Cellular analysis of functional mononuclear cells from chronically inflamed gingival tissue

Functional mononuclear cells from chronically inflamed gingiva of different stages of adult periodontitis (AP) were isolated by using a novel enzymatic dissociation method and extensively characterized at the single cell level. Fresh tissues obtained following surgery were cut into 1-2 mm3 pieces, washed free of residual blood cells and dissociated with the neutral protease enzyme Dispase. Mononuclear cells were then obtained by separation on a mini-Ficoll-Hypaque gradient. This procedure yields an average of 2 x 10(6) cells/400 mg of tissue. The viability of unfractionated cells immediately after enzymatic dissociation was greater than 94%, and after Ficoll-Hypaque gradient separation this was greater than 99%. The relative percentages of lymphocytes, plasma cells, monocytes (MN)/macrophages (M phi) and granulocytes were determined on cytospin preparations by Wright's-Giemsa staining. Cell differential analysis showed that lymphocytes and MN/M phi predominated at all stages of disease, while the frequency of plasma cells increased with the severity of disease. Numbers of plasma cells specific for IgG, IgA, or IgM were determined by immunofluorescence using fluoresceinated anti-heavy chain specific antibodies. The majority of Ig-containing cells were of the IgG isotype, followed by significant numbers of IgA and few IgM-positive cells. IgG, IgA, and IgM secreting cells were also determined by the ELISPOT assay. Total numbers of spot forming cells (SFC) were measured in both the moderate and advanced stages of disease, and the major SFC isotype was IgG followed by IgA; essentially no IgM SFC were detected. Furthermore, higher numbers of IgG and IgA SFC were noted in the advanced stage when compared with the moderate stage of disease.(ABSTRACT TRUNCATED AT 250 WORDS)