The effect of in vivo T helper and T suppressor lymphocyte depletion on wound healing.

The role of T lymphocytes in wound healing is still not well-defined. Because it had been previously shown that in vivo depletion of T cells leads to impaired wound healing, the effect of depleting T cell subsets on subsequent fibroplasia was studied. T helper/effector cells were depleted by the use of the monoclonal antibody GK1.5, reactive against the L3T4 antigen (CD4). T suppressor/cytotoxic lymphocytes were depleted by using the 2.43 monoclonal antibody reactive against the Lyt 2 antigen (CD8). In the first experiment, Balb/c mice were treated with the antibodies starting at 24 hours before wounding was performed, and weekly thereafter. Depletion of the T helper/effector cells had no effect on wound-breaking strength or hydroxyproline deposition in sponge granulomas, whereas depletion of T suppressor/cytotoxic cells significantly enhanced both of these healing parameters. In a second experiment, T cell subset depletion was started on Days 0, 3, 7, 10, and 14 postwounding, and treatments were continued weekly thereafter. Once again, depletion of T helper/effector cells had no effect on wound healing, whereas depletion of T suppressor/cytotoxic cells markedly increased both wound-breaking strength and collagen synthesis. In conclusion, the data show that T suppressor/cytotoxic cells have a counter-regulatory role in wound healing, whereas the T cell subset responsible for up-regulating wound healing remains to be identified.

Stimulation of T cell immunity by arginine enhances survival in peritonitis.

T cell-mediated immunity may play a role in host responses to infection. Arginine is a known thymic and T cell stimulator which enhances host allogenic, mitogenic, and anti-tumor responses. We, therefore, examined the effect of arginine on the survival of rats with severe and lethal peritonitis induced by cecal ligation and double-needle puncture (CLP). In Experiment 1, arginine HCl (100 mg) was given bid by gavage starting immediately after CLP. In Experiment 2, the same dose of arginine was given by gavage bid for 3 days pre-CLP and continued thereafter. In Experiment 3, arginine was administered iv post-CLP (100 mg tid). Arginine had no effect on overall survival in Experiment 1. In Experiments 2 and 3, arginine therapy significantly increased survival at all times. A separate experiment was carried out to determine the reason for the differential response to arginine administered via gavage or iv post-CLP (Experiments 1 and 3). Nonseptic rats showed a 400% increase in plasma arginine 30 min after gavage with 100 mg arginine (P less than 0.001). No rise in plasma arginine was noted when arginine was administered by gavage post-CLP. The impaired intestinal absorption or markedly increased utilization of arginine in this septic model may explain why no improved survival was seen in Experiment 1. The mechanism for the improved survival with arginine therapy seen in Experiments 2 and 3 may be related to its known thymic and T cell immunostimulatory effects.

Suppressor cell generation during normal wound healing.

We have previously shown that 10 day healing wounds in rats contain wound mononuclear cells (WMNC) which inhibit normal lymphocyte mitogenic and allogeneic responses. In the present study we sought to further characterize the WMNC and define their mechanism of action. Polyvinyl alcohol sponges implanted in wounds were harvested and processed 10 days postwounding. The resultant WMNC suspension contained less than 15% macrophages. By FACS analysis, 69.5 +/- 11.4% (mean +/- SD of eight separate experiments) of the cells expressed the all T cell marker (W3/13), while 47.7 +/- 11.9% stained with the T helper/effector marker (W3/25) and 49.5 +/- 18.8% expressed the T suppressor/cytotoxic phenotype (OX8) (Th/Ts ratio = 0.96 +/- 0.13). When various numbers of WMNC were cocultured with 5 X 10(5) PHA-stimulated rat thymic lymphocytes, as few as 500 WMNC inhibit normal blastogenesis. Long-term (72 or 144 hr) culture of WMNC revealed that they maintain their suppressive activity. Furthermore, the conditioned media of long-term cultures also significantly suppressed thymic lymphocyte PHA blastogenesis, suggesting that the WMNC secrete suppressive cytokines. Large doses of human recombinant IL-2 or indomethacin did not abrogate the inhibitory effect of WMNC. We conclude that the healing wound is normally infiltrated by suppressor lymphocytes which generate immune inhibitory cytokines.

Generation of an anti-interleukin 2 factor in healing wounds.

Previously we have noted that fluid obtained from ten-day-old healing wounds noncytotoxically inhibits the blastogenesis of lymphocytes in response to mitogens or antigens. Since these lymphocytic responses are interleukin 2 (IL-2)-mediated, we looked for a specific IL-2 inhibitor in wound fluid. We have found that wound fluid blocks the response of thymic lymphocytes and of two cloned T-helper cell lines (D10 and HT2) to exogenous human recombinant IL-2. The wound fluid enhances fibroblast proliferation, thus demonstrating that its proliferative inhibitory activity is specific for lymphocytes. The findings suggest that wound fluid contains a factor that impairs lymphocyte response to IL-2, probably at the receptor or postreceptor level.

Significance of T-lymphocytes in wound healing.

To determine the importance of T-lymphocytes in wound healing, we examined the effect of T-lymphocyte depletion on the healing of surgical wounds. Thirty Balb/c mice were injected intraperitoneally with 1 mg of rat anti-mouse (IgG2b) cytotoxic monoclonal antibody (30H12) against the Thy1.2 (all T) determinant. Twenty-four hours later animals showed a greater than 95% depletion of Thy1.2 cells in peripheral blood and spleen. Thirty control mice received nonspecific rat immunoglobulin (1 mg). Twenty-four hours after treatment mice underwent a 2.5 cm dorsal skin incision with subcutaneous placement of polyvinyl alcohol sponges. Injections were repeated at weekly intervals. Wound healing was assessed at 2, 3, and 4 weeks by the breaking strength of wound strips and by the hydroxyproline content of sponge granulomas (an index of wound reparative collagen deposition). Thy1.2 depletion at death was 95% to 57% in peripheral blood and 86% to 68% in the spleen. Both groups gained weight equally. We found that T cell depletion significantly impairs wound breaking strength and wound collagen deposition at all times studied. The data strongly suggest that T-lymphocytes modulate fibroblast activity during normal wound healing.