Hypersensitivity to mosquito bites with natural-killer cell lymphocytosis: the possible implication of Epstein-Barr virus reactivation

ARTICLE

Hypersensitivity to mosquito bites (HMB) is characterized by severe local skin reactions which include erythematous swelling with bullae and necrotic ulcerations, then followed by depressed scars. In addition, general symptoms such as high-grade fever, lymphadenopathy, and liver dysfunction occur subsequent to the mosquito bites. This enigmatic disorder has been reported exclusively in restricted areas such as Asia [1-5] and Mexico [6]. Many patients with HMB tend to develop chronic active EBV infection (CAEBV) [4, 5], virus-associated hemophagocytic syndrome (VHAS) [1, 3], and Natural-Killer (NK) cell leukemia/ lymphoma [1-3, 5], occasionally overlapping with hydroa vacciniforme-like eruptions [5, 6].

Recent reports have shown a possible relationship between HMB and EBV infection. In situ hybridization analysis of lesional skin showed that infiltrated lymphoid cells overexpress EBV-encoded small nuclear RNAs (EBER), which are commonly produced in the latent phase of EBV infection [1, 7] and a clonal proliferation of EBV-infected NK cells was shown to be present in peripheral blood [8, 9]. Therefore, HMB has been considered to be one of the characteristic manifestations of EBV-associated NK cell lymphoproliferative disorders [7].

In this report, we describe a typical case of HMB, in which there was a marked increase of EBER-positive lymphoid cells in the lesional skin, as well as peripheral NK cell lymphocytosis after mosquito bites. The importance of monitoring EBV genome copy number, which might contribute to the pathogenesis of HMB, is discussed.

Case report

An 8-year-old Japanese boy was referred to our clinic in June 2000 for severe skin reactions after mosquito bites. For three consecutive summer seasons an erythematous swelling and blister formation occurred immediately after bites on his extremities and this was followed by a deep ulceration which gradually healed leaving a mildly depressed scar. In addition he had high-grade fever, as well as painful lymphadenopathy, which lasted for a few days. His familial and past histories were unremarkable except for chronic sinusitis which he had four years previously.

On examination at the first consultation, freshly erythematous, indurated plaques and blisters were present on his right leg (Fig. 1) and he had a high-grade fever up to 40° C with painful swellings of some superficial lymphnodes and mild palpable hepato-splenomegaly.

Biopsy specimens from the leg lesions showed epidermal spongiotic vesicles and dense perivascular infiltration of lymphoid cells in both dermis and subcutaneous tissue (Fig. 2A). The infiltrates consisted of non-atypical lymphoid cells and eosinophils without angiocentricity and vascular damage (Fig. 2B). Immunohistochemical staining revealed that more than 40% of the infiltrated cells were positive for CD3 and CD45RO, whereas 20% were positive for CD4, CD8, TIA-1, and granzyme B. CD56 positive cells increased to up to approximately 10% of the whole infiltrates (Fig. 2C, arrow). As detected by in situ hybridization (ISH) using an EBER-specific anti-sense oligonucleotide probe, 20% of these infiltrates including CD56⁺ cells expressed EBER (Fig. 2D, arrow).

Laboratory tests are presented in Table I. A mild increase of eosinophils and liver dysfunction were noted. His IgE level was elevated significantly, but the specific IgE against Aedes Communis was slight. The antibody titers against EBV indicated a past infection. The percentage of CD11b⁺ CD16⁺ CD56⁺ NK cells was increased up to approximately 50% of the peripheral lymphocytes. Morphologically they resembled large granular lymphocytes containing azurophilic bodies in the cytoplasm (Fig. 2E). No chromosomal abnormality was detected in cultured peripheral lymphocytes when they were stimulated with 200 units/ml rIL-2 for three days (data not shown). His bone marrow showed normocellularity without hemophagocytic or leukemic features.

To examine whether EBV infection is involved in the development of HMB, we performed a PCR. Genomic DNA was prepared from peripheral blood cells of the patient, two healthy volunteers and patients with EBV-associated disease. PCR primers were designed from within a single copy region, not including internal repeats and satellite motifs, of the EBNA-1 gene. EBNA-1 DNA was easily amplified from the DNA of the patients with EBV-associated disease and HMB (Fig. 3, lane 2, 3 and lane 6, respectively), however, the relative intensities of the PCR products were different. The EBNA-1 product of the patient was more intense than that of the others, indicating a high copy number of the EBV genome in his peri-pheral blood cells. To quantify the viral load, a cloned EBNA-1 plasmid was generated and the amplification step was completed in the linear range. After ethidium bromide staining, the signals for the specific resulting products were analyzed by densitometry (NIH image, Bethesda, MA, USA) and normalized with the results from a control plasmid. The gradual increase of the copy number paralleled the increase in the number of peripheral CD56⁺ NK cells even in winter when he was free of mosquito bites (Fig. 4). Moreover, there was EBNA-1 DNA in the patient's plasma, but not in the plasma of healthy controls (data not shown). These data indicate a lytic cycle of expansion of EBV in the patient.

Discussion

In the present report, we describe a HMB patient with peri-pheral NK cell lymphocytosis associated with EBV infection. In situ hybridization demonstrated that the lesional skin contained EBER positive lymphoid cells and PCR analysis revealed an extremely high EBV-DNA copy number in the peripheral blood. The EBV viral load increased somewhat even in winter in concert with a significant increase in the number of CD56⁺ NK cells.

Numerous reports show that EBER positive cells infiltrate at bite sites [1, 7], HMB occurs in close association with monoclonally EBV-infected NK cell lymphoproliferation in peripheral blood [2, 9], and that HMB presenting with high serum IgE tends to be observed in patients with the NK cell type of CAEBV [10]. These reports clearly indicate that the abnormally high number of EBV-infected peripheral NK cells and infiltrating EBER-positive cells in the lesional skin have pathogenic roles in HMB. Although the source of EBV-infected lymphoid cells has not been examined, from the previous reports and our finding of a parallel increase of peripheral NK cells and EBV copy number (Fig. 4), we consider that most of the EBV infect the NK cell population. More-over, our preliminary PCR analysis showing cell-free EBV in the plasma, which is not usually detected in healthy individuals [11, 12], suggests that repeated mosquito bites may make some of the numerous EBV-infected lymphoid cells enter into lytic cycles and release viral particles [13], against which vigorous cytotoxic T lymphocyte-mediated immune responses may be elicited, similar to the acute phase of infectious mononucleosis [14]. The repeated severe hypersensitivity reactions might induce a continuously elevated EBV load even in mosquito bite-free periods, which would eventually result in malignant EBV clonal expansion. Therefore, the monitoring of EBV viral load in peripheral leukocytes as well as in plasma is useful for evaluating the disease activity and anticipating progression to malignant EBV-associated lymphoproliferative diseases.

It has been shown that HMB is one of the characteristic manifestations of EBV-associated NK cell lymphoproliferation potentially leading to lymphoid malignancy [5, 6], which is similar to hydroa vacciniforme [15, 16]. However, we do not exclude the possibility that HMB might merely be one of the nonspecific symptoms of EBV-associated lymphoproliferation and that insidious progression to overt EBV-associated hematologic neoplasms would happen subclinically independent of mosquito bites. The detailed mechanisms of HMB remain unclear, although similar patients with HMB were endemically reported in Asia and Mexico [1-6]. The following factors may be contributory: 1) a genetic background linked to human leukocyte antigen (HLA) type; 2) environmental factors such as the fact that malaria infection or Euphobia tirucalli are involved in the development of African Burkitt's lymphoma as a cofactor [17]; 3) immunological tolerance induced by exposure to EBV infection early in life, and 4) prevalence of specific EBV subtype with higher tumorigenic potential [18], or lesser immunogenic, mutated EBV-related antigen expression.

We considered the diagnosis of HMB with the NK cell type of CAEBV because recently Kimura et al. proposed that a viral load exceeding 10^2.5 copies/mug of DNA should be used as a diagnostic criterion for CAEBV [10]. Although this patient shows neither symptoms suggesting progression of disease nor abnormal changes in laboratory tests, aggressive therapies such as stem cell transplantation [19, 20] should be considered in accordance with CAEBV therapy protocols, because HMB represents a high risk for progression to VHAS or EBV-associated NK cell lymphoma.

Article accepted on 16/5/02

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