Introduction
There is an emerging body of literature on the role of endotheliotropic viruses, specifically
cytomegalovirus (CMV) and parvovirus B19 (B19), as potentially being causally associated with a myriad of
autoimmune connective tissue disease (CTD) syndromes including Wegener's granulomatosis, microscopic
polyarteritis nodosa , Henoch Schonlein purpura and dermatomyositis. We will present two cases of
classic CTD syndromes where there was an apparent association between B19 and CMV infection and the onset
of collagen vascular disease
Clinical History
A 56 year old male presented with progressive tightening of the lower extremities and proximal arms.
A biopsy revealed morphologic changes typical of eosinophilic fasciitis. However he continued to develop
features of progressive sclerosis including evidence of esophageal dysmotility, sclerodactyly, and
progressive dyspnea, the latter prompting an open lung biopsy. The lung biopsy showed an active
paucicellular capillaritis in concert with vascular drop out and concomitant ensuing septal fibroplasia.
Serology showed a myriad of positive connective tissue disease markers including antibodies to Ro, La,
and RNP. The patient's quantitative IgG antibodies to B19 were markedly elevated although in the absence
of discernible IgM antibodies. B19 DNA was discovered in the patient's lung and skin biopsies.
Case 5 - Figure 1 - There is a striking sclerodermoid tissue reaction involving the dermis characterized by widened collagen bundles arranged parallel to the long axis of the epidermis.
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Case 5 - Figure 2 - C5-C9 - There is prominent deposition of C5b-9 within the microvasculature of the skin corroborative of in vivo activation of the complement cascade sequence and hence indicative of a humorally mediated microangiopathy syndrome mediated by anti-endothelial cell antibodies. RT in situ PCR shows parvovirus B19 and TNF transcript expression in endothelia and in pervascular mononuclear cells (arrows).
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Case 5 - Figure 3 - A lung biopsy shows homogeneous septal expansion with associated vascular drop out.
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Diagnosis
Parvovirus B19 associated Scleroderma
Subsequent Clinical Course
He was treated with prednisone and immuran. He continues to be symptomatic and as well has
experienced a progressive decline in his pulmonary function tests.
Discussion
This case serves as an important one regarding the role of B19 in the evolution of the classic
autoimmune endothelial cell microvascular injury syndrome of scleroderma.
Systemic sclerosis (SSc) is a distinctive idiopathic autoimmune CTD syndrome which differs from the
other CTD's by its relentless and progressive course of collagen overproduction leading to irreversible
morbid alterations of the skin, esophagus, lung, kidney, and heart. Overall the prognosis for this
condition is much worse than other CTD syndromes. Central to the pathology is an endothelial cell injury
syndrome in concert with excessive production of collagen. It is unclear whether the collagen
overproduction is a sequel of endothelial injury or reflects an inherent abnormality of fibroblasts
independent of the vasculopathy. More likely is a synergy between vascular injury and excessive
fibroblastic activation. Potential inciting factors for a combination of endothelial injury and
sclerosis have been explored. A possible role of B19 infection in the evolution of SSc is suggested by
the demonstration of B19 in a high percentage of bone marrow biopsies from SSc patients in the absence of
demonstrable B19 viremia. These patients had a high incidence of anti-B19 NS1 antibodies, a possible
marker of persistent infection. An association of B19 infection with other vascular injury syndromes
including Wegener's granulomatosis, Henoch Schonlein purpura, microscopic polyarteritis nodosa has been
previously documented. Not only is the association in the context of positive serologies but we have
shown B19 transcript localization to endothelial cells, suggesting a direct role for B19 parasitism in
the evolution of vascular injury.
We recently completed a study where we demonstrated a role for parvovirus B19 in the evolution of
systemic scleroderma. Our study group comprised 12 patients who fulfilled clinical criteria for a
diagnosis of systemic sclerosis according to those criteria outlined by the American College of
Rheumatology. All of these patients have been thoroughly evaluated for the presence of telangiectasias,
cutaneous calcinosis, Raynaud's phenomenon, nailfold capillary abnormalities, arthritis, and esophageal,
cardio-pulmonary and renal involvement and other skin signs was performed.
Routine light microscopy
Immunofluorescent studies.
For this test procedure, the following assays were conducted including I gG, IgA, IgM, fibrin, and C3.
An indirect immunofluorescence (IF) methodology with a fluorescein-conjugated rabbit anti-mouse antibody
was used to detect the presence of C5b-9.
Molecular studies
The DNA was extracted from paraffin embedded skin biopsies according to previously published methods
and nested PCR for B19 DNA was performed. The gels were stained with ethidium bromide, DNA bands were
visualized using UV light and the presence of a 286 bp band was taken as an indication of a positive
specimen.
RT in situ PCR
The protocol employed for paraffin-embedded tissues has been previously described . The tissue was
analyzed for B19 RNA using a primer sequence for the VP1 region, indicative of a productive infection.
and for evidence of tumor necrosis factor-α (TNFα);
expression was graded as absent (0), mild (1+), moderate (2+) or intense (3+).
Results
There were 9 women and 3 men aged 25-70 years (mean age 48.9 years). Seven patients were categorized
as having diffuse SSc and 5 as a limited form. With respect to the diffuse SSc group, anti-Scl 70
antibodies were detected in 3, anti-centromere antibodies in 1 and anti-nucleolar antibodies in 3. In the
limited group, anti-centromere antibodies were present in 3 and anti-Scl 70 antibodies in 1. Lung
involvement was present in 11 representing mild disease in 7 patients, moderate disease in 2 patients and
severe disease in 2 patients. The average duration of illness was 8 years (8.7 in the diffuse group and
6.75 years in the limited group). In all cases which had biopsy material available (cases
5,6,7,10,11,12) the papillary dermis was replaced by thickened collagen bundles exhibiting a parallel
disposition to the long axis of the epidermis. There was a marked reduction in the dermal papillae
capillary density.
In cases 5,10,11 and 12 there were conspicuous vasculopathic changes comprising mural edema with
degenerative and proplastic endothelial alterations, the former comprising nuclear pyknosis and
endothelial cell detachment and the latter characterized by endothelial enlargement and hyperplasia. A
perivascular lymphocytic infiltrate surrounded a few of the vessels. There was moderate fibrosis of the
reticular dermis. The adventitial dermis appeared to be preserved. The B19 RT in situ PCR studies
revealed prominent 2+ to 3+ expression in endothelia, perivascular inflammatory cells and fibroblasts. A
roughly parallel pattern both qualitatively and quantitatively was observed for TNFα expression.
In cases 6 and 7 there was prominent hyalinizing fibroplasia of the reticular and adventitial dermis
with atrophy of eccrine structures. Although B19 RNA was identified the staining was weak and focal
(i.e. 1+), being confined to endothelial cells and fibroblasts. A similar staining pattern was observed
for TNFα in case 7; due to unavailability of tissue TNFα studies were not carried out in case
6.
Immunofluorescence
Immunofluorescence testing was carried out in cases 5,6,7,10, and 11; in all cases there was prominent
deposition of C5b-9 within the microvasculature.
Solution phase PCR
Tissue was available in 5 cases; in 3 B19 DNA was detected in the skin tissue (cases 5,6, and 10).
The positive and negative controls gave the expected results. The PCR positive cases could represent
circulating viral DNA unrelated to the histologic changes and PCR negative cases could represent
relatively poor sensitivity as reported in paraffin embedded tissue [11].
Concluding Remarks
In all cases studied there was evidence of B19 RNA expression in endothelium and neighboring
mononuclear cells and fibroblasts through an RT in situ PCR methodology while immunohistology for B19
associated viral antigens showed positivity amidst mononuclear cells and fibroblasts. Based on prior
studies a similar pattern of expression is not seen in control specimens. Cases showing the greatest
degree of viral transcript expression manifested the most severe active endothelial cell injury and
associated perivascular inflammation, events held to be important in the initial phases of SSc. The only
other explanation for this high incidence of B19 infection in our patient population would be an
opportunistic infection in an immunologically incompetent host; in another study control bone marrow
specimens in immunocompromised hosts failed to detect B19 DNA.
A recent study demonstrated B19 genomic sequences in 57% of bone marrow biopsies from unselected SSc
patients compared to controls (57% vs 0, p<.001) as well as in skin biopsies (53% vs 25%)and cultured
skin fibroblasts. Furthermore the authors found antibodies to B19 NS1 protein
in 33% of these patients with its absence in control patients. The only clinical difference between
B19-positive and B19-negative SSc patients was a shorter disease duration in those whose bone marrow was
shown to contain B19. The current study showed no specific differences between those patients with and
without evidence of B19 infection.
B19 has been implicated in other vascular injury syndromes of presumptive immune based etiology.
Specifically, Kawasaki's disease is reported in children expressing B19 specific antibodies and DNA in
peripheral blood. Small vessel vasculitic syndromes associated with B19 infection include cases of WG,
microscopic PAN, and HSP.where B19 DNA has been isolated from the peripheral blood and skeletal muscle.
Such cases have shown a poor response to cyclophosphamide and other immunosuppressant agents, the
conventional standard therapy. One study found an association between histologic evidence of giant cell
arteritis and the presence of B19 DNA in tissue.
In 4 of the patients in whom there was molecular documentation of B19 infection high antibody levels
of IgG were detected in the absence of IgM. Patients in whom we have suspected a role for B19 in the
propagation of other specific CTD's typically had this profile. Sera from these same patients fail to
recognize certain peptide epitopes, suggesting a selective defect in antibody response to viral antigens
that allows persistent B19 infection. Presumably the ability to launch an IgM immune response, critical
for viral clearance, is impaired.
Knowing that lesional skin in SSc patients contains B19 genome and given the localization of viral
transcripts to endothelia, fibroblasts, and inflammatory cells, the question arises as to how B19
parasitism of endothelium might contribute pathogenetically. Endothelial injury has previously been
shown in patients with SSc; patients with SSc have elevated levels of circulating endothelin-1, which,
like Factor 8, correlates with vascular injury. In one study human dermal microvascular and umbilical
vein endothelia were cultured with native and heat inactivated serum from SSc patients and SSc patients
were tested for the presence or absence of antiendothelial antibodies by IIF. The SSc sera manifesting
antiendothelial cell antibodies induced apoptosis of endothelia, an effect mediated by the Fas pathway.
In our cases there was histologic evidence of endothelial cell injury; the observed reduction in the
superficial vascular plexus density was a presumptive sequel of endothelial injury. A similar event
sequence is seen in patients with dermatomyositis; specifically cutaneous endothelialitis in
dermatomyositis is followed by vascular drop out. Furthermore in our cases there was a direct
correlation between the extent of degenerative endothelial cell alterations and the degree of B19 RNA
expression suggesting a causal role of B19 parasitism in the propagation of the endothelial cell
dysfunction.
The B19 receptor is globoside, a neutral glycosphingolipid of red cell membranes referred to as the
blood group P antigen that is also found in megakaryocytes and endothelia. Endothelial injury in
patients infected with B19 likely reflects a combination of direct viral cytotoxicity and humoral
immunity. It has been shown that B19 exerts a cytotoxic effect on infected cells through a
non-structural protein which it encodes designated NS-1; in vitro studies show that NS-1 induces DNA
fragmentation characteristic of apoptosis. Cells infected by B19 are also sensitized to TNF a - induced
apoptosis. Ceramide levels are enhanced by B19 infection and NS-1 expression; ceramide also augments
TNFa mediated apoptosis. TNF a -induced apoptosis is part of the Fas system of cell injury. Antibody
cellular cytotoxicity in patients with SSc is dependent of the Fas system and would be exaggerated by any
upregulation of the Fas pathway as occurs in the setting of enhanced TNF a
expression due to B19 infection. Our cases showing the greatest inflammation and overt endothelial
injury had the highest B19 and TNFa
transcript signals. Accelerated apoptosis leads to the surface displacement of various nuclear and
cytoplasmic based antigens including Ro, LA, and RNP. In addition there may be displacement of certain
plasma membrane based phospholipids to the surface, especially phosphatidylserine and
phosphatidylethanolamine. The exposure of these neoantigens could then evoke antibody production in the
genetically predisposed host and contribute to direct endothelial cell injury. The inherent injury
mediated by the virus could expose cryptic antigens to naïve autoreactive T and/or B cells.
Corroborating a role for autoimmunity as opposed to a nonimmunologic cytolytic event is the deposition of
C5b9 within the cutaneous vasculature in all cases studied; vascular C5b9
deposition characterizes anti-endothelial cell antibody syndromes. It has also been shown that specific
target antigens implicated in SSc (Topoisomerase, U1 sn RNP, and U3 sn RNP) show molecular similarity
with certain viruses such as cytomegalovirus, feline sarcoma virus, and herpes simplex, hence any
endogenous autoantibodies already present in these patients could target cells infected with viruses
manifesting target antigen homology. Perhaps molecular mimicry also plays a role in inducing endothelial
cell injury in the setting of B19 endothelial cell parasitism.
We conclude that direct endothelial, fibroblast and inflammatory cell infection by B19 is observed in
lesional skin of patients with SSc. Whether this indicates a pervasive role of B19 in SSc requires
further study. Conventional immunosuppressive therapy may not be effective in these patients; one could
speculate that this may perpetuate viral infection. There is precedent in the literature for a role for
intravenous γ-globulin in the treatment. Enhanced TNF a
expression raises consideration to anti-TNF therapy in these patients as an ancillary modality.
References
- Ferri C et al. Systemic sclerosis: demographic, clinical and serological features and survival in 1012 patients. Medicine 2002; in press.
- Ferri C, Zakrzewska K, Longombardo G et al. Parvovirus B19 infection of bone marrow in systemic sclerosis patients. Clin Exp Rheum 1999;718-720
- Ferri C, Zakrzewska K, giuggioli D e tal. Parvovirus B19 infection in the skin and bone marrow of systemic sclerosis patients. Arthritis Rheum 2000;42(suppl):abstr ;1491
- Finkel TH, Torok TJ, Ferguson PJ et al. Chronic parvovirus B19 infection and systemic necrotising vasculitis: opportunistic infection or aetiological agent? Lancet. 1994 May 21;343(8908):1255-8.
- Nikkari S, Mertsola J, Korvenranta H. Wegener's granulomatosis and parvovirus B19 infection. Arthritis Rheum. 1994 Nov;37(11):1707-8.
- Veraldi S, Mancuso R, Rizzitelli G et al. Henoch-Schonlein syndrome associated with human Parvovirus B19 primary infection. Eur J Dermatol. 1999 Apr-May;9(3):232-3.
- Crowson AN, Magro CM, Dawood MR. A causal role for parvovirus B19 infection in adult dermatomyositis and other autoimmune syndromes. J Cutan Pathol 2000;27:505-15.
- Magro CM, Crowson AN, Dawood M, Guovo GJ. Parvoviral infection of endothelial cells and its possible role in vasculitis and autoimmune diseases. J Rheumatol 2002;29:1227-1235
- Masiet AT, Rdonan GP Medsger TA Jr et al. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum 1980;23:580-590
- Fridel E, Bekassy AN, Larsson B et al. Polymerase chain reaction with double primer pairs for detection of human parvovirus B19 induced aplastic crises in family outbreaks. Scand J Infect Dis. 1992;24(3):275-82.
- Nuovo GJ, MacConnell PB, Simsir A et al. Correlation of the in situ detection of polymerase chain reaction-amplified metalloproteinase complementary DNAs and their inhibitors with prognosis in cervical carcinoma. Cancer Res. 1995 Jan 15;55(2):267-75.
- Ferri C, Giuggioli D, Sebastiani M, Zakrzewska K, Azzi A, Panfilo S, Abatangelo G: Parvovirus B19 infection of cultured skin fibroblasts from systemic sclerosis patients. Arthritis Rheum 2002: in press.
- Fukushige J, Takahashi N, Ueda K et al. Kawasaki disease and human parvovirus B19 antibody: role of immunoglobulin therapy. Acta Paediatr Jpn. 1995 Dec;37(6):758-60.
- Lhote F, Guillevin L. Polyarteritis nodosa, microscopic polyangiitis, and Churg-Strauss syndrome. Clinical aspects and treatment. Rheum Dis Clin North Am. 1995 Nov;21(4):911-47.
- Magro CM, Dawood MR, Crowson AN. The cutaneous manifestations of human parvovirus B19 infection. Hum Pathol 2000, 31:488-97.
- Diaz F, Collazos J. Glomerulonephritis and Henoch-Schonlein purpura associated with acute parvovirus B19 infection. Clin Nephrol. 2000 Mar;53(3):237-8.
- Watanabe T, Oda Y. Henoch-Schonlein purpura nephritis associated with human parvovirus B19 infection. Pediatr Int. 2000 Feb;42(1):94-6.
- Gabriel SE, Espy M, Erdman DD et al. The role of parvovirus B19 in the pathogenesis of giant cell arteritis: a preliminary evaluation. Arthritis Rheum. 1999 Jun;42(6):1255-8.
- Naides SJ. Rheumatic manifestations of parvovirus B19 infection. Rheum Dis Clin North Am. 1998 May;24(2):375-401.
- Morelli S, Ferri C, DiFrancesco L et al. Plasma endothelin-1 levels, pulmonary hypertension, and lung fibrosis in patients with systemic sclerosis. Am J Med. 1995 Sep;99(3):255-60.
- Ferri C, Latorraca A, Catapano G et al. Increased plasma endothelin-1 immunoreactive levels in vasculitis: a clue to the use of endothelin-1 as a marker of vascular damage? J Hypertens Suppl. 1993 Dec;11 Suppl 5:S142-3.
- Sgonc R, Gruschwitz MS, Boeck G et al. Endothelial cell apoptosis in systemic sclerosis is induced by antibody-dependent cell-mediated cytotoxicity via CD95. Arthritis Rheum. 2000 Nov;43(11):2550-62.
- Crowson AN, Magro CM. The role of microvascular injury in the pathogenesis of cutaneous lesions of dermatomyositis. Hum Pathol. 1996 Jan;27(1):15-9.
- Brown KE, Anderson SM, Young NS. Erythrocyte P antigen: cellular receptor for B19 parvovirus. Science. 1993 Oct 1;262(5130):114-7.
- Cooling LL, Koerner TA, Naides SJ. Multiple glycosphingolipids determine the tissue tropism of parvovirus B19. J Infect Dis. 1995 Nov;172(5):1198-205.
- Sol N, Le Junter J, Vassias I. Possible interactions between the NS-1 protein and tumor necrosis factor alpha pathways in erythroid cell apoptosis induced by human parvovirus B19. J Virol. 1999 Oct;73(10):8762-70.
- Pitonni V, Isenberg D. Apoptosis and antiphospholipid antibodies. Semin Arthritis Rheum 1998 Dec;28(3):163-78. Review.
- Levine JS, Koh J, Suband R, Rauch J. Apoptotic cells as immunogen and antigen in the antiphospholipid syndrome. Exp Mol Pathol. 1999 Apr;66(1):82-98. Review.
- Magro CM, Crowson AN, Regauer S. Mixed connective tissue disease. A clinical, histologic, and immunofluorescence study of eight cases. Am J Dermatopathol. 1997 Jun;19(3):206-13.
- Magro CM, Crowson AN. The immunofluorescent profile of dermatomyositis: a comparative study with lupus erythematosus. J Cutan Pathol. 1997 Oct;24(9):543-52.
- White B. Immunopathogenesis of systemic sclerosis. Rheum Dis Clin North Am. 1996 Nov;22(4):695-708.
