BrrtishJournal oJPIasticSurgery (1990). 43,655662 0 1990 The Trustees of British Association of Plastic Surgeons

The role of lymphocytes C. W. MARTIN

in wound healing

and I. F. K. MUIR

Department of Surgery. University Aberdeen Royal Infirmary

of Aberdeen, and Department

of Plastic and Reconstructive

Surgery,

Summary-In the past, lymphocytes have usually been associated with chronic inflammatory conditions and only recently have animal experiments indicated a possible role in wound healing. The present paper describes a study of lymphocytes in human wounds and scars using monoclonal antibody stains. The results suggest that T lymphocytes may play an important regulatory role in wound healing and scar formation. From the surgical viewpoint lymphocytes have usually been associated with chronic granulomatous conditions such as tuberculosis and chronic ulcers rather than with wound healing. It is true that it has long been recognised that mononuclear cells, previously described rather vaguely as “small round stained cells”, can be seen in conventionally microscopic sections of healing wounds, but it was not possible to identify these cells with certainty and their function was unknown. However, two developments during the last 5 years have altered this picture. Firstly, the introduction of monoclonal antibody staining techniques has made it possible not only to identify lymphocytes with certainty but also to recognise sets and subsets of lymphocytes which have different functions. Secondly, cell culture and biochemical studies have identified and characterised chemical messengers secreted by lymphocytes (lymphokines) which have powerful effects on other cells, particularly macrophages and fibroblasts (Tables 1 and 2). Table I

By the use of monoclonal antibody markers, lymphocytes have been subdivided as follows : B lymphocytes-associated with antibody-mediated immunity T lymphocytes-associated with cell-mediated immunity Subsets-T helper cells T suppressor cells NK lymphocytes-natural killer cells This in vitro work has been extended into animal models, and experiments in animals have shown that lymphocytes may have controlling actions in wound healing (Barbul, 1988; Wahl and Allen, 1988). Recently the opportunity arose to study biopsies of healing wounds in humans.

Material and methods During the course of a project to improve the treatment of patients requesting removal of tattoos,

Lymphokines which directly influence fibroblasts Function

Lymphokine

Acronym

Chemotaxis

Lymphocyte-derived chemotactic factor for fibroblasts Fibroblast inhibition (of

LDCF-F

1

FIF

1

movement) Fibroblast

activation

factor (of

y Interferon

Interleukin-1

Collagen production

factor

(Fibroblast) inhibition proliferation) factor

Transforming

Proliferation

growth

factor b

FAF

t

IF

1

T

IFN

t1

1

TGF

11

t

IL-l

t

655

656 Table 2

BRITISH JOURNAL OF PLASTIC SURGERY Lymphokines

which influence macrophages

Macrophage chemotactic factor

MCF

Macrophage inhibition (of movement) factor

MIF

Macrophage activation factor

MAF

Interleukin-2

IL-2

biopsies were taken to establish the depth of the tattoo pigment. If it was subsequently decided that complete excision of the tattoo was the method of choice then the healing biopsy wound became available for study. Normal wounds Eleven biopsy wounds, all on the arms of young women, were re-excised at different times after the original operation ranging from 1 day to 42 days. In addition 10 scars varying in age from 2 months to 25 years which had been excised in the course of operations were available for study. A few specimens of hypertrophic scars also became available. Since surgical treatment is usually avoided during the active phase of hypertrophy, only six scars were available for studies. Thirteen true keloid scars were also studied. Seven were pre-sternal, one in the deltoid region and five from earlobes.

stage alkaline phosphatase anti-alkaline phosphatase (APAAP) method (Fig. 1). This can be used to demonstrate macrophages and lymphocytes and their subsets (Cordell et al., 1984). The primary reagents were mouse monoclonal antibodies (McAb’s) against human antigens. The secondary reagent was rabbit antibody to mouse antibody. The tertiary reagent was the APAAP complex. (Both were obtained from Dakopatts, Cophenhagen, Denmark). Development was carried out with Napthol AS-MX Phosphate and Fast Red TR salt (Sigma Chemical Co. Ltd) for lo15 minutes, and nuclei were counterstained with haematoxylin (Fig. 1). Any endogenous alkaline phosphatase would give false positive staining and therefore its activity was inhibited by levamisole. The antibodies used are shown in Table 3.

Histological technique All biopsies were frozen immediately after removal and all histochemical staining was done using the APAAP technique.

Evaluation Because of the uneven distribution of lymphocytes in wounds it is difficult to give accurate numbers. It is hoped in the future, by adapting methods used for cell counting in other situations, to achieve more precise measurements but in the present investigation this was not attempted. Therefore, the numbers of lymphocytes were assessed on the arbitrary scale of O-10 (0= no lymphocytes; 10 = very dense infiltrate of lymphocytes). In fact this relatively crude method of assessment showed remarkably consistent variation with regard to time.

Zmmunocytochemistry The method requires a 3-stage procedure-the

Normal skin-controls (Fig. 2) The samples were initially examined using anti-leu

3-

APAAP COMPLEX

Fig. 1 Figure l-Schematic

model of monoclonal antibody technique.

THE ROLE OF LYMPHOCYTES

IN WOUND

657

HEALING

Table 3 Antibody Anti-leu

14

Dilution

Cells stained

I:10

B cells

Anti-leu 4

1:loo

All T cells/pan

Anti-leu

2a

I:10

T suppressor

Anti-leu

3a

I:10

T helper cells

Anti-IL-2 receptor Anti-leu

MS

1:50

IL-2 receptors

I : 10

Macrophages

CD (cell determinant)

number

indicates

CD ciassijicution

Sourre

22

Becton Dickinson

T

3

Becton Dickinson

cells

8

Becton Dickinson

4

Becton Dickinson

25

Dakopatts

Dakopatts

surface

14 (B lymphocyte marker). No B lymphocytes were seen. With the Pan T cell marker only few T lymphocytes were seen in the dermis of all samples of normal skin. These lymphocytes were almost always associated with hair follicles, sebaceous glands and sweat glands or with blood vessels in the upper dermis. None were observed free within the connective tissue of the dermis, or in the epidermis. The majority of lymphocytes were T helper cells. Macrophages were observed in small numbers in some of the biopsies from normal skin, but in most biopsies none were seen. Normal wound healing Results with B lymphocyte stain The first two scars in the series were treated with anti-leu 14, but no B lymphocytes were seen. This antibody was therefore not used on any further sections. All subsequent remarks refer to T lymphocytes. Results with Pan T cell marker The one-day wound (Fig. 3) shows evidence of increased numbers of T lymphocytes around the vessels in the normal undamaged dermis and a small number within the wound itself. At 3 days T lymphocytes are seen within the wound in greater numbers suggesting that the cells are actively migrating into the wound. There is a progressive increase in T lymphocyte numbers in the wound until day 8 (Fig. 4). Intense activity at the wound edge and nearby surrounding normal dermis can be seen in both 7- and 8-day-old wounds. At day 14 lymphocyte numbers are decreasing. The peak in lymphocyte numbers within the wound is therefore between 8 days and 14 days. Examination of the Cweek-old wound (Fig. 5) still shows evidence of increased T lymphocyte

antigen

numbers in the dermis asstiiated with the scar tissue, and in the scar itself. These numbers are obviously less than the young wound but slightly greater than those seen in normal forearm skin. The lymphocytes associated with the scar seem to be present within the new connective tissue. At 2 months, lymphocytes are still present but are unevenly distributed, being concentrated at the edge of the scar. By about 4 months, lymphocyte numbers are still dropping and by 8 months the scar appears to contain fewer lymphocytes than the nearby normal skin, probably because there are no accessory skin structures in the scar, and as mentioned before, the lymphocytes seen in normal skin are associated with these structures. At 20 and 25 years no lymphocytes are seen within the scar, which is by this stage impossible to distinguish microscopically from normal skin. The changes are summarised in Table 4. Results with subset markers When looking at T lymphocyte subsets the enumeration problem becomes more difficult. When serial sections stained by T helper and T suppressor stains are compared, all that can be said is that the majority of lymphocytes observed in the above changes are T helper cells (Figs 6 and 7). Results with Interleukin-2 receptor McAb Interleukin-2 (IL-2) is pivotal for the multiplication of activated T cell subsets. It only acts on cells which express IL-2 receptors. These receptors are not present on resting T cells and the genes for IL-2 receptor are only expressed transiently after activation (Roit, 1988). Therefore even in an active situation only a few cells will be seen expressing receptors at any one time. In the 3-day-old wound Interleukin-2 receptor (IL-2-R) expression was observed in only one T cell; in the 7-day-old wound two cells were seen.

658

BRITISH JOURNAL

Fig. 2

Fig. 3

Fig. 4

Fig. 5

OF PLASTIC SURGERY

Figure t-Normal skin. Pan T cell marker. Few lymphocytes seen round blood vessels and sebaceous glands ( x 40). Figure SOneday-old wound. Pan T cell marker. Lymphocytes are seen within granulation tissue of wound ( x 100). Figure 4.-Eight-day-old wound. Pan T cell marker ( x 40). Figure 5.-Four-week-old wound. Pan T cell marker ( x 40).

The majority of cells did not express IL-2-R. In the one-day scar and scars older than 8 days, there was no evidence of IL-2-R expression on any T lymphocytes. Macrophages

Anti-leu MS was used to detect macrophages. The results showed they were present within the wound from a very early stage (1 day) and peaked in numbers between 3 and 6 days. Macrophages were seen in greater numbers than T lymphocytes at the wound edge in the l-day-old wound, and at 3 days there were more macrophages present within the wound. By 7 days, T cell numbers at the wound edge were far greater than macrophages.

Hypertrophic scars

The few hypertrophic scars were stained for B lymphocytes and none were observed. Pan T cell staining showed a heavy infiltrate of T cells throughout the whole depth of the 4-month and Smonth-old scars. The T lymphocytes seen in the 4-month, 5-month and 9-month-old scars were both scattered diffusely throughout the scar tissue and arranged perivascularly. At 9 months, lymphocyte numbers were decreasing and by 14 months T lymphocytes were only noted perivascularly. In the 2-year-old scar, which is less vascular, fewer lymphocytes were present than in the 14-month-old scar. The results for hypertrophic scars are summarised in Table 5.

THE ROLE OF LYMPHOCYTES

IN WOUND

Table 4 Numbers of lymphocytes staining with the Pan T cell McAb in normal wounds and scars Edge of wound or scar Age of normal scar I day 3 days 5 days 7 days 8 days 14 days 14 days 28 days 28 days 35 days 42 days 2 months 4 months 6 months 8 months 1 year 2 years 3 years 3 years 20 years 25 years

Upper

Lower

Main body of wound or scar

I 2 2 2 2 2 2 3 2 3 2

1 2 2 5 5 3 2 I I

1 2 2 3 4 3 3 2 2

1

1

0 1 1

I 1 0 1 0 I 0 0 0 0 0

2 2

I

0

1 2 1 1 1 0 0

0 0 I 0 0 0 0

659

HEALING

Nearby normal dermis 3 2 3 4 4 3 2 2 1 2 1 1 2 1 2 1 I 1 2 1 1

Keloid scars (Table 6) Scars were initially stained for B lymphocytes but all staining was negative and was therefore discontinued. In contrast to the pattern in normal skin and normal scars, the keloid scars showed much greater T lymphocyte presence. Maximum concentration was seen in the upper dermis of all six earlobe keloids and to a lesser extent in sternal keloids. All keloids, over a wide range of age of scar, showed lymphocytes scattered diffusely throughout the scar tissue. This contrasts with the appearance of normal scars in which lymphocytes were seen in the actual scar only until about 4-5 weeks, after which they decreased rapidly in numbers. Four biopsies taken from the active invading edge of the keloid scars showed much greater concentration of T lymphocytes than three biopsies taken from the static centre of such scars and a striking feature was the concentration of lymphocytes round hair follicles at this invading edge (Fig. 9).

Discussion Figures represent an arbitrary scale f&l0 (0 = no lymphocytes; IO= very dense T cell infiltrate).

Macrophages

The pattern of macrophage involvement followed closely the T lymphocyte pattern although absolute numbers were not as large. Maximum numbers of macrophages were seen in the 4- and 5-month-old scars. Interleukin-2 receptor

A few T lymphocytes were seen to express IL-2-R in the 4- and 5-month-old scars in contrast to none seen in the older scars. Table 5

Lymphocytes in hypertrophic scars Pan T stain

Age ofsrar

EpidermB

Upper scar

Lower scar

4 months 5 months 9months 12 months 14months 2 years

I

5

5

0 0 0 0 0

5 4 3 3 2

5 3 2 2 1

Figures represent an arbitrary scale O-10 (O=no IO= very dense T cell infiltrate).

lymphocytes;

Lymphocyte participation in wound healing

It has long been known that lymphocytes are present in healing wounds (Ross and Benditt, 1969) but their function has been unknown. Monoclonal antibodies have made it possible to identify the subsets of lymphocytes involved in wound healing. The investigations described in this paper have shown the changes in lymphocyte numbers in relation to time in normal wound healing and abnormal scars. It now seems almost certain that lymphocytes do have an important function in normal wound healing and that aberrations of lymphocyte function may in some way be responsible for the abnormal behaviour of some scars. The number of T lymphocytes seen in normal skin was small, and those observed were associated with accessory skin structures. Previous work on rats suggests that lymphocytes do not appear in wounds until as late as 2 days (Castor, 1981), reach peak numbers 7 days postwounding (Fishel et al., 1983) and then progressively fall. No information is available for wounds older than a few weeks. In human wounds studied in this project, a different sequence was seen. Lymphocytes were present within the wound at one day, increased to peak numbers between days 8 and 14 post-wounding and remained present, although

BRITISH JOURNAL OF PLASTIC SURGERY

Fig. 6

i

x

t

’ I

Fig. 8

.

Fig. 9

Figure 6-Sevenday-old wound. T suppressor marker ( x 40). Figure ‘I-Seven-day-old wound. T helper marker ( x 40). Arrows show lymphocytes at wound margin. Note: Figs 6 and 7 are consecutive sections. Figure &Sternal keloid. Pan T cell marker ( x 40). Figure !%-Sternal keloid showing invasion of hair follicle by T helper lymphocytes. T helper marker ( x 100).

in decreasing numbers, in the young scar probably for as long as 4 months. Barbul (1988) presented evidence that lymphocytes are required for optimal wound healing in rats and it may be that lymphocytes are important regulators of fibroblaSt activity both directly and indirectly through the intermediary of macrophages during wound healing. Previous ideas have concentrated on the macrophage as the only cellular inducer of fibroblast activity, but it is now clear that there is a close inter-relationship between the different cells in the wound, in particular macrophages, lymphocytes and fibroblasts. Figure 10 shows the possible lymphocyte macrophage-fibroblast interactions. The direct sequence “Phase 1 factors (i.e. factors produced during the

phase of inflammation)-macrophage-fibroblast” is shown to the left of the diagram. On the right of the diagram the various lymphokines which mediate the interactions between macrophages, lymphocytes and fibroblasts are shown. The probabihty exists that in normal wound healing lymphocytes are involved in the regulation of fibroblast function and the balance of lymphokines is carefully controlled. In hypertrophic and keloid scars, perhaps the continued presence of lymphocytes and an imbalance of these lymphokines is the cause of the excessive fibrosis. The observations described here show that in abnormal scars all the cells shown in Figure 10 (macrophages, T lymphocytes and fibroblasts) persist long after the time when in normal wounds

661

THE ROLE OF LYMPHOCYTES IN WOUND HEALING

Table 6 Keloid scars. Numbers of lymphocytes staining with the Pan T cell McAb Age of lesion (YrJ

Nature of sample

7

Sternal-active

9

Sternal-active edge Sternal-active edge Sternal-static centre Sternal-static centre Sternal-static centre Sternal keloid -active edge -static centre Earlobe Earlobe Earlobe Earlobe Earlobe Deltoid keloid -active edge -static centre

10

IO mo. 4 7 5

4 mo. 2 3 5 10 4

Epidermis

edge

09

1

(B) (B) (B) (B) (B) (El

2

1 1 1 0 2 1

(El (E) (E) (El (El (Ef

1 2 2 2 2

I 0

Superficial scar

Deep scar

3 2

2

5 2 3 3

3 1 2 1

4 3 3 7 I 6 9

3 3 10 4 3 3 4

3 3

3 2

I

B = biopsy; E =excised. Figures represent an arbitrary scale 0- 10 (0 = no lymphocytes; 10 = dense cell infiltrate)

they have become inactive. It is known that in some other chronic fibrotic conditions (such as progressive systemic sclerosis and liver cirrhosis) lymphocytes participate in fibroblast stimulation and multiply in response to an antigenic stimulus. Is it possible that persistent activity of the lymphocytes in abnormal scars is also due to an antigenic stimulus? Both keratin (Mowlem, 1951) and sebum (Yagi et al., 1979) have been suggested as possible antigens. Mowlem (1951) suggested that both hypertrophic and keloid scars are initiated by keratin fragments in the dermis and mentioned specifically hair PHASE 1 PRODueTS

FiBROBLAST

iGFB u I

Fig. 10 Figure l&Flow factors responsible.

diagram

showing

cellular

interactions

and

follicles which became buried in the scar. The keratin of the hairs acted as an antigen and became surrounded by “small round cells” (which were presumably lymphocytes). These observations would fit in with modem immunological theory to provide a hypothesis for the occurrence of keloid scars, if keratin could be shown to be the longacting antigen which activates the T lymphocytes seen in these scars. Mowlem’s theory is a good hypothesis to explain keloid scars. It could be that the antigenic stimulus (i.e. keratin) persists indefinitely in these scars because the scar tissue progressively invades normal tissue and the histological sections show that hair follicles are infiltrated by lymphocytes (Fig. 9). In this way, the antigen is always present in the scar edge. This hypothesis is less credible in explaining the occurrence of hypertrophic scars and in particular the observation that hypertrophic scars show a definite limited period of activity followed invariably by regression. The work of Gillman and his colleagues (1955) suggests a more credible hypothesis for hypertrophic scarring. They showed that epithelial remnants could be trapped in the dermis by surgical incision. They considered that these induce an inflammatory response (rather than an antigen induced response) which subsides as the

BRITISH JOURNAL OF PLASTIC SURGERY

662 keratin is removed by macrophages. As the inflammation subsides, the lymphocyte production of lymphokines ceases and, without further stimulation, the fibroblast activity declines and the scar ultimately regresses. Why apparently similar epithelial remnants should provoke these different reactions is not known. Conclusions

The conclusions of this project are that T lymphocytes have an important role in both normal wound healing and in the genesis of abnormal scars. That there is a complicated interaction between macrophages, lymphocytes and fibroblasts is now well known and, by the secretion of lymphokines, T lymphocytes can exert a regulatory role on wound fibrosis. Although not yet proven, it is becoming more likely that abberations of lymphocyte function may be responsible for abnormal scarring. The value of this expanded hypothesis is that it begins to indicate the points at which the clinician can most usefully intervene to prevent or diminish harmful scarring. For instance, the use of cyclosporin to inhibit T helper cell function in organ transplantation is now well established. If T helper cell overactivity is responsible for continued fibroblast stimulation then T helper cell inhibition by cyclosporin or other compounds with similar properties, which are now under investigation, might be a practical possibility for control of excess scar formation in the future. Acknowledgements We are grateful to Professor 0. Eremin, Regius Professor of Surgery, and Professor F. Walker, Regius Professor of Pathology, in whose departments the laboratory work was carried out. We also thank Dr Angus Thomson, Department of Pathology, for his help and advice and Mr C. R. W. Rayner and Mr 0. M. Fenton for their co-operation.

Wound Healing.

Biological

and Clinical Applications.

New

York: Alan R. Liss Inc. Castor, C. W. (1981). Autacoid regulation of wound healing. In Glynn, L. E. (Ed.) Handbook of Inflammation. Volume 3. Tissue Repair and Regeneration. Amsterdam, New York, Oxford: Elsevier/North Holland Bio Medical Press. Co&II, J. L., FaIini, B., Erber, W. N., Ghosh, A. K., Ahdul-Aziz, Z., McDonald, S., F’ulford, K. A. F., Stein, H. and Masson, D. Y. (1984). Immunoenzymatic labelling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complex). Journal of Histochemistry and Cytochemistry, 31,219. Fishel, R. S., Barb& A., Wasserkrug, H. C., Penberthy, L. T., Rettura, G. and Efron, G. (1983). Cyclosporin A impairs wound healing in rats. Journalof Surgical Research, 34,512. Gillman, T., Penn, J., Bronks, P. and Roux, M. (1955). A reexamination of certain aspects of the histogenesis of the healing of cutaneous wounds. British Journal of Surgery, 43, 141.

Mowlem, R. (I 95 1). Hypertrophic scars. British Journal of Plastic Surgery, 4, 113.

Roit, I. (1988). The acquired immune response. In Essential Immunology. Oxford, London, Edinburgh, Boston : Blackwell Scientific Publications. Ross, R. and Bend&, E. P. (1969). Wound healing and collagen formation. Fine structure in experimental surgery. Journal of Cell Biology, 40, 1042.

Wahl. S. M. and AIlen. J. B. (1988). T-lvmohocvte-deoendent mechanism of fibrosis. In Growth bactois dnd O>her Aspects of Wound

Healing.

Biological

and Clinical Applications.

New

York: Alan R. Loss Inc. Yagi, K. I., Dafalla, A. A. and Osman, A. A. (1979). Does an immune reaction to sebum in wounds cause keloid scars? Beneficial effect of desensitisation. British Journal of Plastic Surgery, 32,223.

The Authors C. W. Martin, B.Med.Biol., now 3rd year medical student, University of Aberdeen. I. F. K. Muir, MBE, MS, FRCSEng, FRCSEd, Honorary Research Fellow and Honorary Consultant in Plastic Surgery. Department of Surgery, University of Aberdeen, and Department of Plastic and Reconstructive Surgery, Aberdeen Royal Infirmary. Requests for reprints to: Mr I. F. K. Muir, Department Surgery,

University

Medical

References

AB9 2ZD.

Barbul, A. (1988). Role of the T cell dependent immune system in wound healing. In Growth Factors and Other Aspects of

Paper received 22 May 1990. Accepted 12 June 1990.

Buildings,

Foresterhill,

Aberdeen,

of