Clin. exp. Immunol. (1991) 85, 515-518

ADONIS

000991049100270V

Immunosuppression follows systemic T lymphocyte activation in the burn patient J. A. TEODORCZYK-INJEYAN, B. G. SPARKES*, G. B. MILLSt & W. J. PETERSI Sunnybrook Health Science Centre, *Defence and Civil Institute of Environmental Medicine, North York, tDepartment of Oncology Research, Toronto General Hospital, and tThe Ross Tilley Burn Centre, Wellesley Hospital, Toronto, Canada

(Accepted for publication 5 April 1991)

SUMMARY A general consensus that thermal injury affects T lymphocyte function adversely is supported particularly by the observation that burned patients' lymphocytes secrete reduced levels of biologically active IL-2 in vitro. In the same patients, however, high serum concentrations of the lowaffinity IL-2 receptor (IL2Ra), a product of an IL-2-activated gene, have been observed. In this study a significant proportion of patients also demonstrated over-physiological levels (from 2 to 500 U/ml) of serum IL-2 ascertained by immunoassay. Increases in serum IL-2 content correlated significantly (P 0-02) with those of serum IL-2Rx during the first week post-burn. Later, serum IL-2Rx levels continued to increase up to 30 days while IL-2 eventually declined. Thus, augmented secretion of IL2Ra appears related to the high serum IL-2 content. Therefore refractoriness to further immune stimulation may be due to early activation of the lymphoid system, rather than to an intrinsic incapacity of T lymphocytes for generating sequential responses.
90)

Partial (range)

Full thickness (range)

Age (range)

Sex M/F

Outcome S/NSt

10+7 5 (2-28)

46 5+ 17 (28-80)

360+ 10 (18-57)

36/7

28/15

Controls 32 + 6 (26-45)

9/3

* Total body surface area. t Survivors/non-survivors.

MATERIALS AND METHODS Forty-three patients (seven women and 36 men) were studied (Table 1). Fifteen patients involved in this study developed sepsis and died between 17 and 45 days after the burn. Forty-one patients were studied two to four times over the hospitalization period, and two patients with total burn surface area (TBSA) injuries over 90% were studied only at admission. Eleven apparently healthy donors (three women and eight men) were taken as normal subjects aged 26 to 45. Heparinized blood and serum samples were collected from patients at admission within 48 h of the burn injury, and subsequently at varying time intervals. Responses of isolated PBMC from blood of these patients were described earlier (Teodorczyk-Injeyan et al., 1986, 1988). In this study of serum IL-2 and IL-2Ra, samples obtained within a week of therapeutic interventions, such as blood transfusion, antibiotics, surgery, terminal sepsis or patient's death, were not considered. Sera were aliquoted, stored at - 70'C and thawed only once before testing. Quantification of IL-2Ra and IL-2 levels in sera from the patients and controls was made using an ELISA based on MoAbs to two different epitopes of the Tac molecule (T Cell Sciences Inc., Cambridge, MA) and Intertest 2 (Genzyme, Boston, MA), respectively. All assays were performed according to the producer's specifications. Statistical significance of differences between means of patients and control data was estimated by Student's t-test. For the comparison of two proportions the x2 test with Yates' correction was used. Regression analysis was used for estimation of the relationship between concentration of serum IL-2Ra and its ligand. RESULTS Preliminary experiments (not shown) established that the addition of soluble purified Tac to human recombinant IL-2 had no effect on the absolute detectable amounts of either of these molecules when assessed in respective immunoassays. This ascertained the validity of subsequent determinations of IL-2 and IL-2Rx levels in the same sample of burn serum. The mean IL-2 level determined in serum of 11 normal donors was 0-6 + 0-8 U/ml. This figure was derived as follows. In sera from six of these individuals no IL-2 was detected. Four others showed a mean of 0-8 + 0 5 and one had 3 U/ml. Taking

10000

E

E

N. a

D

-J

1000

E

C,

C,)

E (n en

*

0

M

w

e"

(n =11)(n=20)2_s 6-9 10-15 20 A c ..

s"

30

-Day after burn

,50

40

-

.

Fig.l. Alterations in serum IL-2 (---) and IL-2Ra (-O-) levels in thermally injured patients. IL-2Ra is depicted as means and standard deviations, with the range in brackets. Serum samples were collected at admission (A) and then at indicated time intervals throughout the postburn period. Control (C) samples obtained from normal donors were assayed simultaneously with patients' sera as described in Materials and Methods.

0-6+0 8 U/ml as representative of normal, sera in this study containing greater than 2 U/ml, with a statistical significance of P < 0.05, were considered to be abnormally elevated. All patients presented in this study had demonstrated a significant suppression of IL-2 secretion by PBMC in vitro, as measured by bioassay on IL-2-dependent cells (TeodorczykInjeyan et al., 1986, 1988). However, a significant (P 500 U/ml serum of IL-2. Between 6 and 15 days post-burn

Immunosuppression follows systemic T lymphocyte activation in the burn patient

2000

0~~~~~~~ 0*

1000

*

0

* 0

*

N

0

200

S 0

0

-~~~~~~

,1

0~~~~1

l0

L-.I/--y/-LL--.

60 ¢ 500 IL-2 (U/mi)

Fig. 2. Concentrations of plasma and serum IL-2 and IL-2R in burn patients studied 2-7 days post-injury. Coefficient of ccorrelation for linear analysis (r) is 0 51 with a significance of P < 0-02.

64% of patients had elevated concentrations of ser um IL-2 from 2-9 to 440 U/ml. Subsequently the proportion of such patients decreased, although in several individuals high IL-' 2 content was still apparent during the later phase of the post- -burn period. Thus, serum IL-2 levels in individual patients fit actuated in a highly variable pattern and are therefore shown in their entirety in Fig. 1. On the other hand a more uniform pattern was observed for the soluble receptor. Serum level is of IL-2Ra showed a gradual increase from the period c )f admission (490 + 310 U/ml) to their highest levels (4985 + 30118 U/ml), with a range from 840 to > 10000 U/ml observed betwe6en 20 and 40 days post-burn (Fig. 1). Convalescent serum IL-2F {a concentration declined to a mean of 1380 + 680 U/ml with a l range of 3002100 U/ml, remaining, nevertheless, significantly higher than that of controls and first-day levels. In view of parallel trends in the serum content of both IL-2 and IL-2Rx (Fig. 2) the relationship of varying lev Fels of IL-2 to its receptor was evaluated by linear regression analysis. In patient sera obtained during the early phase of t 'he post-burn period (2 to 7 days after the injury) increases in IL -2 concentration were correlated with those of IL-2Ra at a sigYnificant level (P < 0-02, coefficient of correlation for linear regresssion analysis r=0.51). No significant correlation between I1L-2 and its receptor was found in patient sera collected later.

517

patients, in spite of inhibited mitogen-induced secretion of bioreactive IL-2 (Teodorczyk-Injeyan et al., 1986), and of a reduced surface IL-2R expression (Teodorczyk-Injeyan et al., 1987), the intrinsic ability to synthesize and/or secrete these molecules is not eradicated. IL-2 production in patients' PBMC can be induced, for example, in the presence of phorbol ester and calcium ionophore, and mitogen-induced IL-2Ra secretion is found to be comparable to control (Teodorczyk-Injeyan et al., 1988). IL-2Ra secretion could also be augmented following deletion of prostaglandin (PG) E2-secreting adherent cells or by supplementation with exogenous IL-2 (Teodorczyk-Injeyan et al., 1991). These observations suggest that in the presence of IL2, or under conditions optimizing its production, the synthesis and release of soluble IL-2Ra is facilitated. Such a mechanism may

be involved in the release of

IL-2Rcx

in vivo given the high

concentrations of endogenous IL-2 in post-burn serum. Increases and peak levels of systemic IL-2 appeared to precede in parallel those of serum IL-2Ra, inclining interpretation toward a causative role for IL-2 in IL-2Ra secretion (Figs. I and 2). This pattern may reflect the kinetics of molecular events in T lymphocyte activation where expression of IL-2 mRNA transcripts is detected before those of IL-2R (Granelli-Pipemo, 1988). The ability of IL-2 to increase transcription of the IL-2Ra gene directly may then provide a mechanism by which increased serum IL-2 levels eventually up-regulate secretion of serum IL2Ra. This could lead to down-regulation of the systemic immune response, as post-burn serum-associated IL-2Ra is known to bind IL-2 (Teodorczyk-Injeyan, Sparkes & Peters, 1989b). Hence, the immunosuppressive nature of post-burn serum, in assays where IL-2 usually participates, indicates that its immunoreactive IL-2 content is biologically unavailable. Cells stimulated by IL-2 demonstrate a reduced capacity for further activation through the T cell receptor (Nau et al., 1987). Thus, immunotherapy with IL-2 has been associated with the development of anergy (Ades et al., 1990), demonstrable in viwo and in vitro by suppressed T cell responses, markedly resembling

the clinical picture of burn patients. IL-2 therapy also induces a pronounced vascular leak syndrome (Rosenstein, Ettinghausen & Rosenberg, 1986) and a high frequency of serious bacterial infections (Kragel, Travis & Feinberg, 1990). Therefore bacterial translocation from the gut (Deitch & Berg, 1987), as well as oedema, electrolyte imbalance and related disturbances (Michie et al., 1988; Anonymous, 1990) in the burn patient, may

all be related to high levels of endogenous IL-2. The present data indicate that major thermal injury induces

rapid and long lasting activation of the T lymphocyte of the immune system. This is reflected by a prolonged presence of high concentrations of two products derived from activated T cells: IL-2 and soluble IL-2Ra. a

compartment

DISCUSSION The present study demonstrates that post-burn seirum levels of both IL-2 and IL-2Ra rise significantly shortly aftetrburn injury, Reports of activated cells in PBMC preparation Is from burn patients are known (Deitch et al., 1989). It has been demonstrated elsewhere that a lipoprotein complex induce d by burning the skin, and possibly composed of T cell receptor proteins and heat shock proteins, could stimulate IL-2 secretio in in PBMC, yet together with mitogenic lectins the compl Vex inhibited bioactive IL-2 production (Sparkes, 1991). These, observations suggest that the impairment of in vitro T lymphoc) yte responses in burns may not so much represent cellular dy rsfunction as refractoriness after a preceding in vivo activati on. In burn

Therapeutic immunomodulation in the burn patient, directed toward replacement and/or stimulation of the 'suppressed' T

lymphocyte function, should therefore be approached with caution.

ACKNOWLEDGMENTS

Supported

from

the

by

Contracts

Defence

&

No.

Civil

DSS

W771 1-6-9483

Institute

of

and

W771 1-8-7053

Environmental

Published by permission of the Department of National

Canada.

Medicine.

Defence,

J. A. Teodorczyk-Injeyan et al.

518 REFERENCES

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