Lymph flow and changes in intracellular enzymes during healing and rejection of rabbit skin grafts

1. Autografts and homografts of full thickness skin were made on a hind limb of rabbits. During the following days the appearance and histological changes of the grafts were studied; the lymph flow from the limb, and the enzyme activities in the supernatant and cell pellet of the lymph after centrifugation were determined, as well as the enzyme activities in the graft roof and the underlying host tissue. It was further examined whether a lymphatic and vascular connexion occurred between graft and host tissue.2. During the first 5 days the grafts changed from pale blue to bright pink, became swollen, soft and had a mild cellular inflammatory exudate. Autografts then became pale, took on the appearance of normal skin with the inflammatory changes subsiding, whereas homografts became firm, showed heavy mononuclear cell infiltration, had a blotchy purple appearance due to thrombosis and haemorrhage, developed widespread necrosis and changed into a black hard scab which was eventually shed. With high dose homografts (6-8 grafts) these changes occurred 1-2 days earlier than with low dose (2-4) grafts.3. The flow of lymph increased during the first 5 days after grafting, then returned to normal with autografts but remained increased with homografts.4. In the supernatant of the lymph the activities of LDH and beta-glucuronidase did not change during the first 5 days but activities of cathepsin, acid phosphatase, GOT and GPT increased. With the autografts the increase in the activities of these four enzymes then subsided, but with the homografts they increased further and there was an increase in the activities of LDH and beta-glucuronidase, even greater than in those of the other four enzymes.5. In the cell pellets of the lymph the activities of the six enzymes did not increase during the first 5 days; with homografts, but not with autografts, they then increased. These increases occurred even though the cell count in the pellet remained unchanged. Thus some of the lymphocytes must have become ;activated' to contain higher enzyme activities.6. The enzyme activities in the roof tissue did not parallel those in lymph. They did not change during the first three days. During the following three days the activities of acid phosphatase, LDH, beta-glucuronidase and cathepsin increased, but not those of GOT and GPT which remained low. From then onwards the behaviour was different with auto- and homografts. With autografts only the activity of acid phosphatase continued to increase, those of LDH, beta-glucuronidase and cathepsin decreased and those of GOT and GPT remained low. With homografts the activities of LDH, beta-glucuronidase and cathepsin continued to increase and became even greater than in the supernatant of lymph, whereas the activities of acid phosphatase, GOT and GPT, remained low.7. In the bed tissue the activities of all six enzymes increased during the first 3 days after grafting, then the activities of GOT and GPT returned towards normal but those of the other four increased further. The only difference between auto- and homografts was that the increase in beta-glucuronidase and LDH activity was much greater with homografts.8. Lymph drainage became established with autografts on day 5 or 6 and then persisted. With homografts the dosage of grafts influenced the result. With low dosage (2-4 grafts) lymph drainage became established in a small percentage of the experiments, also on day 5 or 6, but it persisted for 2-3 days only. With high dosage, no lymph drainage became established. However, when the onset of rejection was delayed by treatment with cyclophosphamide lymph drainage became established also with high dosage homografts.9. Vascularization of the grafts was established on day 3 or 4, and persisted in autografts. In homografts a vascular shut down occurred at about the time of onset of rejection. It therefore occurred later with low than with high dosage and with high dosage on treatment with cyclophosphamide.10. It is concluded that the absence of lymph drainage from homografts is the cause of the small magnitude of increases in enzyme activities of lymph collected during and after their rejection. The increase results from ;activated' small lymphocytes which infiltrate the graft bed and junctional tissue and subsequently undergo necrosis, and that the establishment of a lymphatic connexion between the graft and host tissue is not a prerequisite for rejection.