Analysis of different protein kinase C-dependent events in T cells from allogeneic bone marrow transplantation recipients.
In order to understand the mechanisms underlying the T lymphocyte dysfunction associated to allogeneic bone marrow transplantation (BMT), we assessed two different protein kinase C (PKC) dependent events in T cells from BMT recipients: the PKC-dependent membrane expression and function of the CD69 early activation antigen; and the rapid phorbol ester-induced phosphorylation of PKC protein substrates in lysates from T cells permeabilized with digitonin, in the presence of (gamma-32P)ATP. Most BMT recipient T cells detectably expressed the CD69 surface antigen after 24 h of stimulation with either phorbol 12-myristate 13-acetate (PMA) or anti-CD3 MoAb and PMA, thus indicating that PKC activity is sufficient to induce de novo gene expression. Nevertheless, it is noteworthy that the fluorescent staining intensity with anti-CD69 MoAbs was significantly lower in BMT recipient T cells than in normal T lymphocytes, although no clear-cut correlation was found between the expression of CD69 and the proliferative capacity. However, the pattern of PMA-induced phosphoproteins analysed as early as 1 min after PKC activation in T cells from BMT recipients displaying a low response to mitogenic stimuli, was undistinguishable from that detected in T cells from healthy subjects. In all cases a major 110-kD phosphoprotein was observed, which was inducible with PMA, phorbol 12,13-dibutyrate (PDBu), 1-oleoyl-2-acetylglycerol (OAG) and a phorbol-ester-related activator of PKC (mezerein); moreover, its phosphorylation was blocked by pretreating cells with the PKC inhibitor H-7. Altogether our results suggest that the depressed mitogenic responses, which were also observed in the present study when T cells were stimulated via CD69, cannot be simply attributed to a defective PKC activity.
Balboa
M A
MA
Department of Immunology, Hospital de la Princesa, Universidad Autónoma, Madrid, Spain.
Izquierdo
M
M
Sánchez-Madrid
F
F
Fernández-Rañada
J M
JM
López-Botet
M
M
D015703:Antigens, CD / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism; D016026:Bone Marrow Transplantation / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D037181:Lectins, C-Type; D008213:Lymphocyte Activation / Q000276:immunology; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013601:T-Lymphocytes / Q000201:enzymology*; D014184:Transplantation, Homologous; D000099192:CD69 Antigens
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D037181:Lectins, C-Type; D010750:Phosphoproteins; D011493:Protein Kinase C; D000099192:CD69 Antigens