Membrane and soluble forms of Fas (CD95) in peripheral blood lymphocytes and in serum from burns patients

Burns 27 (2001) 669– 673 www.elsevier.com/locate/burns

Membrane and soluble forms of Fas (CD95) in peripheral blood lymphocytes and in serum from burns patients M. Ju. Lebedev a,*, Ju. S. Ptitsina b, S.A. Vilkov a, S.B. Korablev a, V.V. Novikov b,c a

Laboratory of Immunology, Institute of Traumatology and Orthopaedics, 603155 Verhne Volzhskaya nab. 18, Nizhny No6gorod, Russian Federation b Institute of Epidemiology and Microbiology, Nizhny No6gorod, Russian Federation c Lobache6sky Uni6ersity, Nizhny No6gorod, Russian Federation Accepted 7 March 2001

Abstract Burn trauma results in disorders of regulation of programmed cell death called apoptosis. Apoptosis of immunocytes is associated with the expression of Fas antigen. There are two major forms of Fas molecule, membranous Fas (mFas) and soluble Fas (sFas). The last form is generated by alternative splicing and differs from mFas by lacking 21 amino acid residues containing the transmembrane domain. We determined the expression of mCD3, mCD4, mCD8 and mFas in peripheral blood lymphocytes and the level of the soluble form of Fas in serum in patients with acute thermal trauma (n = 32). As the control blood of healthy volunteers (n=25) was investigated. Compared to healthy volunteers, burn patients showed a remarkable reduction in number of CD3+ lymphocytes in the 24 h following injury, which was accompanied by a decrease in CD4+ but not CD8+ subsets by indirect immunofluorescence method. The decrease of expression of mFas in the patients with acute thermal trauma at all burn disease time was determined simultaneously. We established the decrease of level of sFas during the first (404 925 U/ml) and second (3529 38 U/ml) postburn 10-day periods by the ELISA method. The contents of sFas in serum of healthy volunteers was 5349 31.8 U/ml. There were no relations between the level of membrane Fas expression and contents of the soluble Fas in serum both in clinical manifestation and survival. We suppose that it is impossible to predict outcomes of burn disease by quantity of CD95+ cells and contents of sFas in serum. However, it is probable that significant deviations from the level of sFas may be attributes of non-revealed accompanying pathology. © 2001 Elsevier Science Ltd and ISBI. All rights reserved. Keywords: Burn; Fas/APO-1/CD95 antigen; Apoptosis

1. Introduction Severe thermal trauma is the cause of various disorders of function and regulation of immune system, and it can result in the development of immunosuppression and immunodeficiency. At the same time, it is known that one of the important immune regulatory mechanisms is apoptosis or programmed cell death. The disorders of this mechanism under the influence of an acute thermal trauma can change the cell number during the burn disease [1,2] and may effect the activation processes level of immune system [2].

* Corresponding author. Fax: + 7-8312-360591. E-mail address: [email protected] (M.J. Lebedev).

Fas (APO-1/CD95)-mediated apoptosis plays an important role in apoptosis of immune system cells. Fasantigen, a member of the tumour necrosis receptor superfamily, is 43 kDa surface protein containing a single transmembrane region. Fas cross-linking by its natural ligand (FasL or sFasL) or some monoclonal anti-Fas antibodies results in rapid induction of apoptosis in susceptible cells. Fas is expressed by cells of the immune system (activated T-lymphocytes) and some non-lymphoid tissues. There is a soluble form of Fas antigen (sFas). Functional soluble forms of the Fas receptor are produced by activated peripheral blood lymphocytes and some transformed cell lines. There are three described functional sFas, which are the products of alternative splicing of the Fas mRNA primary transcript [3,4]. Soluble Fas is produced as the form lacking

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21 amino acid residues in the transmembrane and intracellular domains [5]. This sFas blocks apoptosis by inhibiting binding between Fas and FasL or sFasL. That is, Fas, FasL and sFasL induce apoptosis, but sFas is an inhibitor of apoptosis. There is a model according to which to induce the death signal mFas creates a three-dimensional structure. sFas may prevent the formation of this construction [6]. Soluble Fas is present at low level in the serum of healthy persons as a great number of cytokine receptors and cell surface antigens which exist in soluble or secreted form. The serum concentration of sFas significantly increases with tumour [7,8], some autoimmune [9– 11], viral [12–14] and other diseases. The changes of sFas contents in serum may reflect the degree of pathological process activity and can serve as a clinical parameter.

made by the indirect immunofluorescence method using mouse monoclonal antibodies ICO-160 [15] against Fas /APO-1/CD95 antigen, ICO-90 against CD3 antigen, ICO-86 against CD4 antigen and ICO-31 against CD8 antigen. Goat FITC-label monospecific antibodies against mouse immunoglobulins were used as secondary antibodies. The determination of CD3+, CD4+, CD8+ and CD95+ cell was performed using the luminescent microscopy. Intensity of the luminescence was determined visually. Cells with monocyte morphology were not taken into account. In each experiment, the viability of cells with trypan blue (at least 98%) was checked. During the subsequent analysis the immunological parameters of patients were combined into groups, taking postburn time into account.

2.3. Determination of soluble form of Fas-antigen 2. Patients and methods

2.1. Patients Thirty-two patients with acute burns treated at Russian Burn Centre of Institute of Traumatology and Orthopaedics, Nizhny Novgorod during 1998–1999 were investigated. The age of patients varied from 15 to 87 years. The total body surface area burned (TBSAB) was between 15 and 95%. In most cases, the cause of burn was flame. The length of stay was from 2 days to 5 months. All the patients received standard treatment given in the thermal trauma clinic, which include debridement and drug therapy. Patients were classified into two groups: the first group – with moderate burns (n = 8) (TBSAB between 15 and 30% or B 10% subdermal) and second – with major burns (n = 24) (TBSAB \30 or \10% subdermal burns). Nine patients died. All patients were investigated for hepatitis B and C markers, and also lues and HIV. All results of research were negative. Accompanying autoimmune diseases were not revealed. As the control samples of blood of 25 healthy volunteers were investigated. The control investigation was carried out before and after the influenza epidemics period. Sampling of blood for research was taken in the morning with planned analysis simultaneously. Analysis was performed depending on the clinical condition of the patients, but not less then once per 2 weeks up to the last autografting and before discharge from the clinic. For measurement of the soluble form of Fas the serum was used, which was isolated directly after blood had been taken and stored at − 60 °C until analysis.

2.2. Cells population analysis The evaluation of subpopulation cells in PBMC isolated by the gradient centrifugation (1.077 g/ml) was

The content of sFas was determined by ELISA using goat polyclonal antibodies against PBMC superficial antigens, monoclonal antibodies ICO-160 against CD95 antigen and goat polyclonal antibodies against mouse immunoglobulins conjugated with horseradish peroxidase. The plates were sensibilizated during 16–18 h at + 4–8 °C by polyclonal antibodies against PBMCs purified by ammonium sulphate and ion-exchange chromatography on DEAE-cellulose. After washing of the plates samples of patient serums were added and then incubated during 1 h at the room temperature. After this, washing was carried out again. Then the purified monoclonal antibodies ICO-160 and goat monospecific antibodies against mouse immunoglobulins conjugates by peroxidase were added, incubated and washed at the same conditions. For each tested serum, the negative control was made. For this purpose, the polyclonal mouse antibodies were added instead of monoclonal antibodies but in the same concentration. Samples of serum of patients with systemic lupus erythematosus were used as the positive control. After the final washing the reaction was visualized with using of 0.1% o-phenylendiamine in 0.05 M citric buffer (pH 5.0), containing 0.01% hydrogen-peroxide. Reaction was stopped with 1 M H2SO4. The account of results was carried out with using of Multiskan MS (Labsystems, Finland) at the wavelength 492 nm. The results were expressed in U/ml.

2.4. Statistics Results were expressed as mean9 SEM Student’s unpaired t-test or analysis of variance followed by Fisher’s least significant difference test to compare individual means was used. A value PB 0.05 was considered statistically significant. To assay the relationships between CD3+, CD4+, CD8+ and CD95+ cells, Pearson’s coefficients were calculated.

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3. Results

3.1. Influence of a burn injury on expression of CD3, CD4 and CD8 antigens in peripheral blood lymphocytes It has been observed that acute thermal trauma results on the whole in reduction of expression of CD3+ antigen. It was manifested both in decrease of relative number of CD3+ cells and reduction of their intensity of luminescence in the 24 h following injury. The depletion of circulating T-cells could be related with the decreasing of the relative and absolute quantity of CD4+ cells and luminescence intensity, which were reduced at this time also. Further reduction of the number of CD3+ and CD4+ cells was determined in the burn patients at all time stay in clinic. The change of CD8+ cells number and intensity of their luminescence was expressed in a much smaller degree. Authentic differences between number of these cells in the burned patients and control group were received at no stage of burn disease. Results are given in Table 1. CD4+/CD8+ ratio, which is considered as immunoregulatory index, was reduced due to reduction of CD4+ cells number. There were not any differences dependent upon burn localization.

3.2. Influence of a burn injury on expression of mFas in peripheral blood lymphocytes We have found out that acute thermal trauma results in reduction of mFas antigen expression (Fig. 1). It was

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manifested both in decrease of CD95+ cells number (89 1.1%) and reduction of their intensity of luminescence (Table 1). The number of CD95+ cells in the healthy volunteers was 15.7891.5%. The decrease of mFas expression appeared at 48 h after burn. From this time the reduction of the number of CD95+ cells was determined in the burn patients all during their stay in the clinic. In two patients during the whole period of inspection the number of CD95+ cells was significantly higher in comparison with the general parameters of this group and number of CD95+ cells in healthy volunteers. The dynamics of changes of CD95+ cells in patients of the first and second groups and in survivors and non-survivors were similar. There were no distinctions dependent on burns aetiology and localization.

3.3. Influence of a burn injury on sFas le6el in serum Dynamics of the level of sFas in burn patients serum are shown on Fig. 2. We found, that initially in patients with burns and healthy volunteers the average serum sFas levels were not significantly different (451919.8 vs. 534931.8 U/ml, P\ 0.05). However time the content of sFas was much reduced in the burn patients during the second (352938 U/ml) postburn 10-days period or between 4 and 20 days after the burn (3689 22.6 U/ml). Statistical differences of the sFas contents in patients in the first (4269 45.2 U/ml) and second group (434919 U/ml) were not observed. Neither were there any statistically significant differences between survivors (424925.9 U/ml) and non-survivors (4669 21.1 U/ml). There were no distinctions in depending on

Table 1 Expression of CD3, CD4, CD8, and CD95/Fas antigens in peripheral blood lymphocytes and the level of sFas in serum in patients with burns Antigen

Groups

Days after the burns

Healthy volunteers

1–3

4–7

8–14

15–21

22–28

CD3+ cells (106/ml) First Second Total

0.39 9 0.09a 0.249 0.11a 0.32 90.1a

0.059 0.02a 0.469 0.18a 0489 0.8a

0.65 9 0.2a 0.45 9 0.2a 0.55 9 0.2a

0.69 9 0.1a 0.58 9 0.2a 0.63 9 0.1a

0.63 9 0.09a 0.65 90.11a 0.64 9 0.11a

1.3 90.4 1.0 90.4 1.1 90.4

1.5 9 0.3

CD4+ cells (106/ml) First Second Total

0.189 0.07a 0.119 0.03a 0.159 0.01a

0.21 9 0.02a 0.169 0.03a 0.199 0.02a

0.4 90.01a 0.35 90.06a 0.38 9 0.03a

0.4 90.01a 0.28 9 0.01a 0.34 90.01a

0.27 9 0.04a 0.27 9 0.01a 0.27 9 0.06a

0.38 9 0.07a 0.42 9 0.01a 0.4 90.05a

1.1 90.2

CD8+ cells (106/ml) First Second Total

0.39 0.03 0.28 90.07 0.299 0.05

0.29 9 0.01 0.27 9 0.07 0.289 0.07

0.39 9 0.03 0.37 90.05 0.38 9 0.01

0.4 90.06 0.3 90.01 0.35 90.08

0.5 9 0.02 0.46 9 0.06 0.48 9 0.04

0.68 9 0.1 0.68 9 0.14 0.68 9 0.12

0.5 90.03

CD95+ cells (106/ml)

First

0.13 9 0.6a

0.079 0.02a

0.16 90.01a

0.14 9 0.04a

0.08 9 0.02a

0.17 9 0.07a

0.3 90.08

Second Total

0.14 9 0.4 0.14 90.03a

0.07 90.01a 0.079 0.02a

0.17 9 0.02a 0.16 9 0.04a

0.11 9 0.03a 0.13 9 0.03a

0.09 9 0.04a 0.09 9 0.06a

0.17 9 0.09a 0.17 9 0.08a

First Second Total

431 9 25.1a 429 9 24.6 430 9 27.7

398 912.1a 408 911.6a 403 9 14.3a

319 914.1a 284 941.2a 302 923.5a

447 9 11 472 972 466 956

542 994 552 9 112 547 9 105

423 924.1 454 964.9 440 947.2

sFas (U/ml)

a

PB0.05 vs. healthy volunteers.

29–60

534 931.8

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Fig. 1. The relative and absolute quantity of CD95+ lymphocytes in peripheral blood in patients with acute thermal injury.

aetiology and localization of the burn. In patients with considerably increased expression of mFas the contents of sFas in blood did not show any variance. At the same time one patient had a high level of sFas during all the stages of the disease. The expression of mFas on lymphocytes in this patient was not unusual.

4. Discussion Our study shows that the burn injury results in reduction of mFas antigen expression and decrease of the level of serum sFas antigen. Decreased expression of mFas antigen was revealed 1 day after the burn injury and persisted during all time of burn disease. The decrease of the sFas was manifested to a smaller degree and it is reasonable to speak about it only in regard to the period between 4 and 20 days after the burn. In the present study it was not possible to find any relation between the level of mFas expression and the contents of sFas in serum. The increase of CD95+ cells amount was not accompanied by the increase or decrease of the contents of sFas in serum. On the contrary, the increase of sFas contents in blood was not combined with the changes of mFas antigen expression in comparison with expression of mFas antigen. Obtained results confirm that synthesis of membranous and soluble CD95+ antigens are absolutely independent processes and each of them has its own mechanisms of regulation. The influence of burn on these mechanisms should still be clarified. Correlation analysis had shown positive correlations between the relative number of CD8+ cells and CD95+ cells during of the first two weeks from the moment of trauma (RPearson = 0.43, P = 0.02 during the first week

and RPearson = 0.48, P= 0.04 during the second week) and the relative number of CD4+ cells and CD95+ cells during the period between 4 and 14 days after the burn (RPearson = 0.54, P= 0.04). Serum sFas level may be a marker of pathological process activity in chronic glomerulonephritis [16], acute myocardial infarction [17] and other heart diseases [18]. The increase of sFas levels in plasma may be a prognostic parameter for hepatocellular carcinoma [7] and bladder cancer [10]. We showed that the quantity of CD95+ cells and serum sFas level does not depend on aetiology and localization of burn and on the severity and outcomes of burn disease as well. It does not suggest a diagnostic and prognostic importance of measurement of the various forms of Fas antigen for clinical application. At the same time it can be supposed that the significant deviations from the normal level found in some patients can serve as a marker for some latent accompanying pathology. This especially, concerns the increase of the contents of the soluble form of Fas antigen. According to the preliminary data obtained in our laboratory mAb ICO-160 antibody against Fas receptor induces apoptosis of peripheral blood lymphocytes in burn patients, but never in healthy volunteers [19]. Therefore, it should be interesting to investigate the contents of membranous and soluble form of FasL in patients with a thermal trauma to understand the role of Fas–FasL interaction in regulation mechanism of T-lymphocytes apoptosis in thermal trauma.

Acknowledgements We would like to thank Platon Anfimov and Mikl Garmash for technical assistance.

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Fig. 2. The contents of absolute quantity of CS95+ lymphocytes in peripheral blood and soluble form of CD95 antigen in serum in patients with acute thermal injury.

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