188 Burns (1986) 12,18%192 Printedin Great Britain

Cellular immune mechanisms in thermally injured mice

Sudha Mistry, Nerges F. Mistry, N. H. Antia and Swaran Arora Department of Plastic Surgery, J. J. Hospital, Bombay

Summary An attempt was made to establish an animal model for analysing the cellular immune alterations following thermal injury and to correlate the results with those obtained in humans. A standard burn involving 20 per cent of the body surface area was established in CS7 black mice (bl). The results revealed a loss of total T cells until post-burn day 10 and an increase in B cells only during post-burn day 1. The lymphocyte prolifera- tive response to PHA was elevated with the appearance of null cells on post-burn day 10. This observation revealed a generalized depression in the immune re- sponses which was significant until post-burn day 10. However, the results were not comparable with the human studies.

INTRODUCTION PERTURBATIONS in the cell-mediated status of experimentally burned animals have been well documented (Munster et al., 1972, 1973), show- ing a fall in the number of peripheral and thoracic duct lymphocytes (Casson et al., 1968), a marked depression in the mitogen response and a de- creased cytotoxicity response of the spleen and lymph node cells (Markley et al., 1977; Mistry et al., 1981). These changes have been reported to correspond with an enhanced growth of tumours in burn hosts, for example in rats (Ikeuchi et al., 1981). Although much data has been accumu- lated on the cell-mediated immunity (CMI) status of experimental rats, mice have not been extensively used as a model for understanding the immune response following burns. Since sur- face markers for the lymphocyte subpopulation have been well studied in C57/bl mice, an inbred strain of mice was used to establish an animal model for understanding immunological altera- tions, for example, changes in a lymphocyte pop-

ulation and its mitogenic activity. The humoral response was assessed by studying the levels of B cells and their capacity to form antibodies against sheep red blood cells (SRBC). The results ob- tained were compared with those obtained in human patients.

MATERIALS AND METHODS Twenty-five C57/bl inbred adult mice (aged 8 weeks, weighing 20-30g) were selected for the experimental work. These animals were sub- jected to standard full thickness skin loss burns involving 20 per cent body surface area (BSA) (Walker and Mason, 1968). Intraperitoneal fluid resuscitation produced a survival rate of 85 per cent. These animals were monitored on days 5, lo,21 and 1 month following thermal injury. The results were compared with 10 normal controls chosen from the same age-group. Lymph nodes served as the source of cells for all the experi- ments except the plaque-forming assay, where splenocytes were used.

Preparation of cell suspension Animals were killed by cervical dislocation and the lymph nodes from the brachial and inguinal regions and spleen were excised under sterile conditions. The tissues were maintained at 4°C throughout the experimental procedure. The cells were obtained by gently teasing apart the tissue using toothed forceps. A single-layer cell suspension was obtained by passing the suspen- sion through a sieve funnel. The cells were washed three times in (RPMI) 1640 modified media, counted and their viability checked using trypan blue dye.

Mistry et al.: Cellular immune mechanisms in thermally injured mice 189

RPMI-1640 modified media The RPM1 Media was supplemented with argi- nine (200 mg/l), 4 mmol/ml glutamine, 5 per cent fresh human heat-inactivated sera, 100 ug penicil- lin and 1 ml of streptomycin (10 U/ml) and gen- tamycin (10 ug/ml).

Estimation of T-cell percentage The T-cell percentage was enumerated using anti Thy 1.2 sera (Schlesinger, 1970). Anti sera was obtained by courtesy of Dr N. A. Mitchison (University College, London). In brief, lXIOh cells were suspended in RPMI-1640 modified media and then incubated for 30 min at 37 “C, in 0.5 ml of anti sera (1.3Odil.). Following the incubation the cell pellet was washed three times and incubated with 0.5 ml of guinea-pig comple- ment (1:30dil.) for 30 min at 37°C. The cells were washed again and suspended on in 0.5 ml of RPMI-1640 with 3 drops of 0.25 per cent trypan blue solution and allowed to stand for l&15 min. The T ceils appeared dark blue in colour. A minimum of 200 cells were scored for enumera- tion of total T-ceil percentage.

Quantitation of B cells B cells were enumerated using erythrocyte- activated complement rosette (EAC) technique. In brief, SRBC were activated with antibodies to SRBC (Amboceptor, 1 : 3000 haemolysing titre obtained from Haffkine Institute, Bombay). These sensitized cells were washed three times in RPMI-1640 and resuspended in 0.5 ml of guinea- pig complement (1 : 30 dilution) and further incu- bated at 37°C for 1 h. The cells were washed three times in media and 1~10~ lymphoyctes obtained from the lymph node suspension were added and further incubated for 30 min. The cells were then fixed and enumerated for EAC roset- tes. A minimum of 200 ceils were scored for enumeration of total B-cell percentage.

Lymphocyte blastogenic response The method utilized was as described by Adler et al. (1970). In brief, 1-2~10’ cells were sus- pended in 1 ml of modified RPMI-1640 media and incubated with PHA for 72 h. The dose of PHA added was 20 ug/l X 10’ cells. This dose was found to give optimum stimulation in a dose- response curve (Fig. 1). On day 4 the cultures were pulsed with 1 uCi/ml of ‘H-thymidine (sp. activity 1 uCi/mM) and incubated for a further period of 18 h. The reaction was terminated, the cells were harvested and the radioactivity was

1o.w 20 Ir9 30 Gl 40 F9 PHA P

Fig. 1. Lymphocyte transformation: PHA dose- response curve.

counted on a liquid scintillation counter (Beck- man LS 100). The results were expressed as the stimulation index.

Plaque-forming cells The assay was performed using Cunningham’s (1965) technique. Each mouse was sensitized with 0.1 ml of 5 per cent SRBC given sub- cutaneously at 1 or 24 h or on day 5 or 15 and on day 25 after injury, beginning on day 5 after injection of SRBC. Five animals from each group were killed. Spleen cell suspension was prepared from each mouse and the RBCs were lysed with 0.83 per cent ammonium chloride suspension. The leucocyte pellet was washed three times in RPMI-1640 media and 3000 cells were suspended in 5Oyl of 10 per cent fetal calf serum. To this suspension was added 50 ul of 5% SRBC (indica- tor cells) and 50 u of (1 : 40 dilution) guinea-pig complement. The mixture was incubated at 37”C, and 5 ui was suspended in the chamber which was sealed and incubated for 34 h at 37 “C. Plaques appeared on monolayers and were directly counted under the microscope with a low power objective. The results were expressed as the number of plaques per 1~10~ cells.

Enumeration of null cell percentage This was calculated by subtraction of T+B cell percentage from 100. In control population, %T+B tended towards 100. Statistical analysis was performed for each assay on different days using Student’s unpaired t test.

RESULTS Total T-cell percentage The mean T-ceil value in the control was 70.2 per centk12.18, the levels ranged between 56 per

Burns (1986) Vol. 12/No. 3 190

80

60

_-_----------- normal ranae

0 5 10 21 30 Post-burn days

Fig. 2. Total T-cell percentage

. 7 2

20.

0 5 10 21 30 Post-burn days

Fig. 4. Null-cell percentage.

cent and 89 per cent. After the onset of injury the values significantly (RO.001) decreased until day 10. However, a rising trend was observed on post-burn day 21 (Fig. 2).

B-cell percentage The mean B-cell value in the controls was 21.8 per centk10.09. The levels ranged between 12 per cent and 43 per cent. After the onset of injury, in contrast to T-cells, a significant eleva- tion in the B cells was observed during post-burn day 1 (P<O.OOl). However, the levels fell on post-burn day 5, and on post-burn day 10 the values reverted to within normal range (Fig. 3).

Null-cell percentage In controls the null cells averaged at 7.2 per centk7.34. The values ranged between 0 per cent and 26 per cent. After the onset of injury be- tween time 0 and post-burn day 5, the null-cell percentage showed a rising trend and a maximum elevation was observed on post-burn day 10. However, on day 21, the T- and B-cell percen-

.

t -----------L- 1

d 6 lb il 30 Post-burn days

Fig. 3. Total B-cell percentage.

normal range

. L J

0 5 10 21 30 Post-burn days

Fig. 5. Lymphocyte blastogenic response to PHA (20mg/ml).

tages were within normal limits, thereby reducing the null-cell percentage. At 1 month the null-cell levels were within the normal range (Fig. 4).

Lymphocyte blastogenic response In controls the mean stimulation index obtained was 2.38k1.48. The levels ranged between 1.80 and 4.66. Following burns, a significant increase in the stimulation index was obtained until day 10. By day 21 there was a gradual decrease and at 1 month the levels were within normal range (Fig. 5).

Plaque-forming cells (PFC) In controls, the number of plaques averaged 22.33+4.98/1x10h cells. At the onset of injury until post-burn day 10 a significant reduction (P~O.001) in the number of plaques was observed. This decrease showed a rising trend and at 1 month the levels were within normal range (Fig. 6).

Mistry et al.: Cellular immune mechanisms in thermally injured mice 191

Fig. 6. (2. Plaque-forming cells (10X).

DISCUSSION It is evident from the results that thermal injury induces a marked decrease in the amount of total circulating T cells. This observation corroborates with lymphocytopenia particularly of the T cells in the thoracic duct of rats (Casson et al., 1968). spleen of mice (Markley et al., 1977) and peripheral blood in humans following thermal injury (Neilan and Taddeini, 1977; Antonacci et al.. 1984). Further, significant inhibition in natu- ral killer and antibody-dependent cytotoxicity within 24 to 48 h after major burns is also evident (Antonacci et al., 1983). In our study T-cell mitogenic response to PHA was augmented. Similar observations were reported by Daniels et al. (lY70). Leguit et al. (1973) and Mahler and Batchelor (1971). This converse association of low total T cells and an increase in PHA response could probably mean an increase in blastoid lym- phocytes stimulated by substances released fol- lowing injury (Ikeuchi et al., 1981). These observations also suggest that T-cell immuno- regulatory mechanisms are sensitive to autologous tissue antigen load and may be responsible for the induction of burn-induced immunologic suppression directly or through redistribution of specific functional lymphocyte subpopulations among various lymphoid compartments.

In contrast to the T-cell depression the B-cell level was significantly elevated during the first post-burn day and an inversion of T : B cell ratio was observed during the initial phase. However, a sudden return of B cells to normal was observed on post-burn day IO. Similar results have been observed in burn patients (Mistry and Antia, lY78).

The second important feature observed was loss of generation of antibody-forming cells to sheep red blood cells until post-burn day 10. This was most pronounced in the first 24 h after ther- mal injury. The increased B-cell count following

z3 8

c -------------

b normal range -I

i:;j

5 10 21 30 Post-burn days

h, Plaque (I x IO” cells).

injury did not seem to be activated by the pro- duction of antibody to a T-cell dependent anti- gen. This loss could be due to a selective fall in the T helper T suppressor cell ratio (Miller and Claudy, lY7Y). A marked increase in the T sup- pressor cell ratio to T helper has been well documented (Antonacci et al., 1982, 1984). It can therefore be stated with relative certainty that alterations in the number or proportion of regulatory T cells, observed early in the course of severe burn trauma, may be responsible for the induction of burn-induced immunological suppression. These changes correlated linearly and were significant at 24-48 h post-burn.

The total T- plus B-cell percentage in controls ranged between 74 per cent and I02 per cent. Considering the standard deviation for the re- spective values, the sensitivity of the test pre- cluded identification of null cells or double-label cells (Anthony et al., 1975a). T- plus B-cell percentages from burned mice showed a marked variation from a total of 100 per cent. Since repeated washings of the lymphocytes did not improve the T- plus B-cell count, it could bc stated that the rise in the T- plus B-cell count may not be due to non-specific coating of lympho- cytes. These cells were deleted as early as during post-burn day I. No null cells have been reported so far in animal experiments. The possible ex- planation for this fact could be attributed to the high cortisol secretion associated with adaptation to the injury-induced stress.

Since mice are relatively sensitive to cortico- steroids, the initial lymphocytopenia might be the result of a self-compensatory mechanism which in turn could be due to lack of organization in lymph nodes or due to burns giving rise to a class of immature lymphocytes termed null ceils. The appearance of these cells has been reported in human burns patients (Volenec et al., 1977; Mistry and Antia. 1978), patients with carcinoma

192 Burns (1986) Vol. lZ/No. 3

of the bronchus (Anthony et al., 1!475b) and patients with acute rheumatic fever (Leuker et al., 1975). It should be further noted that the full thickness skin wound in these animals healed faster despite bacterial infection with Pseudo- monas aeruginosa. This clinical feature corre- lated with the sudden return of the immunologic- ally altered patterns towards the normal range by the tenth post-burn day. Therefore it appears that, at least in our experimental animal model, the immunological alterations did not completely correlate with the human system (Mistry and Antia 1982). Hence this mouse model may not be suitable for direct comparison with the results obtained in humans.

Acknowledgements This work was conducted in the Department of Plastic Surgery, under the auspices of the Re- search Society of Grant Medical College and J. J. Hospital and with a grant from the Tata Trusts.

REFERENCES Adler W. H., Takiguchi T., Marsh, B. et al. (1970)

Recognition by mouse lymphocyte in vitro-1 Defini- tion of a new technique and results on stimulation by PHA-P and specific antigens. J. Exp. Med. 131, 1049.

Anthony H. M., Kirk J. A. and Madson K. E. (1975a) EAC rosetting lymphocytes in patients with carcino- ma of the bronchus II, a sequential study of thirty patients. Effect of BCG. Clin. Exp. Immunol. 20,29.

Anthony H. M., Kirk J. A. and Madson K. E. (1975b) Patients with carcinoma of the bronchus I, some parameters of the test and of its prognostic signi- ficance. Clin. Exp. Immunol. 20, 29.

Antonacci A. C., Calvano S. E. and Shires G. T. (1983) Restoration of autologuus mixed lymphocyte response following thermal injury with T cell growth factor. Fed. Proc. 42, 951.

Antonacci A. C., Good R. A. and Gupta S. (1982) T cell subpopulations following thermal injury. Surg. Gynecol. Obsret. 155, 1.

Antonacci A. C., Reaves L. E., Calvano S. E. et al. (1984) Flow cytometric analysis of lymphocyte -sub- populations after thermal injury in human beings. Surg. Gynecol. Obstet. 159, 1.

Casson P. R., Genser B. M. and Converse J. M. (1968) Immunosuppressive sequelae of thermal injury. Surg. Forum 19, 509.

Cunningham A. J. (1965) A method of increased sensi- tivity for detecting single antibody forming cells. Nature 207, 1106.

Daniels J. C., Cobbs E. K., Lynch J. B. et al. (1970) Increased nucleic acid synthesis in lymphocytes from patients with thermal burns. Surg. Gynecol. Obstet. 130, 783.

Ikeuchi S., Aikawa N., Okuda M. et al. (1981) Change in cell mediated immunity and tumour growth after thermal injury. Burns 7, 400.

Leguit P., Meineez A., Zeiglemaker W. P. et al. (1973) Immunological studies in burns patients I, lympho- cyte transformation in vitro. Inl. Arch. Allergy Appl. Immunol. 44, 101.

Leuker R. D., Addin Z. H. and Williams J. C. (1975) Peripheral blood T and B lymphocytes during‘acute pneumatic fever. J. Clin. Invesr. 55. 375.

Mghler B. and Batchelor J. R. (1971) PHA transforma- tion of lymphocytes in burned patients. Transplanta- tion 12, 409.

Markley K., Smallman E. T. and John L. A. (1977) The effect of thermal trauma in mice on cytotoxicity of lymphocytes. Proc. Sot. Exp. Biol. Med. 154,72.

Miller C. L. and Claudy B. J. (1979) Suppressor T cell activity induced as a result of thermal injury. Cell. Immunol. 44,201.

Mistry N. F. and Antia N. H. (1978) Quoted from MSc. thesis of Mistry N. F., Bombay University, India.

Mistry S. and Antia N. H. (1982) Cellular Immune mechanisms following thermal iniurv. PhD Thesis. Bombay University, India. . .

Mistrv N. F. and Antia N. H. (19781 Ouoted from MSc. of immunological alterations in thermal injury in mouse. J. J. J. Group Hosp. Grant Med. CON. XXVI, 11.

Munster A. M., Eurenius K. and Katz R. (1973) Cell mediated immunity after thermal injury. Ann. Surg. 177, 139.

Munster A. M., Eurenius K., Mortensen R. F. et al. (1972) Ability of splenic lymphocytes from injured rats to induce a graft versus host reaction. Trans- plantation 14, 106.

Neilan B. and Taddeini L. (1977) T lymphocyte rosette formation after major burns. JAMA 238, 493.

Schlesinger M. (1970) Anti t3 antibodies for detecting thymus dependent lymphocytes in the immune re- sp&se of mice to &Be. Nkure 226, 1254.

Volenec F. J.. Mani M. M.. Clark G. M. et al. 11977) Peripheral blood T and B lymphocytes in patients with burns. II Sequential rosette analyses considering burn severity and pseudomonas sepsis. Burns 4, 7.

Walker M. L. and Mason A. D. (1968) A standard animal burn. J. Trauma 8. 1049.

Paper accepted 26 September 1985.

Correspondence should be uddressed to: Dr Swaran Arora, Department of Plastic Surgery, J. J. Hospital, Bombay - 8, India.