ARTICLE IN PRESS Acta histochemica 106 (2004) 107–110
www.elsevier.de/acthis
A novel pattern of pp65-positive cytomegalic endothelial cells circulating in peripheral blood from a renal transplant recipient Qing Yea, Guanghua Luoa, Xiaozhou Hea, Lu Zhenga, Xuan Donga, Xianlin Xua, Jinsheng Gaob, Peter Nilsson-Ehlec, Ning Xuc,* a
Comprehensive laboratory, The Third Affiliated Hospital, Suzhou University, Changzhou 213003, China Heredity Institute, Medical College of Suzhou University, Suzhou 215001, China c Department of Clinical Chemistry, Institute of Laboratory Medicine, University Hospital of Lund, Lund S-221 85, Sweden b
Received 25 November 2003; received in revised form 29 January 2004; accepted 30 January 2004
KEYWORDS Human cytomegalovirus; Cytomegalic endothelial cells; Immunohistochemistry; Kidney transplantation
Summary The present study reports a novel pattern of cytomegalic endothelial cells (CEC) in peripheral blood from a female renal transplant recipient infected with human cytomegalovirus (HCMV), which has not been reported previously. Localization of specific early antigen of HCMV, pp65 antigen, was examined by immunohistochemistry. Staining of an endothelial cell marker (CD34) was used to characterize endothelial cells. It is demonstrated that many leukocytes surrounded and adhered to a proteinlike material, in which pp65-positive CEC were detected. The composition and function of this protein-like material are yet unknown. The patient lacked clinical symptoms of HCMV disease. Furthermore, similar localization patterns were found in other renal transplant recipients suffering from HCMV infections as determined by real-time PCR to detect HCMV DNA in blood. These patients showed no or only minor clinical symptoms of HCMV infection. It is suggested that these novel localization patterns of CEC may play a role in the host defense in patients infected with HCMV, but the exact relation between HCMV infection and CEC formation needs further investigation. & 2004 Elsevier GmbH. All rights reserved.
Introduction Human cytomegalovirus (HCMV) infection remains a major health threat in immunocompromised patients, such as those who have undergone solid
organ transplantations (Dummer et al., 1985; Singh et al., 1988; Kano and Shiohara, 2000) and in those suffering from AIDS (Fiala et al., 1986, 1991; Gerna et al., 1998). In renal transplant recipients, the clinical symptoms related to HCMV disease and the
*Corresponding author. Tel.: þ 46-173-462; fax: þ 46-130-064. E-mail address:
[email protected] (N. Xu). 0065-1281/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2004.01.003
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prevention of HCMV infection show variations among patient populations, depending mainly on the intensity of immunosuppression (Sia and Patel, 2000). Patients with active HCMV infection may show subtle disturbances of organ function, even without clinical symptoms of HCMV infection. One of the indicators of subtle disturbances is a decrease in pulmonary diffusion of carbon monoxide (Van Son et al., 1987). Endothelial cells (ECs), together with epithelial cells, fibroblasts, and smooth muscle cells are important targets for the virus (Sinzger et al., 1995). In particular, HCMV has been shown to infect and fully replicate in ECs in immunocompromised patients (Kas-Deelen et al., 2001). In disseminated infection, cytomegalic endothelial cells (CEC) may also circulate in peripheral blood (Percivalle et al., 1993), and virus dissemination is mediated by leukocytes carrying virus acquired from infected endothelium and these cells transmit the infection to uninfected EC (Waldman et al., 1995, 1998; Grundy et al., 1998). In the present study, peripheral blood samples from a renal transplant recipient infected with HCMV were tested for the presence of pp65, a specific antigen of HCMV. An interesting finding is that many leukocytes were surrounding and adhering to a protein-like material in which several pp65positive EC were detected. However, the patient lacked clinical symptoms of HCMV infections.
Material and methods A 55-year-old female patient who received kidney transplantation in the Third Affiliated Hospital of SuZhou University on 9 April 2002 was the subject of the present study. Both donor and recipient were positive for HCMV-IgG. HCMV pp65 antigen was detected using the CMV POt Kit (IQ Products, Groningen, The Netherlands). Briefly, 10 ml blood was drawn from a cubital vein via venipuncture and K2-EDTA was used as anticoagulant. After dextran separation and erythrocyte lysis, leukocytes were centrifuged onto two slides, fixed, and permeabilized to allow subsequent detection of CMV pp65 antigen. Positive staining of the HCMV pp65 antigen was detected by C10/C11 monoclonal antibodies, followed by a secondary horseradish peroxidaselabeled antibody and was visualized by using the substrate 3-amino-9-ethylcarbazole, according to the manufacturer’s instruction. After staining, slides were quickly stained by using Mayer’s haematoxylin solution. HCMV antigen-positive cells exhibited homogeneous red-brown nuclear staining
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and negative leukocytes appeared as blue-purple under the light microscope. HCMV-infected EC were identified by using CD34 monoclonal antibodies, which detect an EC marker (Silvestri et al., 1992; Hristov et al., 2003). The procedure was similar to that of the HCMV pp65 antigen assay except that the CD34 primary monoclonal antibody was used. EC exhibited homogeneous red-brown staining and leukocytes appeared blue-purple under the light microscope. Negative controls in the absence of primary antibodies were included in the experiments.
Results and discussion The HCMV pp65 antigen assay was applied to blood samples of the patient in the 13th week posttransplantation. A new pattern of CEC was found in peripheral blood. As shown in Fig. 1, many leukocytes surrounded and adhered to a proteinlike material that contained several pp65-positive CEC. The type of these cells was confirmed by staining with CD34 antibodies (Fig. 2), that identifies EC (Hristov et al., 2003). Grefte et al. (1993, 1995) described large CMV-infected EC (diameter 30–35 mm) in peripheral blood of patients with active CMV infections. These EC were present as single cells in the circulation. However, we found a unique localization pattern of CEC that has not been reported previously: several pp65-positive CEC being included in protein-like material surrounded by many leukocytes that adhere to it (Figs. 1 and 2). This protein-like material may be a result of interactions between leukocytes and HCMVinfected CEC, although the detailed mechanism of this phenomenon is not yet understood. According to previous reports, adhesion of polymorphonuclear leukocytes to human umbilical vein EC occurs via interactions of CD18 molecules (integrins) on the surface of leukocytes and intercellular adhesion molecule-1 on the surface of human umbilical vein EC (Gerna et al., 2000). The composition and function of the protein-like material observed in the present study are unclear. Furthermore, most of the leukocytes surrounding and adhering to the protein-like material are lymphocytes. The proteinlike material may be secreted by lymphocytes to prevent dissemination of HCMV. The presence of HCMV-infected EC in peripheral blood of patients with an active HCMV infection indicates that such an infection may be accompanied by widespread vascular damage. These EC have been found to progressively enlarge until detaching from small vessel walls and entering the
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Figure 2. CEC with CD34-positive staining (red-brown staining) are present in a protein-like material surrounded by adhering leukocytes. Original magnification, 200.
eight patients had fever and were clinically diagnosed as having pneumonia, but the CEC localization pattern was not found in these eight patients (unpublished data). Therefore, we propose that the presence of this novel localization pattern of CEC may play a role in the host defense in patients infected with HCMV. However, the specific mechanism and clinical relevance of CEC inclusion in protein-like material needs further study.
Acknowledgements
Figure 1. CEC with pp65-positive staining (red-brown staining) are present in a protein-like material (panels A– C) surrounded by adhering leukocytes. Original magnifications, (A) 100; (B) 200; and (C) 400.
circulation. Thus they represent a systemic parameter suitable for diagnosis of HCMV infection and for the study of pathogenesis of disseminated infections (Percivalle et al., 1993). A recent study suggested that CEC levels may be related to subclinical HCMV pneumonitis (Kas-Deelen et al., 2001). However, in the present study the patient did not have anti-HCMV therapy because clinical HCMV disease symptoms were not manifest. Furthermore, we examined 31 other patients and a similar localization pattern of CEC was found in seven patients. All patients did not show signs of active disease during the experiments. However,
Jiang Zhu, Qing-Feng Mo, Min Tan and Yan Tan provided excellent technical assistance. We thank Yaping Lu and Xuping Li for collection of samples. This study was supported by a research grant from the Jiangsu province and a research grant of the Third Affiliated Hospital of the Suzhou University.
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