—  SPECIALTY CONFERENCE  —

Dermatopathology

Case 6 - CMV Associated Microscopic Polyarteritis Nodosa with Skin, Kidney and Lung Involvement

Cynthia M. Magro
Ohio State University
Columbus, OH


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Clinical History
A 49 year old woman presented with a 2 week history of fever, vasculitic lesions on the lower extremities. In addition she subsequently developed shortness of breath. Bronchoalveolar lavage revealed extensive hemorrhage. Other laboratory investigations revealed a low haemoglobin along with moderate thrombocytopenia and lymphopenia, a positive rheumatoid factor, and depressed complement. Cytomegalovirus antibodies of both IgM and IgG isotype were positive. In addition CMV DNA was isolated from the blood. A skin biopsy was performed and revealed a striking pandermal vascular reaction characterized by nodular aggregates of neutrophils and mononuclear cells surrounding and permeating the cutaneous vasculature accompanied by extensive fibrin deposition. Many of the endothelial cells appeared proplastic, manifesting a hobnailed nuclear contour. In situ PCR studies for CMV revealed staining of endothelial cells for CMV. A few scattered cells within the interstitium were also positive. Although initially she was given exclusively immunsuppressive therapy which lead to worsening of her symptoms eventually she was given a combination of gancylcovir and prednisone. This combined therapy over the course of several weeks lead to resolution of her symptoms.


Case 6 - Figure 1 - There is a necrotizing neutrophil rich vasculitis involving the skin.
Case 6 - Figure 2 - An open lung biopsy shows a necrotizing capillaritis
Case 6 - Figure 3 - In situ hybridization studies for CMV RNA transcript expression reveals positive staining of endothelial cells.

Diagnosis :
CMV Associated Microscopic Polyarteritis Nodosa with Skin, Kidney and Lung Involvement.

Discussion
This case underscores the importance of viruses in the propagation of autoimmune disease. Cytomgegalovirus is emerging as an important trigger in the provocation of syndromic complexes that exactly recapitulate classic autoimmune disease. Human cytomegalovirus (CMV) can be associated with a state of acute and chronic infection in immunocompetent hosts. In chronic infections the patients harbor latent virus in permissive cells such as mononuclear cells and endothelial cells with periods of reactivation during which active replication leads to cell lysis and viral shedding. Although in the immunocompetent host, primary CMV infection or periods of reactivation may be completely subclinical, in immunocompromised hosts, there is considerable morbidity and mortality. In immunocompetent hosts, CMV has been implicated in the genesis of atherosclerotic lesions, rapidly progressive coronary artery disease and endothelialitis in cardiac transplant patients, coronary re-stenosis after angioplasty and inflammatory aortic disease. In immunocompromised hosts CMV endothelialitis with associated cytopathic changes is well described.

Viruses have been invoked as a potential basis for certain vasculopathic conditions of autoimmune based etiology. Perhaps the most widely accepted virally mediated vasculitic disease being hepatitis C associated mixed cryoglobulinemia. These diseases are defined by the tissue expression of an Arthus type III immune complex reaction, namely a leukocytoclastic vasculitis. We have recently reported on the role of parvovirus B19 in the evolution of various autoimmune syndromes proposing direct parasitism of endothelium, as opposed to immune complex formation, as a key event in the propagation of these disorders. In light of the established tropism of CMV for vascular endothelium, we hypothesized that CMV could be equally implicated in a similar spectrum of endothelial based systemic disorders. We report a series of 9 immunocompetent previously healthy adults who developed a significant micro and macroscopic – vasculopathic disease syndrome, which in 8 of the cases closely mimicked classic autoimmune disease. In each case there was a relatively abrupt onset of symptoms temporally associated with positive IgM and IgG CMV antibodies, +/- positive CMV cultures. The clinical features, pathologic spectrum and treatment modalities for CMV associated autoimmune disease in these 9 cases are described.

Materials and methods
Nine cases were prospectively available for study. In none of the cases was a role for CMV suspected at the time of clinical presentation. Rather, clinical evaluation for CMV was prompted by the pathologic findings identified at the time of the biopsy. Routine light microscopic examination was conducted on each case using formalin fixed paraffin embedded tissue . In addition, in each case an in situ hybridization technique to detect CMV mRNA was employed.

In Situ Hybridization
The protocol used in this study for paraffin embedded, formalin fixed tissues has been previously described. The tissue was analyzed for cytomegalovirus mRNA using a cocktail of oligonucleotide probes, labeled with fluorescein, directed at CMV mRNA. After incubation with an anti-fluorescein-alkaline phosphatase conjugate, the target-probe complex was detected after precipitation of the chromogen NBT/BCIP (nitroblue tetrazolium and bromochloroindolyl phosphate); the signal was blue as compared to the counterstain nuclear fast red, which colors the negative cells a light pink. Cases deemed positive were those manifesting cellular staining. The latter was defined by a moderately intense cytoplasmic and/or nuclear blue signal over background (histologically normal lung), which was either diffuse homogeneous, granular or assumed a perinuclear dark blue dot-like pattern.

Results

Clinical findings
The patient population comprised 9 previously healthy adults (6 women and 3 males) ranging in age from 31 to 72; mean age 53 years. Seven patients presented with an acute syndromic complex of days to weeks in duration (patients 1,2,4-8) while in one patient the presentation was subacute, over 3 months (patient 9). Finally one patient had a more insidious clinical course over 2 years (patient 3). In three cases, a dominant and or striking component of the patients' presentations was that of pulmonary hemorrhage (patients 1,5, and 9) which in two patients was accompanied by renal failure (patients 1 and 5), clinically indistinguishable from other classic pulmonary renal syndromes (i.e. Goodpasture's syndrome and microscopic polyarteritis nodosa). In one of these patients there was concomitant palpable purpura (patient 1). Two patients had dominant skin findings of purpura, which in one patient was accompanied by Raynaud's phenomenon and oligoarthritis (patient 6). One patient presented with hepatosplenomegaly and generalized constitutional symptoms including fever and malaise (patient 8). Two patients had serious multiorgan thrombotic disease (patients 4 and 9). One patient developed accelerated scleroderma (patient 3).

Four patients had thrombocytopenia (patients 1,2,3,7). Five patients had an elevated ESR. Antiphospholipid antibodies were positive in 3 out of 3 patients tested (patients 7-9) one in whom the dominant clinical presentation was that of recurrent pulmonary thromboembolism and multiorgan thrombosis. The remaining patients did not have an antiphospholipid workup. A positive ANA was seen in 3 patients including one patient who also had antibodies to Ro, La, and RNP (patients 1,8,9). Three patients had a positive rheumatoid factor (patients 1, 6 and 8), two patients had depressed complement (patients 1 and 2) and one patient had a positive ANCA (patient 7). In two patients in whom factor VIII levels were measured (patient 7 and 9) as an index of microvascular injury the values were significantly increased at 187 and 365 respectively. Resolution and or improvement occurred in all 6 patients who received Ganciclovir either subsequently to or concurrently with a regimen of prednisone. One patient with multiorgan thrombosis (patient 4) died abruptly, prior to antiviral treatment.

Serologic testing conducted in 7 of the patients confirmed the presence of active CMV infection by virtue of high-level antibody titers for both CMV specific IgM and IgG. In the two patients in whom there was only positive antibodies of IgG isotype for CMV, the cultures were positive. There were four additional patients in whom CMV infection was documented by culture. Specifically, blood cultures were positive for CMV in patients 1, 2, 8 and 9. Cultures of bronchoalveolar lavage fluid from patients 1 and 5 as well as postmortem lung tissue from patient 4 were positive for CMV.

Pathologic findings

Routine Light Microscopy
Biopsies of patients with cutaneous vasculitis revealed a classic necrotizing neutrophil rich pattern in patient 1(see figure 1), a lymphocytic vasculitis in patient 2 and a granulomatous vasculopathy in patient 6 (see figure 2). Other features of the skin biopsy in these three patients included foci of palisading interstitial histiocytic infiltration with variable piecemeal fragmentation of collagen and focal interface dermatitis. Finally, the lining endothelial cells manifested striking proplastic alterations imparting a hobnail protuberant morphology to the endothelial cells although without diagnostic viral inclusions. One patient with scleroderma demonstrated a subtle sclerosing reaction involving the adventitial dermis and papillary dermis. The endothelial cells and eccrine ductular cells manifested enlargement with an activated appearing heterochromatin and focal mural edema.

Lung biopsies performed on 2 of the three patients with hemoptysis revealed two disparate patterns. In one patient there was striking pauci-cellular septal capillary necrosis with vascular thrombosis (patient 9, see figure 3). The second patient (patient 5) manifested extensive alveolar hemorrhage with concomitant hemosiderin deposition and homogeneous expansion of the septa by collagen with associated vascular drop out. There was disruption of the basement membranes with foci of reduplication along with proplastic endothelial cell changes. Very occasional septal capillaries demonstrated mural and luminal thrombosis, and very focal infiltration of the septa by neutrophils was observed. Direct immunofluorescent studies revealed granular deposits along the alveolar lining and focally within the septal capillaries defining an immunofluorescent pattern that has been described as a granular variant of Goodpasture's syndrome.

Kidney biopsies performed in patients 1 and 5 respectively were those of a proliferative glomerulonephritis with significant deposits of immunoglobulin and complement within the glomerular capillaries and mesangium.

In patient 8, biopsies of the lymph nodes and bone marrow revealed distinctive ringed granulomas comprising epithelioid histiocytes with admixed giant cells and concomitant extensive fibrin deposition recapitulating in part the morphology of a Churg Strauss granuloma.

In patients 4 and 7, there was pauci-inflammatory thrombogenic vasculopathy involving large caliber veins. Striking granular deposits of C5b-9 and IgM were observed within the endothelial cells of a medium caliber lower leg vein in patient 7. Classic cytopathic changes of CMV were not identified in any of the cases.

Molecular studies
Cells staining positively for CMV mRNA were identified in cases 1-5,7, and 8 (see figures 4 and 5). Cells infected by CMV included endothelial cells, type II pneumocytes and eccrine ductular cells. In comparison to classic CMV infection of the lung in an immunocompromised patient in which many classic "owl-eyes" inclusion are seen, the number of positive cells was very small (less than 1% of those seen in the productive viral infection marked by many inclusions) and certainly at disparity with rather striking pathologic abnormalities. The staining pattern was diffuse homogeneous nuclear and cytoplasmic one with some superimposed granularity and much less intense than evident in control lung tissues where typical CMV inclusions were seen, indicative of a low viral copy number.

Concluding Remarks regarding CMV associated autoimmune disease
We have described 9 previously healthy adults who developed serious multiorgan/vital organ disease temporally associated with serologic evidence of recent CMV infection. In addition, all patients had clinical and serologic features indicative of an autoimmune diathesis, specifically a high erythrocyte sedimentation rate, low complement levels, polyclonal hypergammaglobulinemia, positive rheumatoid factor, high C reactive protein, lymphopenia, and/or autoimmune thrombocytopenia. In two of the cases the features defined a pulmonary renal syndrome the spectrum of which comprises Wegener's granulomatosis and microscopic polyarteritis nodosa, Goodpasture's syndrome, mixed cryoglobulinemia and Henoch Schönlein purpura. In two other patients the clinical presentation closely resembled primary antiphospholipid antibody syndrome by virtue of the presence of large venous thrombosis which in one of the patients was associated with a positive lupus anticoagulant. Finally, one patient presented with scleroderma, the prototypic connective tissue disease syndrome where endothelial cell injury is of cardinal importance.

These clinical presentations, although varied, underscore the importance of a vasculopathy in the propagation of their disease. This is not surprising as the endothelium is a permissive cell for CMV infection and reactivation [6, 7, 8, 9, 13] . There were three distinct reaction patterns: a leukocytoclastic vasculitis, a necrotizing lymphocytic vasculitis and/or pauci-inflammatory thrombotic occlusive phenomenon. A clue to the viral based etiology was the unusual cytopathic alterations of the vascular lining cells accompanied by variable mononuclear cell infiltration albeit without frank cytopathic changes held to be diagnostic of cytomegalovirus infection. The endothelium often showed degenerative features, the latter included in the context of endothelial cell detachment. The significant elevation of factor VIII levels in all patients tested was serologic confirmation of endothelial cell injury. Blood and lung cultures documented the presence of replicating CMV in most of the cases. Interestingly, in situ hybridization for viral RNA demonstrated that relatively few cells were infected, that most of these were endothelial cells, and that a given infected cell had low level viral load. The reasons why the level of viral RNA synthesis in endothelial cells in this condition is low will require further analysis, but is in marked contrast to CMV infection with classic owl-eye inclusions, where viral RNA and DNA levels are high, often greater than 500 per infected cell. One may speculate that host factor(s) are partly controlling viral replication but the host is not able to completely eradicate the virus, leading to a relatively protracted, multi-organ disease process similar to what has been described with EBV and chronic fatigue states.

Immunofluorescent studies were corroborative of a role for humoral immunity by virtue of the presence of prominent deposits of C5b-9 within the vasculature. In two cases there was direct capillary endothelial cell localization compatible with antiendothelial cell antibody activity (i.e. a Gell and Comb's type II immune reaction). In the kidney and lung respectively the granular pattern of complement deposition within the capillaries and mesangium and the granular deposition along the alveolar basement membrane also suggested a potential role for immune complexes.

The concept of vasculitis developing in the setting of CMV infection without classic CMV cytopathic alterations is not without precedent. There are reports of patients typically with some underlying immune dysregulatory state in whom there was a necrotizing neutrophil rich vasculitis without viral inclusions [14, 15] . However, this concept is not a widely recognized, and CMV is only rarely considered pathogenetic in the evolution of autoimmune disease. The possibility that the findings related to CMV were epiphenomena and not causal to the immune mediated injury must be raised. While we do not have "pre-illness" CMV serologies for the patients, the data supports an acute onset of infection rather than reactivation of a prior CMV infection by virtue of the positive IgM serologies which are not seen in CMV reactivation. In addition, the IgM antibodies are indeed specific to CMV and not due to a polyclonal B cell response as evidenced by the presence of actual CMV organisms (determined through culture and in situ hybridization). While the reactivation of a prior CMV infection may have the same mechanistic effects in the induction of an autoimmune-like vascular injury, the initiating/ causative role of CMV would be more difficult to ascertain, as CMV reactivation has been known to occur in hospitalized patients. Thus, we included only patients with a known acute CMV infection by virtue of positive IgM serologies.

One must address the potential mechanisms by which this virus can evoke micro and macrovascular disease. The endotheliotropic properties of CMV are well established. The viral infection in our patients was in the context of low level infectivity in an immunocompetent host unassociated with classic cytopathic changes of CMV infection. While fully expressed CMV infection is anti-apoptotic for endothelia via the mechanism of cytoplasmic sequestration of p53, accelerated apoptosis of endothelium can occur in the setting of low-level or latent infectivity unassociated with cytopathic change. The proposed mechanisms include: 1. IgG antibodies directed at the human CMV late protein UL94 inducing endothelial cell apoptosis; 2. Upregulation of nuclear p53 in the earliest phase of reactivation which promotes endothelial apoptosis; and 3. Enhancement of antigenicity of uninfected endothelia through increased adhesion molecule and HLA DR expression mediated by the local cytokine milieu generated by cellular immune response to low level infection or by reactivation.

One potential sequel of accelerated apoptosis is the rearrangement within the plasma membrane of the following phospholipids: phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine. The two most critical phospholipids in the induction of antiphospholipid antibodies in this setting are phosphatidylserine and phosphatidylethanolamine as they are displaced from the inner aspect of the plasma membrane to the outer surface, presenting naïve antigen to circulating T and B cells. Furthermore, beta-2 glycoprotein binds to its co-factor phosphatidylserine upon displacement of this phospholipid to the surface of the cells. Only after binding to phosphatidylserine does it become an antigenic target. Antibodies to these phospholipids potentially displaced during the apoptotic process as well as the co-factor beta-2 glycoprotein were the dominant and almost exclusive ones identified in our patients. In addition, the acceleration of apoptosis also leads to the surface displacement of RO, La, and RNP. All three antibodies were present in one patient. Therefore, accelerated apoptosis results in the displacement of these aforesaid neoantigens (i.e. extractable nuclear antigens and antiphospholipids) and in the genetically predisposed host the result may be one of antibody formation with subsequent endothelial cell injury.

The precipitation of scleroderma in one patient could be explained on the basis of CMV associated endothelial cell injury. Endothelial cell injury is held to be critical in the pathogenesis of scleroderma whereby elevations in thrombomodulin, elevations in angiotensin converting enzyme, and elevated Von Willebrand factor are held to be serologic indices of endothelial injury. It is likely that a latent infection in which immediate early (IE1 and IE2) CMV genes drive host gene expression is implicated. SSc skin lesions have been shown to contain mononuclear cells which express TGF beta alongside interstitial fibroblasts which express alpha 1 collagen mRNA; both genes are induced by CMV. Perhaps the most convincing link implicating CMV in scleroderma is the presence of high-titer IgG antibodies to the polyglycine motifs of CMV.

A final proposed mechanism is that of molecular mimicry. Anti-topoisomerase II autoantibodies cross-react with a peptide sequence of the UL70 protein of CMV. Thus, in predisposed individuals CMV may be an inciting role in the induction of an autoantibody response.

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