of Surgery, Sinai Hospital of Baltimore,


21215; and Johns Hopkins Medical Institutions,




Presented at the Annual Meeting of the Association for Academic Surgery, Louisville, Kentucky, November l&18,1989

cytotoxic subset actually enhances wound healing [3]. In order to ascertain the identity of the lymphocyte subset whose depletion results in impaired wound healing, we examined the effect of simultaneous depletion of Thelper/effecter and T-suppressor/cytotoxic lymphocytes on fibroplasia and compared it with all T-lymphocyte depletion.

T-cell depletion leads to impaired wound healing. We studied the effect of combined T-helper and T-suppressor lymphocyte depletion on wound healing and compared it with the effect of all T-cell depletion. Groups of 10 male balb/c mice, 8 weeks old, underwent a 2.5~cm skin incision and subcutaneous implantation of polyvinyl alcohol sponges. Twenty-four hours prior to wounding one group was treated with 30H12, a rat anti-mouse monoclonal antibody against the Thy-l.2 antigen present on all Tcells (1 mg); another group received 1 mg each of GK1.5 (anti-L3T4, CD4; anti-helper/effecter subset) and 2.43 (anti-Lyt 2.1, CDS; anti-suppressor/cytotoxic subset). All monoclonal antibodies are cytotoxic in viuo. Controls received 1 mg of nonspecific rat IgG. Treatments were repeated weekly. Animals were sacrificed at 2 and 4 weeks postwounding. Equal depletion of all T- and Th- and Tssubsets in peripheral blood and spleens was noted in the two experimental groups at sacrifice. Depleting Thy-l.2 cells (all T-cells) impaired wound healing as assessed by wound breaking strength and collagen synthesis. Combined anti-T-helper/effecter and T-suppressor/cytotoxic depletion resulted in improved wound-healing parameters. This suggests that there is a Thy-1.2+, L3T4-, Lyt2subpopulation of T lymphocytes which normally stimulates wound healing. o 1990 Academic press, IW.


Wounding Wounding was performed under pentobarbital anesthesia (4 mg/kg BW intraperitoneal). The backs of the mice were shaved and cleansed with an organic iodine solution, and a 2.5-cm midline dorsal skin incision was made down to the level of the dorsal fascia. Two subcutaneous pockets were created at the upper poles of the wound and preweighted, sterile, saline-moistened polyvinyl alcohol sponges were inserted (Unipoint Industries, Unipoint, NC). The wounds were then closed with running 5-O nylon sutures. All sutures were removed after 2 weeks. Generation of Mabs

Supported by NIH Grant lR29GM38650 r To whom correspondence and reprint requests should be addressed at Department of Surgery, Sinai Hospital, 2401 W. Belvedere Avenue, Baltimore, MD 21215 460 Inc. reserved.


Male balb/c mice, 7 to 8 weeks old, were obtained from a commercial breeder (Harlan Sprague-Dawley, Indianapolis, IN). The animals were housed five/cage, kept at a constant temperature and humidity, and exposed to a 12-hr light/dark cycle. The mice were fed a pelleted diet (Teklad LM 485 Mouse/Rat Diet, Winfield, IA) which supports normal growth, reproduction and longevity, and they drank tap water. Both food and water were offered ad Zibitum. All animals were acclimatized to our laboratory conditions for at least 1 week prior to use in the experiments. The mice were weighed at the start of the experiments and then weekly, until sacrifice.

The importance of lymphocytes in the intricate process of wound healing has begun to be elucidated only in the past few years. The specific role played by lymphocytes in the healing process has yet to be delineated fully. Lymphokines, secretory products of activated lymphocytes, influence in vitro fibroblast replication, migration, and collagen synthesis [l]. In uiuo depletion of T-lymphocytes by specific monoclonal antibodies (Mab) results in impaired wound healing, as measured by wound breaking strength and collagen synthesis [2]. However, depletion of the T-helper/effecter lymphocyte subset has no effect on the healing wound while depletion of the T-suppressor/

0022-4804/90 $1.50 Copyright 0 1990 by Academic Press, All rights of reproduction in any form


In oiuo T-lymphocyte depletion was achieved by the administration of highly specific Mabs. In order to deplete all T-cells we used the 30H12 Mab which binds to the Thy-l.2 antigenic determinant found on all T-lymphocytes [4]. T-helper/effecter lymphocytes were depleted




TABLE Percentage








in Viuo Cytotoxic



Gk1.5 + 2.43

30H12 Thyl.2 Blood Spleen

2 4 2 4

week week week week

74.7 79.5 75.9 66.7



f 6.2 ? 14.8 f 8.3 f 13.2

61.8 76.3 74.7 51.6

Note. Mean f SEM of four separate determinations

+ f ?I +


14.6 16.8 11.7 10.7

76.3 50.3 80.2 71.1


+ 4 f 8.8 _+ 1.2 f 8.9

84.9 92.9 89.3 91.0

(see text for details on calculating

using the GK1.5 Mab which reacts with the L3T4 (CD4) antigenic determinant [5], while Mab 2.43, which reacts against the Lyt2 antigen found on suppressor/cytotoxic T-cells (CD8) was used to deplete this subset [6]. These three rat anti-mouse Mabs are of the IgG2s subtype and, therefore, are cytotoxic in viva The hybridoma cells (American Type Culture Collection, Rockville, MD) were grown in culture and then injected intraperitoneally into Pristane-primed male nude (nu/nu) balb/c mice. Ascites fluids containing the Mabs were precipitated with 50% saturated ammonium sulfate, resuspended, and extensively dialyzed against PBS. Mab concentrations were estimated from the total protein concentration by immune electrophoresis. Nonspecific rat immunoglobulins were obtained by treating rat serum similar to the ascites fluid.

+ f f f

L3T4 3.7 2.8 3.5 3.5

94.5 82.8 81.7 96.1

f 2.5 f 5.8 + 8.3 _+ 1.2

LYQ 88.6 50.4 81.9 95.8

k + + *

4.6 4.6 5.2 1.3

percentage depletion).

pooled and the mononuclear cells were isolated by density centrifugation over Lympholyte M (Cedar Lane Labs, Hornby, Ontario). Spleens from two to three animals were also pooled, minced, treated with ammonium chloride (0.83%) to lyse red blood cells, and then were macrophagedepleted by Petri dish adherence. The resulting blood and spleen mononuclear cells were stained with fluorescein (FIT0labeled 30H12 (anti-Thy 1.2, against all T-cells), phycoerythrin-labeled GK1.5 (anti-L3T4; CD4) against helper/effecter T-cells, and FITC-labeled anti-Lyt 2(CD8) against suppressor/cytotoxic cells (all from Beckton Dickinson, Mountain View, CA). Percentage staining was determined in a fluorescence-activated cell sorter (EPICS, Coulter Corp., Hialeah, FL) and these values were used to calculate the depletion of T-cells and subsets in treated mice [7] using the formula:

Depletion of T-Lymphocytes Mice were treated the day before wounding and once a week thereafter until sacrifice. Twenty mice were injected intraperitoneally with 30H12 at a dose of -1 mg/mouse; 20 mice received combined treatments with Gk1.5 and 2.43, each at a dose of 1 mg/mouse; controls were injected 1 mg/mouse of rat serum immunoglobin. Ten mice in each group were sacrificed at 2 and 4 weeks postwounding, respectively. The efficacy of the Mab treatments was assessed by determining the depletion of the T-lymphocytes and their subsets in the spleen and the peripheral blood. At sacrifice heparinized cardiac blood from two or three mice was

U-D T 100 - D = i% ’ where U = percentage of cells in untreated mice, T = percentage of cells in treated mice, and D = percentage of T-cell depleted in treated mice as a percentage of total lymphocytes in untreated mice. The values for U and T were determined by fluorescence analysis and were used to calculated D, which in turn is employed to calculate the percentage of subset depletion according to the equation




% depletion


of Mice Treated



Wound breaking strength (g f SEM) Time of sacrifice


2 weeks 4 weeks

311 -c 15 443 f 19

* P = 0.001 vs controls;

= D/U X 100.

Antibodies Hydroxyproline (@g/100 g sponge f SEM)


Gk1.5 + 2.43



Gk1.5 + 2.43

235 f 13* 377 f 17*

394 + 27*-# 505 f 22**#

1692 f 111 3679 f 164

969 f 86* 2722 f 121*

2271 f Sl*,# 4380 IL 91*,#

#P = 0.001 vs 30H12; all by ANOVA.





Assessment of Wound Healing At sacrifice, the dorsal pelt containing the healing scar was removed and cut into two to three strips on a multibladed guillotine, each strip being centered by an equal segment of the scar. The strips were kept in saline solution for less than 60 min prior to measurement of wound breaking strength using a constant speed tensiometer. The inplanted sponges were harvested, cleared of all surrounding fibrous tissue, and frozen at -26°C for subsequent determination of hydroxyproline content by the method of Woessner [8]. The hydroxyproline content is used as an index of reparative collagen synthesis at the wound site. Analysis of Data All data are reported as Means f SEM. Analysis of variance tests were applied for comparisons between groups using the StatView II statistical package (Abacus Concepts, Berkeley, CA) on a Macintosh II computer. Statistical significance was achieved at a P -c 0.05. RESULTS

The animals gained weight equally in all groups (data not shown). There were no wound infections or other complications. The 30H12 antibody, whether administered for 2 or 4 weeks, effectively depleted peripheral blood and spleen Thy-1.2+ lymphocytes and also helper/effecter and suppressor/cytotoxic T-cells (Table 1). Similarly, simultaneous administration of Gk1.5 and 2.43 caused depletion of T-helper/effecter and suppressor/cytotoxic cells. The combined monoclonal antibody treatment (Gk1.5 + 2.43) was more effective than 30H12 treatment in depleting the Thy-1.2+ population (all T-cells) (Table 1). Depletion of T-lymphocytes by the administration of 30H12 markedly impaired wound healing, as assessed by both the wound breaking strength and the hydroxyproline accumulation in subcutaneously implanted polyvinyl alcohol sponges (Table 2). The impairment in wound healing was noted both at 2 and 4 weeks postwounding. By contrast, combined administration of Gk1.5 and 2.43 resulted in statistically significant enhancement of the wound healing parameters both at 2 and 4 weeks postwounding (Table 2). DISCUSSION

The data from these experiments confirm previous findings that depletion of Thy-1.2+ lymphocytes profoundly impairs wound healing, as assessed by wound breaking strength and collagen synthesis [2]. However, simultaneous depletion of T-helper/effecter and T-suppressor/cytotoxic T-cells leads to enhancement of woundhealing parameters, similar to depletion of the suppressor/ cytotoxic subset alone [3]. These findings support the hypothesis that T-cells play a major role in modulating

VOL. 48, NO. 5, MAY


wound healing. The different effects of the two monoclonal treatments on wound healing cannot be explained on the basis of differential effects in depleting T-lymphocytes or subsets. The depletion data (Table 1) clearly shows that the 30H12 and the combined Gk1.5 + 2.43 treatments led to equal depletion of all T-cells and subsets in the peripheral blood and spleens. Therefore, three must be smaller subpopulations which are differentially affected by the treatments and yet these differences may account for the widely divergent effects on wound healing parameters. There is a major question which thus far remains unanswered and which cannot be answered on the basis of these experiments. What is the nature of Thy-1.2+ cell subpopulation which is depleted by treatment with the 30H12 Mab leading to impaired wound healing, but which must be L3T4-, LytB- since it is not depleted by simultaneous administration of specific Mabs. The combined Mab administration depletes peripheral blood and spleen T-lymphocytes , but clearly it is not depleting a subset which is sensitive, however, to the anti Thy-l.2 (30H12) treatment. This Thy-1.2+, L3T4-, Lyt2- cell population obviously has a major stimulatory role in wound repair since its depletion causes marked perturbations in the repair process. Several cells, other than lymphocytes, bear the Thy-l antigen and potentially could be affected by the 30H12 treatments. Murine fibroblast cell lines express the Thy1 antigen and, in the case of embryonic fibroblasts, antiThy-l serum is cytotoxic in vitro [9]. There is no evidence that the 30H12 Mab is cytotoxic in viva against fibroblasts. Furthermore, administration of 30H12 in nude mice does not result in impaired wound healing, suggesting that the antibody does not affect fibroblasts [lo]. Another Thy-1.2+ element is the dendritic epidermal cell. This bone marrow derived cell population is Thy-l+, Ia-, L3T4-, Lyt-2- [ll, 121 and proliferates in response to Tcell mitogens and IL 2 [13]. These cells are immunologically active and influence the activity of other dermal elements, namely keratinocytes, which participate in epidermal healing. We are now in the process of addressing the issue of whether the epidermal dendritic cells and their activity are affected by in vivo administration of 30H12. In conclusion, wound healing is strongly regulated by immune lymphocytes. Positive regulation of wound healing is carried out by a Thy-l+ cell population whose identity remains to be clarified. REFERENCES 1.


Barbul, A. Immune regulation of wound healing. In E. Faist, D. R. Green, and J. Ninnemann (Eds.), Immune Consequences of Trauma, Shock and Sepsis. Berlin: Springer-Verlag, 1989. Pp 339349. Peterson, J. M., Barbul, A., Breslin, R. J., Wasserkrug, H. L., and Efron, G. Significance of T lymphocytes in wound healing. Surgery





3. Barbul, A., Breslin, R. J., Woodyard, J. P., Wasserkrug, H. L., and Efron, G. The effect of in vivo T helper and T suppressor lymphocyte depletion on wound healing. Ann. Surg. 209: 479, 1989. 4. Ledbetter, J. A., and Herzenberg, L. A. Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens. Zmmunol. Rev. 47: 63,1979. 5. Wilde, D. B., Marrack, P., Kappler, J., Dyalinas, D. P., and Fitch, F. W. Evidence implicating L3T4 in class II MHC antigen reactivity; monoclonal antibody GK1.5 (anti-LBT4a) blocks class II MHC antigen-specific proliferation, release of lymphokines and binding of cloned murine helper T lymphocyte lines. J. Zmmunol.





Woessner, J. The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch. Biochem. Biaphys. 93: 440,196l.


Stern, P. L. 0 alloantigen (London) 246: 76, 1973.

on mouse and rat fibroblasts.



Barbul, A., Shawe, T., Rotter, S. M., Efron, J. E., Wasserkrug, H. L., and Badawy, S. B. Wound healing in nude mice: A study on the regulatory role of lymphocytes in fibroplasia. Surgery 105:


Bergstresser, P. R., Tigelaar, R. E., and Streilein, J. W. Thy-l antigen-bearing dendritic cells in murine epidermis are derived from bone marrow precursors. J. Znuest. Dermatol. 83: 83, 1984.


Takashima, A., Nixon-Fulton, J. L., Bergstresser, P. R., and Tigelaar, R. E. Thy-l+ dendritic epidermal cells in mice: Precursor frequency analysis and cloning of concanavalin A-reactive cells. J. Invest. Dermatol. 90: 671, 1988.


Nixon-Fulton, J. L., Bergstresser, P. R., and Tigelaar, R. E. Thyl+ epidermal cells proliferate in response to concanavalin A and interleukin 2. J. Zmmunol. 136: 2776, 1986.


131:2178,1983. 6. Sarmiento, M., Dyalinas, D. P., Lancki, D. W., Wall, K. A., Lorber, M. I., Loken, M. R., and Fitch, F. W. Cloned T lymphocytes and monoclonal antibodies as probes for cell surface molecules active in T cell-mediated cytolysis. Zmmunol. Reu. 68: 135, 1982. Seaman, W. E., Wofsy, D., Greenspan, J. S., and Ledbetter, J. A. Treatment of autoimmune MRL/lpr mice with monoclonal antibody to Thyl.2: a single injection has sustained effects on lymphoproliferation and renal disease. J. Zmmunol. 130: 1713, 1983.


Wound healing and T-lymphocytes.

T-cell depletion leads to impaired wound healing. We studied the effect of combined T-helper and T-suppressor lymphocyte depletion on wound healing an...
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