International Immunopharmacology 22 (2014) 273–276

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Cimetidine effects on the immunosuppression induced by burn injury Parviz Kokhaei a,d,⁎, Mahdieh Shokrollahi Barough a,c, Zuhair M. Hassan b a

Department of Immunology, Semnan University of Medical Sciences, Semnan, Iran Department of Immunology, Medical School, Tarbiat Modarres University, Tehran, Iran c Student's Research Committee, Semnan University of Medical Sciences, Semnan, Iran d Immune Gene therapy Lab, CCK, Karolinska University Hospital Solna, Stockholm, Sweden b

a r t i c l e

i n f o

Article history: Received 1 June 2014 Received in revised form 4 July 2014 Accepted 5 July 2014 Available online 16 July 2014 Keywords: T lymphocyte B lymphocyte Burn injury Cimetidine

a b s t r a c t Although many studies on the immune response following burn injuries have been reported, more attention has been given to the immunosuppression mechanism and mediators that shape the process of immune suppression. Specifically, information is not available concerning the immunomodulatory effects of the drugs which are involved in the immune response restoration. In this study, we investigated the effects of Cimetidine on the modulation of immune response in patients with burn injury of 20–60%. Two groups of patients were involved in this study; the patients in one group were treated with 15 mg/kg per day of Cimetidine while the patients in the other group were treated with placebo. Peripheral blood mononuclear cell (PBMC) expressing CD3, CD4, CD8, CD19 and CD3/HLA-DR was analyzed by flow cytometry. Cell proliferation assay using H3 thymidine was performed on PBMC samples. The proliferation assay showed a significant suppression of cell proliferation rate in postburn patients (p = 0.001). We observed a significant reduction in the lymphocyte count (p = 0.001) and frequency of CD3 (p = 0.007) and CD4 (p = 0.001) T cells in post-burn patients. Also, the frequency of CD 19+ and HLA DR+ cells was increased compare to normal donors following burn injury. Treatment with Cimetidine increased the frequency of CD8 + T cells in the patient's peripheral blood. The PBMC proliferation rate was restored following the treatment with Cimetidine (p = 0.02). Our data indicates that Cimetidine may have beneficial effects on cell mediated immunity following burn injury. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Infection complication represents a major cause of death in patients with extensive burn injury [1,2]. Extensive burn injury causes profound alterations in normal host immune response. Thus, the main aim of treatment after resuscitation is to improve patient's immune response [3,4]. Reports have shown that the main contributors are impaired leukocyte and cytokine imbalance [5,6]. Bystrova, NA et al. described the failure of T cells to respond to T-dependent antigens following burn injury [7]. Several investigators have observed immunosuppression effects of low molecular weight peptides found in the serum of burn and trauma patients [7]. The impairment of T helper cell function due to polarization toward T helper 2-type response has been suggested as a major cause for post-traumatic immunodeficiency [8]. Improvement of the immune competence in post burn patients through different immunotherapeutic strategies has been reported; IFN

⁎ Corresponding author at: Department of Immunology, Semnan University of Medical Sciences, Semnan, Iran. Tel.: +98 23 33654362; fax: +98 23 33654361. E-mail address: [email protected] (P. Kokhaei).

http://dx.doi.org/10.1016/j.intimp.2014.07.003 1567-5769/© 2014 Elsevier B.V. All rights reserved.

gamma has been recognized as an effective antimicrobial prophylactic cytokine in trauma [9]. Effects of fat intake [10] and Ornithine Alpha-Ketoglutarate preteen have been investigated in modulation of immune response [11]. Cimetidine was administrated in this study because the drug is well documented with recognized immune potentiating properties [12]. Cimetidine is a histamine receptor antagonist which blocks H2histamine receptor and usually is used to reduce gastric acid secretion and gastric ulcer treatment [13]. Cimetidine also blocks the activation of regulatory T cells and facilitates cell-mediated immunity (CMI) by CD8+ T cells [14]. On the other hand, histamine induces the production of inflammatory cytokines such as IL-6 and IL-1β. Furthermore, H4 and H2 receptors of histamine control histamine-induced interleukin-16 release from human CD8+ T cells [15]. Thus, Cimetidine may modulate the immunomodulatory effects of histamine [16]. In thermal injuries Cimetidine modulates edema formation by blocking of burn induced inflammation [17,18]. T cell activation and regulation, which have very important roles in prevention of infection and wound healing, are impaired in thermal injuries [19]. However, CMI boosting could be useful in the treatment of burn wounds and Cimetidine may be a good candidate for this strategy [20]. In this study, we investigated the effects of Cimetidine on T cell proliferation, activation and other features of immune system in post-burn patients.

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% of total body surface area

Burn injury severity 50 45 40 35 30 25 20 15 10 5 0

2.4. Flow cytometry analysis

p=0.135

Cell samples were analyzed on a Coulter flow cytometer with serial filter configuration. The analysis was focused on the lymphoid areas of the forward and side scatters. Double stained cells were analyzed using Coulter software. 2.5. Proliferation assay

Treated

Non treated

Fig. 1. Burn injury severity measured by total body surface area that defined by Wallace Rule of Nines, treated patients have received Cimetidine. Treated and nonretreated groups are match in terms of burn severity.

2. Material and methods 2.1. Samples Peripheral blood samples were collected from twenty two healthy subjects and thirty-seven patients exposed to burn injuries that had severe acute phase burn injury with 20–60% of total body surface area. 18 of the patients were treated with Cimetidine and 19 untreated.

Five ml of peripheral heparinized bloods from Cimetidine treated and non-treated patients were collected and the lymphocyte was isolated by Ficoll hypaque. PBMC proliferation was measured by incorporation of [3H]-thymidine. 2 × 105 PBMC from patients and healthy controls were stimulated with 5 μg/ml phytohemagglutinin (PHA) in RPMI 1640 medium supplemented with 2 mM/l glutamine, 10% heat inactivated AB+, 100 IU/ml penicillin and 100 mg/ml streptomycin in 96-well U-bottomed tissue culture plates and incubated in humidified CO2 5% at 37 °C. After 3 days, 1 μCi [3H]-thymidine was added to each well followed by further incubation for 18 h. The plates were harvested using Skatron A.S automatic harvester Flow Labs, Rockville MD and uptake of [3H]-thymidine was determined in liquid Scintillation counter (Bekman). Proliferation responses were calculated by Stimulation Index ¼ the increase in cpm in test=the increase in cpm of base lineCpm ¼ count per minute:

2.6. Statistics 2.2. Drug Cimetidine was purchased from Chemidarou Co. (Tehran, Iran). 15 mg/kg was chosen according to the standard procedures [21]. The drug was administered per 24 h for four days.

In this study one-way analysis of variation (ANOVA) and Kruskal– Wallis nonparametric test and T-test analysis were employed using SPSS software. 3. Results

2.3. Immunophenotyping For cell surface phenotyping, anti CD4, CD8, CD4/CD8, CD19, and HLA-DR (DAKO, Denmark) were used. We established the reference immunophenotypic pattern using standard procedures. In this study 100 μ of blood sample was treated as follows; each sample was immunostained with 10 μ mAbs directly conjugated with Fluorescein Isothiocyanate (FITC) or R-Phycoerythrin (RPE) in Q-Prep apparatus afterwards, in which three immunopreps were added automatically, 0.7 ml immunoprep A (Formic Acid 1.2 ml/L), 0.32 ml immunoprep B (Sodium Carbonate 6.0 g/L, Sodium Chloride 14.5 g/L, Sodium Sulfate 31.3 g/L) and 0.14 ml immunoprep C (Paraformaldehyde 10.0 g/L, Phosphate Buffer) 9Coulter. Each sample was then kept in 2–8 °C and dark for about 24 h [22].

The initial stage of this work consisted an efforts aimed at obtaining an effective and acceptable stage of burn injuries which clearly displayed clinical signs of burn injury. The stage of burn used in this study was divided in two group so that the groups were matched for the extend of burn injury (Fig. 1). The results showed a significant suppression of immune system as indicated in Fig. 2. Injured patients display a significant reduction in the lymphocyte number comparing to control group as well as a significant change in surface markers of the immune cells comparing to the control group (Table 1). 3.1. Effect of Cimetidine on burn injury induced B cell immunosuppression In order to assess the effect of Cimetidine on B cell status following burn injury, the protocol shown in Table 1 was employed for 37 patients

p=0.0001

40

% of WBC count

35 30 25

p-=0.342

20 15 10 5 0

Normal

Treated

Non treated

Fig. 2. WBC percent in two groups of patients and normal controls. Patients in treated and nontreated group showed significant WBC reduction.

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Table 1 Comparison of normal donors and nontreated burn patients. Groups/markers

CD3

CD4

CD8

CD19

HLA-DR

CD4/CD8

CD3/HLA-DR

Nontreated patients Normal p value

57.85 ± 13.24 67.04 ± 5.51 0.007

32.5714 ± 8.061 43.45 ± 7.35 0.001

21.5 ± 3.84 24.04 ± 5.11 0.094

33.25 ± 5.3 15.5 ± 4.23 0.001

24.44 ± 7.97 18.35 ± 6.07 0.05

1.46 ± 0.11 1.94 ± 0.57 0.012

1.83 ± 1.72 1.366 ± 0.605 0.388

and 22 controls. Results indicate that the percentage of CD19 (p b 0. 001) and HLA-DR positive cells were significantly (p b 0. 05) increased in burninjured patients. Cimetidine failed to exhibit a significant effect in the percentage of B cells comparing to untreated patients (Table 3). 3.2. Effect of Cimetidine on burn injury induced T cell immunosuppression In order to evaluate the effect of Cimetidine on T cell status following burn injury, the protocol shown in Table 2 was employed for 37 patients. Results indicate that the percentage of CD8 cells was significantly (p b 0.01) increase in the Cimetidine treated burn-injured patient. Cimetidine failed to exhibit a significant increase in the percentage of CD3+, and CD4+ T cells comparing to untreated patients. 3.3. Effect of Cimetidine on the lymphocyte proliferation In order to assess the T cell proliferation in normal donors and Cimetidine treated and untreated patients, 37 patients were divided in two groups and the stimulation index was evaluated following PHA stimulation. The results in Fig. 3 indicate that the stimulation index in Cimetidine treated were significantly (p b 0.027) higher than untreated group. The proliferation rate of the control group was considered 100, and other groups were compared with the control group. 4. Discussion One of the most threatening effects of burn injuries has been determined by many clinicians and investigators, to be severe suppression of the immune system which can lead to sever opportunistic infection (resistant to all conventional antibiotics). Our understanding in this regard come from ordinary burn injuries as well as skin exposure to sulfur mustard [23,24]. The initial stage of this work consists of efforts aimed to understand the immune status of patients. Our results indicated a severe suppression of total WBC as well as significant decrease in the CD3, CD4 but not CD8 cells comparing to the normal control group. These results are in line with the other reports, which indicate severe suppression of the immune system [5,7]. The ratio of CD4/CD8 have also significantly (p b 0.01) decreased in comparison with normal cases and two groups of treated and non-treated patients in compare to

each other. This status indicates Cimetidine effects on CMI and the ratio is compatible with an increase in the percentage of CD8 + cells in treated patients in comparison with non-treated patients. Cimetidine has a significant role in cell mediated immunity progression and reduces impairment of CD8+ cells in injuries such as surgeries [25] Recently in DNA vaccine clinical trials Cimetidine used such an adjuvant for CD8+ T cells enhancing [26,27]. One of the drugs in atopic rhinitis treatment is Cimetidine, Yang PC, et al showed after 4 week treatment with Cimetidine, CD4/CD8 ratio is reversed and the numbers of helper T cells with CD4 marker fell and the numbers of CTL cells increased in the patients with allergic rhinitis [28]. The status of CD19 showed a significant (p b 0.001) increased after burn exposure. This observation probably is due to microbial infections and is documented with an increase of CD3/HLA-DR. A decrease in expression of CD25 (the B-cell activation marker) both before and after cytokine stimulation of burn patient is reported [29]. Mitogen-induced immunoglobulin production in burn patient was elevated initially but suppressed at 3–4 weeks after injury [30]. Studies on the ability of Cimetidine to reverse burn injury immunosuppression resulted in noticeable observation. Cimetidine is a histamine (H2) antagonist widely used for treatment of duodenal ulcers and other gastric hyper secretory conditions [31]. Recently it has been acknowledged that Cimetidine can reverse histamine induce suppression of the immune response [32]. Cimetidine has also been implicated in augmentation of cell-mediated cytotoxicity and in the abrogation of suppressor T-cell function [14,33]. Cimetidine, at the dose similar to those employed in our work, has been proved to abrogate the immunosuppression caused by burn in mice [33]. Cimetidine has been effective in countering the immunosuppressive conditions produced after burn, common variable hypogammaglobulinemia and AIDS in the clinic [33–35]. Cimetidine effects on CD8+ T cell progression are very important in viral infection and tumor immunity [36,37]. Cimetidine, administrated daily to the patient could augment CD3 to the normal rang but did not have any significant effect on CD4. Results showed no change in the CD19 and CD3/HLA-DR in patients before and after treatment with Cimetidine but could significantly increase (p b 0.027) the proliferation of lymphocyte in the group of treated patients comparing to untreated patients. Cimetidine enhances delayed type hypersensitivity response and it is very important in cell mediated

Table 2 Comparison of Cimetidine treated and nontreated burn patients. Groups/markers

CD3

CD4

CD8

CD19

HLA-DR

CD4/CD8

CD3/HLA-DR

Nontreated Treated p value

57.85 ± 13.24 58.5 ± 10.21 0.887

32.5714 ± 8.061 28.2778 ± 7.06 0.132

21.5 ± 3.84 26.93 ± 6.76 0.012

33.25 ± 5.3 32 ± 14 0.871

24.44 ± 7.97 23.09 ± 10.79 0.757

1.46 ± 0.11 1.13 ± 0.36 0.039

1.83 ± 1.72 1.48 ± 1.04 0.611

Table 3 Comparison of normal donors and treated patients. Groups/markers

CD3

CD4

CD8

CD19

HLA-DR

CD4/CD8

CD3/HLA-DR

Treated Normal p value

58.5 ± 10.21 67.04 ± 5.51 0.003

28.2778 ± 7.06 43.45 ± 7.35 0.001

26.93 ± 6.76 24.04 ± 5.11 0.09

32 ± 14 15.5 ± 4.23 0.001

23.09 ± 10.79 18.35 ± 6.07 0.17

1.13 ± 0.36 1.94 ± 0.57 0.001

1.48 ± 1.04 1.366 ± 0.605 0.74

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120

Stimulation Index

p=0.0001 100 80

p=0.027

60 40 20 0 Normal

Treated Groups

Non treated

Fig. 3. PBMC of patients and controls were cultured for 72 h and [3H]-thymidine added in last 18 h of culture. Cimetidine treated patients show better proliferation index compare to nontreated patients.

immunity [38,39]. This treatment was also applied on the patients exposed to sulfur mustard and we got a significant improvement in the immune response. (Unpublished results). Up to now several immunomodulator agents have provided to be promising in the cancer and AIDS therapy and they may also be effective in restoring burn injury immunosuppression and preventing the grave consequences [40]. Further experimental and clinical studies may elucidate a possible role for immunomodulators in this regard. In conclusion our data indicates that Cimetidine may have beneficial effects on lymphocyte proliferation and cell mediated immunity following burn injury. Acknowledgment The authors would like to thank Tarbiat Modarres University for financial support of the study and Iranian Blood Transfusions Organization (1370 D 11 IM) for providing Lab. and technical facilities. References [1] Ioannovich JD, Hinzmann RD, Deichsel G, Steinmann GG. Rationale, design and performance of a clinical trial to investigate interferon-gamma (Imukin) in the prophylactic treatment of severe burns-related infections. Intensive Care Med 1996;22(Suppl. 4):S468–73. [2] Brusselaers N, Monstrey S, Snoeij T, Vandijck D, Lizy C, Hoste E, et al. Morbidity and mortality of bloodstream infections in patients with severe burn injury. Am J Crit Care 2010;19:e81–7. [3] Zimmer S, Pollard V, Marshall G, Garofalo R, Traber D, Prough D, et al. The 1996 Moyer Award: effects of endotoxin on the Th1/Th2 response in humans. J Burn Care Res 1996;17:491–6. [4] Barrow RE, Jeschke MG, Herndon DN. Early fluid resuscitation improves outcomes in severely burned children. Resuscitation 2000;45:91–6. [5] Peter F, Schuschke D, Barker J, Fleishcher-Peter B, Pierangeli S, Vogt P, et al. The effect of severe burn injury on proinflammatory cytokines and leukocyte behavior: its modulation with granulocyte colony-stimulating factor. Burns 1999;25:477–86. [6] Murphy TJ, Choileain NN, Zang Y, Mannick JA, Lederer JA. CD4+ CD25+ regulatory T cells control innate immune reactivity after injury. J Immunol 2005;174:2957–63. [7] Bystrova NA, Miasnikov AD, Prokopenko LG. Development of immunosuppression and its correction with cell membrane stabilizers in burn trauma. Patol Fiziol Eksp Ter 1995:24–7. [8] Zedler S, Faist E, Ostermeier B, von Donnersmarck GH, Schildberg FW. Postburn constitutional changes in T-cell reactivity occur in CD8+ rather than in CD4+ cells. J Trauma 1997;42:872–80 [discussion 80–1]. [9] Murray HW. Current and future clinical applications of interferon-gamma in host antimicrobial defense. Intensive Care Med 1996;22(Suppl. 4):S456–61. [10] Mi L. Effects of fat intake on immunomodulation in burned patients. Zhonghua Zheng Xing Shao Shang Wai Ke Za Zhi 1992;8:257–60 [327]. [11] Le Boucher J, Farges M-C, Minet R, Vasson M-P, Cynober L. Modulation of immune response with ornithine A-ketoglutarate in burn injury: an arginine or glutamine dependency? Nutrition 1999;15:773–7.

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