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Alveolar Hemorrhage and Renal Microangiopathy in Systemic Lupus Erythematosus.

Complex Small Vascular Injury With Apoptosis

Acute alveolar hemorrhage (AH) in systemic lupus erythematosus (SLE) that is attributed to lupus and not to a secondary complication, such as infection, congestive heart failure, or uremia, is rare.[1] Alveolar hemorrhage occurs in less than 2% of lupus patients. For patients developing the condition, it carries a poor prognosis with reported mortality rates of 70% to 90%.[1-4] When examined histologically, the lungs of 13% of these lupus patients show a neutrophilic capillaritis, and in another 13% of the lupus patients, the pulmonary pathology consists of diffuse alveolar damage.[1,5] In more than 70% of cases there are few identifiable changes other than alveolar hemorrhage, a finding that has been referred to as "bland" pulmonary hemorrhage.[1,2,4,6-9]

In a review of the literature, 20 cases of AH in SLE patients were found in which lung tissue was studied for immune complex deposits.[5,7,10-19] Fifteen of these cases could be described as showing bland pulmonary hemorrhage and 5 as having an alveolar capillaritis. Alveolar wall immune complex deposits were found in 11 cases with bland histologic changes and in 3 cases having an alveolar capillaritis, providing evidence that most SLE-associated AH is immune complex mediated.[5,7,10,2-7,19] The mechanism by which the alveolar walls are injured by a capillaritis is readily apparent, but is obscure in those cases that either have no inflammation or inflammation that is rare and widely dispersed.

In most cases, AH in lupus presents as a pulmonaryrenal syndrome with an active form of lupus nephritis. In a series of 14 SLE patients with AH reported by Zamora et al,[1] 13 (93%) had a simultaneously occurring lupus nephritis. Renal biopsies were performed in most of these patients, but detailed results were not provided. Among 12 previously reported cases of lupus with AH in which the renal pathology was described, 8 demonstrated a World Health Organization (WHO) class IV, 2 a WHO class III, and 2 a WHO class V lupus nephritis.[2-4,10,12-16,19] Immune-mediated vascular lesions frequently accompany a proliferative lupus nephritis. In the kidney, these vascular lesions have been classified as (1) uncomplicated vascular immune complex deposits and (2) noninflammatory necrotizing microangiopathy, also referred to as lupus microangiopathy.[20-27]

Uncomplicated vascular immune complex deposits consist of a deposition of immunoglobulins and complement primarily in afferent arterioles.[20] The involved vessels show no evidence of injury and appear normal by light microscopy or contain subendothelial collections of eosinophilic material.[20] The lupus microangiopathy is reported in 6% to 17% of renal biopsies and in 27% of autopsies on lupus patients.[21,23-27] Like uncomplicated deposits, this microangiopathy primarily involves afferent arterioles, and large amounts of immunoglobulin and complement are found in arteriolar walls by immunofluorescence microscopy. By light microscopy, the arteriolar walls are effaced by amorphous eosinophilic material.[20-25,27] A notable feature of a lupus microangiopathy is the absence or near absence of inflammation in the injured vessel.[20-25,27] This absence of inflammation is similar to the pulmonary findings in patients with bland AH.

Recent studies have indicated that apoptosis may play a central role in initiating and sustaining the autoimmune response in lupus-prone individuals.[28,29] These studies also suggested that the stimulated immune response can increase apoptotic cell death and amplify the progression of the disease.[28,29] Apoptosis is a form of cell death that results in the shrinkage of cells and their fragmentation into smaller membrane-bound vesicles.[28-30] Unlike necrosis, in which cells are lysed and the cytoplasmic contents spill into adjacent tissues, provoking an acute inflammatory reaction, the membrane-bound cell particles derived from apoptosis are rapidly removed by macrophages.[28,30] This removal prevents the dying cells from eliciting an inflammatory response; it is additionally thought that the macrophages actively inhibit inflammation by secreting anti-inflammatory cytokines.[28-30]

This study was undertaken to investigate the relationships and the mechanisms of small vascular injury in the lung and kidney of 2 lupus patients who died of diffuse AH. The findings are compared with those from autopsies on lupus patients who did not have SLE-related pulmonary hemorrhage. Because the histopathologic changes in the cases of AH were distinctly noninflammatory, studies were performed to investigate whether apoptosis plays a role in the pathogenesis of the vascular pathology.

MATERIALS AND METHODS

Case Collection

Specimens from patient 1 consisted of a transbronchial lung biopsy and a renal biopsy that were performed 2 days apart. Specimens from patients 2 through 6 were obtained from autopsies of lupus patients. Renal biopsies were performed on patients 2 and 6 before death. Cases 1 and 2 represent the authors' experience with the pathology of a lupus-associated pulmonaryrenal syndrome in which the pulmonary pathology was characterized by acute severe alveolar hemorrhage. Cases 3 through 6 comprise autopsies of patients who died with active SLE, and lung and kidney tissues were examined for the presence of immune complex deposits. All of the patients in this report had been clinically diagnosed as having lupus because they manifested 4 or more of the revised criteria of the American Rheumatism Association for SLE.[31] The clinical and pathologic findings in these cases are summarized in the Table, which lists renal disease diagnoses based on the modified WHO classification of lupus nephritis.[27]
Lupus Patients With and Without Alveolar Hemorrhage:
Summary of Clinical and Pathological Findings

Patient Age, y/
No. Sex Diagnosis Cause of Death

1 28/F SLE Alveolar hemorrhage

2 22/F SLE Alveolar hemorrhage

3 34/F SLE Mesenteric artery thrombosis,
 small intestinal infarction,
 septicemia

4 19/M Overlap Colonic perforation,
 syndrome, peritonitis, septicemia
 SLE-scleroderma

5 49/M SLE Diffuse alveolar damage,
 aspergillosis

6 21/M SLE Herpes simplex pneumonia

Patient
No. Pulmonary Pathology Renal Pathology[dagger]

1 Alveolar hemorrhage, bland Class IVb lupus nephritis
 alveolar wall changes, IgG, (with segmental necrotizing
 IgM, IgA, and EM alveolar lesions), lupus
 wall deposits microangiopathy

2 Alveolar hemorrhage, bland Class IVc lupus nephritis
 alveolar wall changes, EM (with segmental necrotizing
 alveolar wall EM deposits and sclerosing lesions),
 lupus microangiopathy

3 Pulmonary congestion, Class IIb lupus nephritis
 edema; no EM of IF alveolar (mesangial
 wall deposits hypercellularity),
 arteriolosclerosis

4 Bronchopneumonia; no EM Class Va lupus nephritis
 alveolar wall deposits (pure membranous
 nephropathy)

5 Hyaline membranes, invasive Class IVa lupus nephritis
 aspergillosis; no EM of IF (mesangiocapillary
 alveolar wall deposits proliferation without
 segmental lesions),
 arteriolosclerosis

6 Herpes simplex pneumonia; Class Vc lupus nephritis
 no EM or IF alveolar wall (membranous
 deposits; pulmonary glomerulonephritis
 fibrosis, interstitial and associated with focal and
 alveolar; pleural fibrosis segmental endocapillary
 hypercellularity,
 crescents, and sclerosis),
 uncomplicated IgG, IgM, C3,
 and C1q arteriolar deposits

(*) SLE indicates systemic lupus erythematosus; EM, electron
microscopy; and IF, immunofluorescence microscopy.

[dagger] Renal disease diagnoses based on the modified
WHO classification of lupus nephritis.


Tissue was processed by routine methods for light, immunofluorescence, and electron microscopy. Immunohistochemistry for CD68 (Dako Corporation, Carpinteria, Calif), S100 (Ventana Medical Systems, Inc, Tucson, Ariz), and myeloperoxidase (Dako) was performed on paraffin sections of lung and kidney using a streptavidin-biotin-peroxidase method with a Ventana Nexus (Ventana) automated immunostainer. The number of myeloperoxidase and CD68-positive cells in the alveolar walls of sections of normal control lung and the lungs of patients with AH were counted in 20 nonoverlapping, high-power, x400 microscopic fields (HPFs) and recorded as cells per HPF.

Apoptosis was examined on 4-[micro]m-thick paraffin sections using the ApopTag kit (Intergen Co, Purchase, NY) for terminal deoxynucleotidyl transferase (TdT)-mediated digoxigenin-diethylnitrophenyl thiophosphate (dNTP) nick-end labeling (TUNEL) of DNA. Deparaffinized, rehydrated sections were treated by proteinase K digestion (20 [micro]g/mL, Sigma, St Louis, Mo). Sections were then incubated with working-strength TdT enzyme solution in a humidified chamber at 37 [degrees] C for 1 hour, followed by antidigoxigenin peroxidase conjugate and staining with 3,3'-diaminobenzidine (Zymed Laboratories, San Francisco, Calif). Counterstaining was with methyl green (Sigma). Prior to TUNEL reactions, endogenous peroxidase was quenched in fresh 3% hydrogen peroxide in phosphate-buffered saline. Quenching for immunoperoxidase reactions used 3% hydrogen peroxide in 50% methanol and phosphate-buffered saline. The positive control for all reactions was tonsil, the negative control consisted of tonsil incubated without TdT enzyme or primary antibody. For immunohistochemistry and TUNEL reactions, control tissue also consisted of sections of normal kidney and lung from autopsies of patients without kidney or lung disease. To test for the selectivity of the TUNEL labeling, tonsil and normal kidney and lung sections were treated prior to the TUNEL reactions with DNase I (Sigma).

Clinical Summaries of Patients With AH

Patient 1.--At 28 years of age, this Hispanic woman was diagnosed as having SLE after presenting with a malar skin rash, arthritis, proteinuria of 13.2 g/24 h, renal insufficiency, hemoptysis, a positive antinuclear antibody test (ANA), and an antibody to double-stranded DNA (dsDNA). Renal and transbronchial biopsies were performed. The patient was treated with prednisone and cyclophosphamide, and responded with improvement of her renal disease and cessation of the pulmonary hemorrhage. She was followed for the next 6 years at a renal outpatient clinic, where she was treated with cyclophosphamide. She then developed a sudden deterioration of renal function, left-sided chest pain, fever, and a cough productive of thick sputum. She was admitted to the intensive care unit and treated with pulse steroids and antibiotics, at which time she experienced a sudden onset of pulmonary hemorrhage. Oxygenation progressively deteriorated, and the patient died of acute respiratory insufficiency. The final clinical diagnoses were pulmonary hemorrhage, left lower lobe pneumonia, and acute renal failure. An autopsy was declined.

Patient 2.--A 22-year-old African American woman was diagnosed as having SLE 5 years previously, when she developed a malar skin rash, renal disease, a positive ANA, and an antibody to dsDNA. She was being treated with prednisone and antihypertensive medication when she experienced the sudden onset of severe muscular weakness. She had increasing proteinuria and a rise in serum creatinine from 171 to 441 [micro]mol/L. A renal biopsy was performed, and intravenous prednisone and cyclophosphamide were administered. As an outpatient, she suffered an acute gastrointestinal hemorrhage from a gastric ulcer. She was hospitalized and appeared stable until the fifth hospital day, when she became short of breath and experienced an acute episode of hemoptysis. A chest radiograph showed bilateral alveolar infiltrates. A pulmonary arteriograph showed no evidence of pulmonary emboli. The patient was hemodialyzed with ultrafiltration, but she continued to have hemoptysis and died on the 35th day of hospitalization with deteriorating arterial blood oxygen levels. White blood cell and neutrophil counts were within normal limits or were mildly elevated during the hospital course. An autopsy was performed.

RESULTS

Pathologic Findings in Patients With AH

Patient 1.--Renal Biopsy.--By light microscopy, the glomeruli disclosed a diffuse proliferation of endocapillary cells, segmental fibrinoid necrosis, and infiltrates of polymorphonuclear leukocytes. Many glomerular capillary wire loop lesions and a few cellular crescents were present. The walls of afferent arterioles were effaced by smudgy eosinophilic material that severely narrowed the vessel lumens, and nuclear pyknosis and karyorrhexis were seen within the altered arteriolar walls (Figure 1). These arterioles showed no inflammation. In some arteries, karyorrhexis was identified in individual shrunken cells within the media (Figure 2). Immunofluorescence microscopy revealed intense deposition of immunoglobulin (Ig) G, IgM, IgA, C1q, C4, and C3 in glomeruli in the mesangium and capillary walls, as well as in afferent arterioles (Figure 3). Electron microscopy demonstrated numerous mesangial and subendothelial electron-dense deposits. Endothelial tubuloreticular inclusions were present. An afferent arteriole was sampled that showed subendothelial and intramural dense deposits and loss of the structural integrity of the media. Degenerating vascular cells showed a condensation of cytoplasm and the formation of membrane-bound vesicles containing dense osmiophilic material.

[Figures 1-3 ILLUSTRATION OMITTED]

Transbronchial Lung Biopsy.--Immunofluorescence microscopy showed intense granular to pseudolinear deposits of IgG along the alveolar walls (Figure 4). There was moderate reactivity for IgM and IgA. Only weak and focal deposits of C3, C1q, and C4 were seen. Light microscopy revealed alveolar hemorrhage. The alveolar walls were not inflamed. Hyaline thickening of alveolar capillaries and small arteries was not seen. Karyorrhexis was identified in shrunken cells in alveolar spaces, and pyknotic nuclear fragments, cell debris, red cells, and hemosiderin were present in alveolar macrophages. Part of the paraffin block was processed for electron microscopy, and electron-dense deposits were found in the basement membranes of the alveolar walls.

[Figure 4 ILLUSTRATION OMITTED]

Patient 2.--Renal Biopsy.--A renal biopsy was performed 75 days before death and 35 days before the onset of pulmonary hemorrhage. Light microscopy showed glomeruli with moderate thickening of glomerular capillary walls. There was no appreciable glomerular hypercellularity. Prominent spikes and holes were seen in the thickened glomerular capillary basement membranes with the periodic acid-Schiff and periodic acid-methenamine silver stains. The glomeruli showed no polymorphonuclear leukocyte infiltrates, hyaline thrombi, crescents, or segmental necrotizing lesions. The afferent arterioles were unremarkable. Subendothelial arteriolar deposits were not identified. There was advanced interstitial fibrosis and tubular atrophy, and one fourth of the glomeruli demonstrated global or segmental sclerosis. Immunofluorescence microscopy, revealed intense deposits of IgG in the glomerular capillary walls in a granular pattern and moderate fluorescence for IgM, IgA, C4, C1q, and C3. Electron microscopy disclosed numerous, large, subepithelial and intramembranous electron-dense deposits along the glomerular capillary walls. Prominent mesangial and rare, small, subendothelial, dense deposits were present. Endothelial tubuloreticular inclusions were not seen.

Autopsy.--The light microscopic sections of kidney showed glomeruli with thickened glomerular basement membranes and a superimposed proliferation of endocapillary cells. Polymorphonuclear leukocyte infiltrates were present in the glomerular tufts, and many glomeruli displayed segmental necrosis. Numerous glomerular wireloop lesions and hyaline capillary loop thrombi were seen. Approximately one third of the glomeruli were globally or segmentally sclerotic. The afferent arterioles showed amorphous eosinophilic material effacing the media. These arterioles frequently showed continuity at the glomerular hilum with segmental necrotizing glomerular lesions. Nuclear pyknosis and karyorrhexis were present in the amorphous material in the arteriolar walls (Figure 5). Shrunken smooth muscle cells with pyknotic nuclei were seen in the media of afferent arterioles proximal to the segments showing the amorphous change. Karyorrhexis was sometimes identified in these individual cells. Electron microscopy disclosed numerous mesangial, epimembranous, and intramembranous dense deposits and large subendothelial deposits in many glomerular capillary loops. Numerous tubuloreticular endothelial inclusions were present.

[Figure 5 ILLUSTRATION OMITTED]

The lungs demonstrated extensive and multifocal areas of alveolar hemorrhage filling alveolar spaces (Figure 6). This consisted of fresh blood and focal intra-alveolar collections of fibrinous material. Large macrophages containing red blood cells, hemosiderin, proteinaceous debris, and pyknotic nuclear fragments were present within the alveoli, and hemosiderin was focally deposited within alveolar walls. Rare, widely dispersed collections of neutrophils were seen in some alveolar capillaries and in some alveoli, but most alveoli and alveolar walls showed no inflammation. Individual shrunken cells showing karyorrhexis were present within some alveolar walls (Figure 7). Pyknotic nuclear fragments and shrunken cells showing karyorrhexis were also found outside the alveolar walls within areas of alveolar hemorrhage. Alveolar walls and pulmonary arterioles did not show any eosinophilic thickening resembling that seen in the renal arterioles.

[Figures 6-7 ILLUSTRATION OMITTED]

Electron microscopy showed numerous electron-dense deposits in the alveolar walls (Figure 8). Electron-dense deposits were located directly beneath endothelium and alveolar epithelium, but were most frequently seen within the basement membrane. The bone marrow was unremarkable and specifically did not show reduced numbers of myeloid cells.

[Figure 8 ILLUSTRATION OMITTED]

Clinical and Pathologic Summaries of Patients Without Lupus-Associated Pulmonary Hemorrhage

Patient 3 was a 34-year-old white woman who had been hospitalized at the state psychiatric hospital for many years. She was diagnosed with lupus at 25 years of age on the basis of a discoid skin rash, arthritis, proteinuria, a positive ANA (speckled pattern), an anti-Ro antibody, and antibodies to single-stranded DNA. Patient 4 was a 19-year-old African American man who had a 3-year history of SLE that was diagnosed on the basis of a malar skin rash, proteinuria, an ANA, and an antibody to dsDNA. Patient 5 was a 49-year-old white man with a 20-year history of SLE diagnosed on the basis of photosensitivity, mucositis, polyarthritis, autoimmune hemolytic anemia, thrombocytopenia, glomerulonephritis, an ANA, an antibody to dsDNA, and antiphospholipid antibodies. He was hospitalized with a fever of 102 [degrees] F and worsening anemia. He died 4 days after admission with marked respiratory distress, hypoxemia, and bilateral upper lobe alveolar infiltrates. Patient 6 was a 21-year-old African American man who was diagnosed as having SLE at age 11 years, when he developed arthritis, pleuritis, a facial rash, proteinuria, and positive anti-dsDNA, anti-Smith, and anti-ribonucleoprotein antibodies. A renal biopsy taken 1 month before this patient's death demonstrated a membranous glomerulonephritis with segmental glomerulosclerosis. Immunofluorescence microscopy of the biopsy showed arteriolar deposits of immunoglobulins and complement, but no lupus vasculopathy was seen by light microscopy. The patient was being treated with prednisone when he developed shortness of breath. A chest radiogram showed bilateral confluent pulmonary infiltrates. He was intubated and administered antibiotics, but died with decreasing oxygen saturation 9 days later.

An autopsy was performed on all of the patients who did not experience pulmonary hemorrhage. Alveolar wall and renal arteriolar immune deposits were not found in cases 3, 4, or 5. Patient 5 had pulmonary pathology consisting of diffuse alveolar damage and invasive pulmonary aspergillosis. Patients 3, 4, and 5 all had a lupus nephritis, but in none of the cases was there a notable degree of histologic activity. Specifically, segmental necrotizing glomerular lesions, hyaline glomerular thrombi, wire-loop lesions, glomerular polymorphonuclear leukocyte infiltrates, vasculitis, and lupus microangiopathy were not found.

For patient 6, immunofluorescence microscopy revealed granular deposits of IgG, IgM, C1q, C3, and C4 in the glomeruli along the glomerular capillary loops and in afferent arterioles. Electron microscopy demonstrated numerous epimembranous and intramembranous electrondense deposits and small mesangial deposits. By light microscopy, glomeruli showed glomerular basement membrane thickening. Approximately 1 glomerulus in 20 revealed a segmental crescent, and 1 in 15 showed segmental sclerosis. Afferent arterioles were histologically unremarkable or in rare instances showed small accumulations of subendothelial hyaline and periodic acid-Schiff-positive material. No alveolar wall deposits or electron-dense deposits were found in the lung by immunofluorescence or electron microscopy, respectively. The lungs of patient 6 were involved by a severe herpes simplex pneumonia.

Immunohistochemical and TUNEL Studies

In patients 1 and 2, TUNEL-positive cells were found within the alveolar walls, and many cells within the alveolar spaces were TUNEL positive (Figure 9). The intraalveolar TUNEL-positive cells consisted of alveolar macrophages and other smaller cells. In the kidneys, afferent arterioles involved by a lupus microangiopathy contained pyknotic nuclear material that stained prominently with the TUNEL technique. In some arterioles, positive staining could be localized to individual smooth muscle cells (Figure 10). TUNEL-positive staining was also prominent in the segmental necrotizing lesions of the glomerular tufts. In patient 6, TUNEL positivity was not identified in afferent arterioles that in serial sections showed immunoperoxidase staining for IgG but no light microscopic evidence of the structural injury of a lupus microangiopathy. A few TUNEL-positive cells were seen in the glomeruli of patients 3, 4, 5, and 6. TUNEL-positive cells were not seen in normal kidney or lung; in renal arterioles of patients 3, 4, and 5; or in lung of patients 3 and 4. TUNEL-positive staining was found in association with diffuse alveolar damage in patient 5 and in the herpes simplex pneumonia of patient 6. All nuclei within the control sections showed TUNEL-positive staining when the reactions were performed after treatment with DNase I. Without DNase I treatment, only selected cells within germinal centers of tonsil and none of the cells in normal kidney and lung sections were TUNEL positive.

[Figures 9-10 ILLUSTRATION OMITTED]

Granulocytes, identified morphologically as polymorphonuclear leukocytes and immunohistochemically by staining for myeloperoxidase, were absent or were only rarely identified in arterioles involved by a lupus microangiopathy. This was in contrast to the numerous myeloperoxidase-positive polymorphonuclear leukocytes that were present in glomeruli. CD68-positive cells were found in renal blood vessels involved by a lupus microangiopathy (Figure 11). The CD68-positive cells consisted of large monocytes in the lumens of the arterioles and of dendritic cells located along the outside of the vessels. The dendritic cells were negative for S100. Numerous CD68-positive monocytes were present in the glomeruli of patients 1 and 2. A few CD68-positive cells were present in the glomeruli, but not in the arterioles of patients 3, 4, 5, and 6. Alveolar macrophages in the lungs of patients 1 and 2 were noted to be myeloperoxidase positive, but there was no increase in the number of myeloperoxidase-positive cells in the alveolar walls above the number found in normal control lung (alveolar wall myeloperoxidase-positive cells: normal control, 28.10 [+ or -] 8.91 cells/HPF; AH case 1, 22.60 [+ or -] 6.25/HPF; AH case 2, 21.70 [+ or -] 6.29/HPF). In the lungs of patients 1 and 2, there were large numbers of CD68-positive macrophages within the alveolar spaces, and there was a marked increase above normal control lung in the number of CD68-positive monocytes within the alveolar walls (alveolar wall CD68-positive cells: normal control, 2.00 [+ or -] 2.15/HPF; AH case 1, 33.25 [+ or -] 8.26/HPF; AH case 2, 31.80 [+ or -] 8.49/HPF) (Figure 12).

[Figures 11-12 ILLUSTRATION OMITTED]

COMMENT

The cases of AH in this report indicate that lupus vasculopathy of the kidney and immune complex-mediated AH with bland histologic features have a similar pathogenesis. The vascular changes in kidney and lung are notable for the absence of any significant participation of granulocytes and occur as part of a pulmonary-renal syndrome with severely active forms of lupus nephritis. By morphologic examination and by the histochemistry of TUNEL labeling, findings consistent with apoptosis can be identified in alveolar walls containing immune complex deposits and in renal arterioles involved by the lupus microangiopathy. The TUNEL assay provides evidence of internucleosomal DNA degradation in individual arteriolar and alveolar wall cells.[32] The best morphologic evidence of apoptosis is considered to be karyorrhexis in individual cells, and this is demonstrated in the lupus microangiopathy and in alveolar walls in association with AH.[32,33] In addition, the immunohistochemical identification of increased numbers of CD68-positive monocytes in the lupus microangiopathy and in alveolar walls in areas of AH is consistent with proposed mechanisms of phagocytic clearance of apoptotic cells by the monocyte-macrophage system and the suppression of acute inflammation during apoptotic cell death.[28-30]

There are histologic differences between the renal microangiopathy and the alveolar wall injury. The alveolar walls do not show the homogeneous eosinophilic alteration seen in the afferent arterioles. This is probably because the alveolar walls are thin and lack a media in which apoptosis is seen in the renal arterioles. In the lung, apoptotic cells are found on the outside of the alveolar walls, and in some alveoli numerous apoptotic bodies are present. Although we have not been able to identify the type of cell involved by apoptosis, some may be alveolar epithelial cells. External injury to the alveolar wall may be the counterpart of arteriolar smooth muscle injury.

In the cases of AH, the CD68-positive alveolar macrophages are strongly myeloperoxidase positive. This is a distinct change from the alveolar macrophages of normal lung and is different from the CD68-positive monocytes found in alveolar walls with AH and in the lupus microangiopathy. The mononuclear cells within the alveolar walls and in the lumens of renal arterioles show no apparent myeloperoxidase staining. The myeloperoxidase in the alveolar macrophages is likely to be the result of their pronounced ingestion of red blood cells and cell debris and the accumulation in phagosomes of increased amounts of macrophage-derived myeloperoxidase.[34]

The presence of alveolar wall immune complex deposits uncomplicated by AH was investigated by a study of lung tissue from 4 lupus patients who had various lung disorders other than AH and who also had inactive or mildly active forms of lupus nephritis. Alveolar wall deposits identified by electron or immunofluorescence microscopy were not found in any case. Among these 4 patients, case 6 showed immune complex deposition in renal arterioles that was uncomplicated by the vascular wall necrosis of a lupus microangiopathy. The patient had a mildly active, mixed membranous, and focal proliferative (modified WHO class Vc) lupus nephritis in which no subendothelial glomerular electron-dense deposits were identified. This contrasts with the electron microscopic findings in the cases of AH with alveolar wall immune complex deposits and a renal lupus microangiopathy, in which large, subendothelial, glomerular capillary basement membrane--dense deposits were found. Subendothelial electron-dense deposits are characteristic features of WHO class III and class IV lupus nephritis, and large subendothelial deposits are found in the most severe disease.[27]

Findings of immunofluorescence microscopy have been reported on lung tissue from 16 SLE patients who had lung disease other than AH.[35-41] Eight cases showed no alveolar wall immunoglobulins.[41] Eight cases, however, did demonstrate pulmonary vascular immunoglobulin deposits.[35-39,41] In 6 cases, the deposits were present in the alveolar walls and were associated with a mononuclear cell interstitial pneumonia (4 cases), a pseudolymphoma (1 case), and interstitial fibrosis (1 case). Two of the 8 cases with pulmonary vascular immunoglobulin deposits had IgG and C1q in the small pulmonary arteries but not in alveolar walls, and the pulmonary pathology consisted of plexigenic pulmonary hypertension.[41] Four of these 8 cases had a class IV lupus nephritis, while the remainder had mild clinical renal disease.[35-39,41] It would probably not be appropriate to consider alveolar wall immunoglobulin deposits in cases of interstitial pneumonia uncomplicated. The interstitial pneumonia of lupus in humans may be equivalent to the interstitial pneumonia of New Zealand black/white hybrid mice, which develops with the appearance of alveolar wall and glomerular immunoglobulin deposits.[42] Nevertheless, the reports do show that alveolar wall immune complex deposits can be found in the absence of AH, with or without active renal disease.

The factors that influence immune complex vascular injury in the lung and kidney appear to be related to those factors mediating the intensity and location of deposits in a glomerulonephritis. This is indicated by clinical studies in which the lupus microangiopathy in the great majority of cases is found with an active WHO class III or IV lupus nephritis and infrequently with an inactive class II (mesangial) or V (membranous) lupus nephritis.[20-25] Similarly, AH with bland alveolar wall changes usually occurs as a pulmonary-renal syndrome with proliferative forms of glomerulonephritis and rarely with a class II or class V lupus nephritis or in patients with no clinical renal disease.[7,10-25] A change in the pattern of immune complex deposition and its possible relationship to microvascular injury of the kidney and lung is shown in patient 2 in this report. During a period of 40 days, there was a transformation from a class V lupus nephritis without renal vascular lesions to a severe necrotizing class IV nephritis with a lupus microangiopathy. At the same time, the patient developed immune complex-mediated AH.

A major difference between the glomerular and the vascular lesions is the common recruitment of neutrophils into an active glomerulonephritis and the infrequent finding of granulocytes in the microangiopathy. In the lung, the difference between AH showing a capillaritis and AH with bland histologic features may not be clear-cut in some cases. Churg et al[17] and Segal et al[19] reported cases of SLE with immune complex-associated pulmonary hemorrhage that showed primarily alveolar hemorrhage without alveolar wall inflammation, but the authors stated that rare, widely scattered foci of inflammation of the alveolar walls by polymorphonuclear leukocytes were present. Similar findings of rare collections of neutrophils within alveolar capillaries were seen in our autopsy of patient 2, and some observers might classify such cases as an example of capillaritis. The lung tissue from patient 1 consisted of a transbronchial biopsy. The presence of a capillaritis in areas of the lung not sampled in the biopsy obviously cannot be ruled out. Nevertheless, several studies of lung biopsies have illustrated and described the neutrophilic infiltration of alveolar walls, which appears to be quite different than cases with predominantly bland AH and focal inflammation.[1,5,12]

Patient 2 was treated with cyclophosphamide and prednisone for 30 days before an autopsy showed diffuse AH with bland alveolar wall changes. The therapy could have modulated the cellular response to the microvascular injury. The patient, however, was not myelosuppressed during this period, and in the kidney, many neutrophils were present in glomeruli but not in the microangiopathy of the afferent arterioles. Similarly, the lungs showed granulocytes within the alveolar capillaries but not in excess numbers above that seen in normal lung. This finding suggests that the immune complex-mediated lung and renal microvascular injury is not chemotactic to neutrophils and that there is an absence of firm neutrophil adhesion. The pathology of this acellular lesion appears to be pathogenetically different from experimental immune complex-mediated lung disease that characteristically produces a neutrophilic capillaritis with hemorrhage.[43]

Monocytes and dendritic interstitial cells seem to play an important role in these microvascular changes. The identification of monocytes in the injured vessels suggests that they function in the clearance of apoptotic cells and the inhibition of local inflammation[28-30] The dendritic cells that are located on the outside of the affected arterioles are CD68 positive and S100 negative. This is an antigenic expression of interstitial macrophages rather than specialized antigen-presenting cells. These cells are likely to be primarily phagocytic, although they may have an additional role in antigen presentation for a secondary immune response.[44]

On the basis of our histopathologic and immunopathologic findings, as well as the results of the TUNEL method for apoptosis, we propose that bland alveolar hemorrhage in lupus patients is a form of alveolar capillary injury that is analogous to the lupus microangiopathy of the kidney. We further propose that this form of pathology involves apoptosis that follows the deposition of immune complexes in the small vessels. In autoimmune disorders, dysregulation of apoptosis is thought to prevent the depletion of autoreactive T cells during clonal selection.[28,29] Systemic lupus erythematosus is hypothesized to be the result of a breakdown in self-tolerance, in which nucleosomes as well as other cell constituents derived from apoptotic cells serve as immunogens that activate the autoimmune response.[28,29] The findings presented here also suggest that the autoimmune response can induce apoptosis.

Financial support was provided by grants from Kidney Care Foundation, Inc, Jackson, Miss, and the University of Mississippi Institutional Review Board, Jackson, Miss. Illustrations were prepared by John Coleman, DO.

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Accepted for publication November 30, 2000.

From the Departments of Pathology (Drs Hughson, He, and Henegar) and Medicine, Division of Rheumatology (Dr McMurray), University of Mississippi Medical Center, Jackson, Miss.

Presented at the Annual Meeting of the United States and Canadian Academy of Pathologists, New Orleans, La, March 28, 2000.

Reprints: Michael D. Hughson, MD, Department of Pathology, University of Mississippi Medical Center, 2500 N State St, Jackson, MS 39216-4505 (e-mail: mhughson@pathology.umsmed.edu).
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Author:Hughson, Michael D.; He, Zhi; Henegar, Jeffrey; McMurray, Robert
Publication:Archives of Pathology & Laboratory Medicine
Geographic Code:1USA
Date:Apr 1, 2001
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