, prior to filtration through mesh. Dermal cell suspensions thus obtained were stained using specific monoclonal antibodies, and analysed by fluorescence microscopy or flow cytometry. Mean values were as follows: CD45+ leucocytes 39%, HLA-DR+ cells 39%, Ulex europaeus agglutinin I+ endothelial cells 26%, CD1a+ cells 3.9%, CD11b+ cells 16%, CD11c+ cells 6%. Mitomycin C-treated crude dermal cell suspensions induced allostimulation of peripheral blood mononuclear cells in a 7-day culture, as assessed by 3H-TdR incorporation. Depletion of CD1a+ Langerhans-like cells from the dermal cell preparation, by 95, 74 and 90% in three separate experiments using immunomagnetic beads, reduced 3H-TdR incorporation at optimal responder-to-stimulator cell ratios by 90, 64, and 87%, respectively. Our findings suggest that, in normal human dermis, the great majority of the alloantigen-presenting capacity resides in the CD1a+ Langerhans cell-like dendritic antigen-presenting cell population, and not to any great extent in either CD1a- macrophage-like cells, or HLA-DR+ endothelial cells. The relationship of the CD1a+ dermal antigen-presenting cells to the Langerhans cell lineage remains to be determined.