Viral Infections Endothelial damage caused by ... - Nature

Bone Marrow Transplantation (2003) 31, 475–479 & 2003 Nature Publishing Group All rights reserved 0268-3369/03 $25.00

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Viral Infections Endothelial damage caused by cytomegalovirus and human herpesvirus-6 H Takatsuka1, T Wakae1, A Mori1, M Okada1, Y Fujimori1, Y Takemoto1, T Okamoto1, A Kanamaru2 and E Kakishita1 1 Second Department of Internal Medicine, Hyogo College of Medicine, Hyogo, Japan; and 2Third Department of Internal Medicine, Kinki University School of Medicine, Osaka, Japan

Summary: Infection with cytomegalovirus (CMV) or human herpesvirus-6 (HHV-6) may have a role in vascular endothelial damage after bone marrow transplantation (BMT). In total, 41 patients who underwent BMT were classified into four groups (12, 10, 7, and 12 patients who were infected with both CMV and HHV-6, CMV alone, HHV-6, and neither virus, respectively). Levels of thrombomodulin, plasminogen activator inhibitor-1, and cyclic GMP were 7.571.7 FU/ml, 76.4724.1 ng/ml, and 9.5171.1 pmol/ ml, respectively, in the patients with both viruses, while the respective values were 2.970.67 FU/ml, 33.878.09 ng/ ml, and 2.9071.4 pmol/ml in patients infected with CMV alone, 4.870.96 FU/ml, 47.779.21 ng/ml, and 5.4870.55 pmol/ml in patients with HHV-6 alone, and 1.670.39, 17.577.88 ng/ml, and 0.4570.3 in those with neither virus. All three markers were significantly higher in the three groups with at least one virus than in the uninfected patients (Po0.05), and were also higher in patients with HHV-6 alone than in those with CMV alone (Po0.05). These results suggest that infection by CMV or HHV-6 causes vascular endothelial injury, with HHV-6 having a stronger effect than CMV, and combined infection having a stronger effect than either virus alone. Such viral infection may be a cause of thrombotic microangiopathy after BMT. Bone Marrow Transplantation (2003) 31, 475–479. doi:10.1038/sj.bmt.1703879 Keywords: cytomegalovirus; human herpesvirus-6; vascular endothelial damage

Thrombotic microangiopathy (TMA) has recently been attracting increasing attention as a complication of bone marrow transplantation (BMT).1 Immunosuppressants, acute graft-versus-host disease (GVHD), viral infection, and steroids have been reported as possible risk factors for secondary clinical manifestations of TMA after BMT, but the pathogenesis is still unknown.2–6 However, some

Correspondence: Dr H Takatsuka, Second Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinimiya, Hyogo 663-8501, Japan Received 15 October 2001; accepted 25 May 2002

information concerning possible secondary clinical manifestations of TMA7–10 and an association between vascular endothelium damage and increased levels of cytokines11 as well as chemokines12,13 has been reported. We investigated the impact of viral infection, particularly cytomegalovirus (CMV) and human herpesvirus (HHV)-6, on the vascular endothelium after BMT, because these two viruses are common infections post-BMT. The possibility that such viral infections could be a factor in causing TMA after BMT was also examined.

Materials and methods In total, 41 consecutive patients who were seropositive for CMV and/or HHV-6 or whose donors were seropositive and did not have reactivation of CMV or HHV-6 before BMT were studied. They underwent allogeneic BMT at our institution from October 1996 through September 1999. Table 1 shows the age, sex, underlying disease, and donor type, as well as conditioning regimen and GVHD prophylaxis. We made a diagnosis of TMA when the reticulocyte count increased and the platelet count decreased in addition to presence of criteria corresponding to grade 2 or higher in the classification system of Zeigler et al2 (fragmented erythrocytes comprising at least 1.3% of 2000 peripheral blood cells counted and elevation of LDH). The basic conditioning regimen was busulfan (American Home Products Corporation Madison, USA), cyclophosphamide (Bristol-Myers Squibb, NY, USA), and total body irradiation (busulfan at a dose of 4 mg/kg/day 2 days, cyclophosphamide at 30 mg/kg/day 2 days, and irradiation at 3 Gy 4) or busulfan and cyclophosphamide (busulfan at 4 mg/kg/day 4 days and cyclophosphamide at 60 mg/kg/day 2 days). GVHD prophylaxis was with cyclosporin (CsA) (Novartis, Basel, Switzerland) plus methotrexate (American Home Products Corporation Madison, USA), with or without methylprednisolone (Pharmacia Corporation, Peapack, USA).14 CsA was infused from day 1 for 24 h, and was kept at a target blood level of 400–600 ng/ml until marrow engraftment, after which the dosage was varied depending on the symptoms of GVHD. If GVHD became worse, the doses of CsA and methylprednisolone were increased or CsA was replaced with FK506 (Tacrolimus

Endothelial damage by CMV and HHV-6 H Takatsuka et al

476 Table 1

Clinical characteristics

Age, range (median) Sex, M : F

Both viruses (n=12)

CMV only (n=10)

HHV-6 only (n=7)

Uninfected (n=12)

16–44 (31) 6:6

17–41 (31) 4:6

25–49 (31) 5:2

25–44 (34) 7:5

1 2 3

4 4

2 1

3 4

Underlying disease AML ALL MDS SAA LBL CML

1

1

1

2 4

1

3

4

Donor type R UR

3 9

3 7

5 2

10 2

10 2

8 2

2 4 1

7 4 1

9 3

8 2

4 3

7 5

Conditioning regimen BU/CY/TBI BU/CY CY/TLI GVHD prophylaxis CsA/MTX/mPSL CsA/MTX

AML: acute myelogenous leukemia, ALL: acute lymphoblastic leukemia, MDS: myelodysplastic syndrome, SAA: severe aplastic anemia, LBL: lymphoblastic lymphoma, CML: chronic myelogenous leukemia, R: related, UR: unrelated, BU: busulfan, CY: cyclophosphamide, TBI, total body irradiation, TLI: total lymphoid irradiation, CsA: cyclosporine, MTX: methotrexate, mPSL: methylprednisolone, CMV: cytomegalovirus, HHV: human herpesvirus.

hydrate) (Fujisawa Pharmacetical CO., LTD, Osaka, Japan). Screening of bronchoalveolar lavage fluid (BALF) and peripheral blood lymphocytes was performed by polymerase chain reaction (PCR) before and after BMT for detection of CMV and HHV-6 infection.15 BALF and peripheral blood samples were collected on day 35 after BMT, and peripheral blood alone was collected once weekly until day 100. CMV and HHV-6 primers used and the amplification conditions were the same as reported previously.16,17 The direct immunoperoxidase method was also used for CMV antigen screening, employing a peroxidase-labelled monoclonal antibody for viral matrix protein p65 (HRP-C7).18 The immunoperoxidase method was used for CMV antigen screening of BALF on day 35 and for screening of peripheral blood once weekly until day 100, and then every 2–4 weeks depending on the clinical manifestations until discharge from hospital. Acyclovir (GlaxoSmithKline plc, Brentford, UK) (250 mg three times daily) was administered from day 7 to approximately day 30 for CMV prophylaxis. All patients received 12.5 g of immunoglobulin containing a high titer of anti-CMV antibodies every 2 weeks until 3 months after transplantation. To detect CMV infection, both the BALF and peripheral blood were checked for CMV–DNA on approximately day 35 after BMT by the PCR method.19 If either the peripheral blood or BALF was positive for CMV by at least one method, ganciclovir was administered according to the schedule proposed by Singhal et al20 (5 mg/ kg twice a day in week 1 and once a day in weeks 2 and 3). After 3 weeks, the dose was adjusted depending on the CMV–DNA level and the C7-HRP antigen level in Bone Marrow Transplantation

peripheral blood as well as the extent of myelosuppression in weeks 2 and 3. Blood samples were collected into tubes containing 3.8% citric acid at a ratio of 1 : 9. After separation by centrifugation at 1500 rpm for 10 min at 41C, plasma was stored at 801C. Plasminogen activator inhibitor type 1(PAI-1) and thrombomodulin (TM) levels were determined as markers of vascular endothelial damage. PAI-1 was measured using a commercially available chromogenic assay (Biopool, CA, USA) (normal; 4–43 ng/ml) and soluble TM was measured by a commercially available sandwich enzyme immunoassay based on monoclonal antibodies (Fuji-rebio Co. Ltd., Tokyo, Japan) (normal; 4.5 FU/ml).21 Cyclic GMP was measured by a commercially available sandwich enzyme immunoassay (R&D Systems, Inc. Minneapolis, MN, USA) (normalp0.08 pmol/ml) as an alternative to measurement of nitric oxide. These parameters were determined at the same times as the tests for CMV and HHV-6 infection were carried out. Statistical analysis was done using the Mann–Whitney U-test and Po0.05 was considered to indicate a significant difference.

Results Among the 41 patients who underwent BMT, 12 were infected with both CMV and HHV-6 infection was symptomatic in three and two patients, respectively (all with symptoms of interstitial pneumonia), 10 were infected with CMV-alone infection was symptomatic in three (with symptoms of interstitial pneumonia in one and a fever in the other two), seven were infected with HHV-6 alone


infection was symptomatic in one (who only developed fever), and 12 were not infected with either virus. In addition, VOD was not recognized in any one of the infected patients. However, acute GVHD occurred in all 12 patients infected with both viruses, seven of the 10 patients infected with CMV alone, five of the seven patients infected with HHV-6 alone, and two of the 12 uninfected patients. Table 1 summarizes the clinical characteristics of each group of patients. The four groups were comparable with regard to age, sex, underlying disease, conditioning regimen, GVHD prophylaxis, and donor type. Levels of thrombomodulin, PAI-1, and cyclic GMP were 7.57 1.7 FU/ml, 76.4724.1 ng/ml, and 9.5171.1 pmol/ml, respectively, in the patients with both viruses, while the respective values were 2.970.67 FU/ml, 33.878.09 ng/ml, and 2.907 1.4 pmol/ml in patients infected with CMV alone, 4.87 0.96 FU/ml, 47.779.21 ng/ml, and 5.4870.55 pmol/ml in patients with HHV-6 alone, and 1.670.39, 17.577.88 ng/ml, and 0.4570.3 in patients with neither virus. Levels of TM in patients infected with CMV alone, HHV-6 alone, and both viruses were significantly higher than those in uninfected patients (P ¼ 0.00078, ¼ 0.00034, and o0.00001, respectively). This was also the case with PAI1 and cGMP. The P-values for the three infected patient groups in the same order as above were P ¼ 0.00024, 0.00017, and 0.0001, respectively, for PAI-1, and those for cGMP, P ¼ 0.014, 0.007, and 0.00025. The levels of TM, PAI-1, and cGMP were also significantly higher in patients infected with HHV-6 alone than in those infected with CMV alone (P ¼ 0.012, 0.0049, and 0.042, respectively). Levels of TM, PAI-1, and cGMP in the patients infected with both agents were significantly higher than those in patients infected with CMV alone (P ¼ 0.00047, 0.0029, and 0.0039, respectively), and with HHV-6 alone (P ¼ 0.01, 0.011 and 0.022). Among the four groups, the levels of these three parameters were the highest in the patients infected with both viruses (Figures 1–3). In all patients, the levels of TM, PAI-1, and cGMP were normal before BMT. There was no simple association between the appearance of symptoms and levels of markers of vascular endothelial injury. Patients revealing the highest level of any marker

477

Figure 2 PAI-1 levels in patients infected with CMV alone, HHV-6 alone, both viruses, and neither virus. *Mann–Whitney U-test.

Figure 3 Cyclic GMP levels in patients infected with CMV alone, HHV-6 alone, both viruses, and neither virus. *Mann–Whitney U-test.

of vascular endothelial injury did not necessarily have the severest symptoms. Since the PCR test is qualitative, the correlation between number of copies of virus and levels of TM and PAI-1 cannot be delineated by this test. The titer of CMV antigen as determined by the C7-HRP method was not significantly related to the level of TM or PAI-1, however. Among the 41 patients, four patients who were infected with both viruses developed TMA following BMT.

Discussion

Figure 1 Soluble TM levels in patients infected with CMV alone, HHV-6 alone, both viruses, and neither virus. *Mann–Whitney U-test.

TMA has been reported to occur in 3–71% (mean: 14%) of patients after allogeneic BMT. Thus, the incidence can be high, but it varies widely. The mortality rate from TMA also varies widely from 0 to 100%.1,2 A diverse etiology and consequent variations in severity may account for the marked variability of the incidence, clinical manifestations, and prognosis of TMA after BMT. In general, the risk factors for TMA after BMT seem to be intensive conditioning (irradiation/chemotherapy), use of immunosuppressants such as cyclosporine (CsA) and FK506, GVHD, and infection.1–6 It has also been reported that Bone Marrow Transplantation


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fungal infection, diffuse alveolar hemorrhage, hepatic venoocclusive disease, GVHD, and CMV infection are frequently associated with TMA.3 In fact, vascular endothelial CMV infection can be detected in about 50% of patients who develop TMA secondary to human immunodeficiency virus infection.22 In patients undergoing kidney transplantation, ganciclovir was reported to be effective for TMA after plasmapheresis failed to achieve a response.23 With regard to HHV-6, it has been reported that infection of vascular endothelial cells with this virus leads to cell damage or activation, which may predispose to the development of TMA.24 The findings obtained in the present study suggest that vascular endothelial cells are both stimulated and injured by infection with CMV and HHV-6. The levels of these parameters might have increased secondary to their reactivation rather than to infection with these viruses. Although details are not given in figures, the levels of TM and PAI-1 were increased by reactivation of HHV-6 or CMV in other cases, and in other cases, they were decreased when HHV-6 or CMV resolved. In some other cases, the levels of TM and PAI-1 changed in association with an increase and decrease in level of C7HRP. Our findings also indicated that vascular endothelial injury caused by HHV-6 was significantly more severe than that caused by CMV infection, while the damage arising from infection with both viruses was significantly more severe than that caused by a single virus. In the present cases, infection with either CMV or HHV-6 manifested only with fever and interstitial pneumonia. Neither gastritis nor enteritis as reported in the literature could be demonstrated in any of our patients. In addition, the vascular endothelial injury was somewhat more severe in patients infected with HHV-6 than in those with CMV, but symptoms experienced did not vary according to virus. These data suggest that viral infection should not be overlooked when the etiology of TMA after BMT is discussed. As described above, GVHD and VOD predispose patients who have undergone BMT to TMA. In the present study, VOD occurred in none of the patients. The occurrence of GVHD was increased significantly in patients infected with a virus or viruses over uninfected patients. The occurrence was also significantly increased in patients infected with two viruses as compared with that in patients infected with either virus alone. The patients infected with CMV and HHV-6 were not significantly different from each other in terms of occurrence and severity of GVHD. We have previously reported that systemic inflammatory response syndrome may be involved in the onset of complications after BMT.25 Systemic inflammatory response syndrome is related to various inflammatory cytokines and to the vascular endothelial injury caused by these agents.26,27 Data obtained in the present study suggest that viral infection may lead to elevation of cytokine levels. In the present study, the level of any cytokine was not determined. The relation between CMV infection and levels of inflammatory cytokines after BMT has been reported recently,28 which then causes vascular endothelial damage, or else that viral infection causes direct injury to vascular endothelial cells and the consequent elevation of TM and PAI-1 levels, thus leading to the onset of TMA. Our data also suggest that nitric oxide may be involved in the

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pathogenesis of TMA, although cyclic GMP was measured instead of directly monitoring nitric oxide. In conclusion, viral infection may induce vascular endothelial damage after BMT, the severity of which depends on the type of virus. Infection with HHV-6 seems to cause more severe vascular endothelial injury than does infection with CMV. In addition, there is a possibility that infection with these viruses may contribute to the onset of TMA after BMT.

Acknowledgements We thank Ms R Tanaka for her expert technical assistance.

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