Effector T cells and macrophages in urine as a hallmark of systemic vasculitis accompanied by crescentic glomerulonephritis

Minoru Sakatsume and Fumitake Gejyo

Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan

Keywords: crescentic glomerulonephritis; effector T cells; macrophages; urine; vasculitis



   Introduction
 Top
 Introduction
 Subjects and methods
 Cases
 Results
 Discussion
 References
 
T cells and macrophages appear in the urine of patients with glomerulonephritis (GN), accompanied by active cellular infiltration such as cellular crescent formation and diffuse interstitial cell infiltration, but not in the urine of patients with GN without the active inflammatory lesions. The urine T cells are mainly of the effector type (CD62L-CD45RO+RA-), which is the same as the phenotype of infiltrating T cells in and around glomeruli. Moreover, these T cells express T helper lymphocyte 1 (Th1)-type cytokines [1]. The appearance of these immune effector cells in urine can be considered to reflect cellular immune responses in inflammatory renal lesions.

The detection of these effector cells in the urine of patients can be used as a new clinical method to assume the presence of renal inflammatory lesions. We report here on two patients with rapidly progressive renal dysfunction, proteinuria and haematuria, who could not undergo renal biopsy because of severe complications in other organs, and who had numerous effector T cells and macrophages in their urine. Subsequent autopsies on them revealed the presence of crescentic GN. The detection of immune effectors in the urine of patients is very informative with respect to the diagnosis of inflammatory renal involvement, including cellular crescent formation.



   Subjects and methods
 Top
 Introduction
 Subjects and methods
 Cases
 Results
 Discussion
 References
 
Flow cytometric analysis of urinary cells and peripheral blood mononuclear cells (PBMNCs)
Urine and blood samples from our patients were prepared as previously described [1]. Briefly, fresh urine samples were centrifuged and the pellets were incubated with either fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated monoclonal antibodies, or both. Isotype-matched FITC- or PE-conjugated non-specific antibodies were also used for negative controls. Two-colour flow cytometric analysis was performed on a FACScanTM (Becton Dickinson, Franklin Lakes, NJ). Dead cells were excluded by forward scatter, side scatter and propidium iodide gating. The gates for mononuclear cells and lymphocytic cells were set using a previously published method [2,3]. The counts for T lymphocytes or monocytes/macrophages were determined by multiplying the number of viable cells in the gated mononuclear cell region in each sample by the percentages of CD3+ or CD14+ cells in the population. The counts for cells were standardized in accordance with the osmotic pressure of the urine; isotonic pressure, 280 mOsm/kgl, was designated as 1. PBMNCs were separated from heparinized blood samples with a Ficoll-gradient method using LymphoprepTM (Nycomed Pharma AS, Oslo, Norway) and treated in the same manner as cells from the urine samples. The urine samples of both patients showed more mononuclear cells than neutrophils on FACS analysis, and did not disclose a significant number of bacteria (>1x104 colonies/ml urine) on cultures, whereby the possibility of bacterial infection in the urinary system could be ruled out.



   Cases
 Top
 Introduction
 Subjects and methods
 Cases
 Results
 Discussion
 References
 
Case 1. MPO (myeloperoxidase) anti-neutrophil cytoplasmic antigen (ANCA)-related systemic vasculitis
A 76-year-old Japanese man was admitted to Niigata University Hospital on 21 November 1999 because of fever, proteinuria and haematuria. His admitting diagnosis was fever of unknown origin. He consulted us on 8 December 1999 for renal problems; his serum creatinine had gradually increased to 2.6 mg/dl. Urinalysis showed proteinuria (0.6 g/day) and haematuria (21–30 red cells/field), and his creatinine clearance (Ccr) was 25 ml/min. C-reactive protein (CRP) was elevated (22 mg/dl). MPO-ANCA was positive at 107 U/ml (normal range <10 U/ml), whereas PR3-ANCA was negative. Bronchoalveolar lavage from the right lobes showed many haemosiderin-loaded macrophages, compatible with alveolar haemorrhage. A flow cytometric analysis of urine and blood was performed at that time. Working under the diagnosis of ANCA-related systemic vasculitis, we began pulse therapy with methylprednisolone (15 mg/kg for 3 days) and intravenous cyclophosphamide (14 mg/kg), and followed that with additional pulses of methylprednisolone and plasma exchange, because of uncontrollable alveolar bleeding. One and half months after the start of the treatment, the patient died from respiratory failure induced by pulmonary infection.

Case 2. ANCA-negative systemic vaculitis
A 66-year-old Japanese man was admitted to Niigata University Hospital on 20 May 2001 because of proteinuria, haematuria, abdominal pain and bloody stools. His condition had been diagnosed as pneumonia or pneumonitis, and treatment with antibiotics and 30 mg of prednisolone had been ineffective. On admission, he had purpura on the lower extremities. Urinalysis revealed proteinuria (2.0 g/day) and haematuria (15–20 red cells/field). Laboratory tests showed serum creatinine 0.9 mg/dl, IgG 857 mg/dl, IgA 791 mg/dl, IgM 72 mg/dl and CRP 5.3 mg/dl. Complement system components were within normal ranges. MPO-, PR3-ANCA and anti-nuclear antibodies were absent. Pathological examination of skin lesions biopsied on the lower extremities showed leukocytoclastic vasculitis. Several days after admission, the patient's serum creatine gradually increased to 2.3 mg/dl, and at the same time he began to have massive bloody stools. A flow cytometric analysis of his urine and blood was performed at that time. Working under the diagnosis of systemic vasculitis syndrome, we began pulse therapy with methylprednisolone (20 mg/kg for 3 days), and followed that with intravenous cyclophosphamide (10 mg/kg) and another pulse of methylprednisolone because of uncontrollable bleeding from the lower alimentary tract. One month after the start of the initial treatment followed by oral administration of prednisolone (1 mg/kg p.o.) and cyclophosphamide (0.8 mg/kg p.o.), the bleeding stopped and his condition improved. One month later, however, the patient died from cerebral bleeding and opportunistic pulmonary infection.



   Results
 Top
 Introduction
 Subjects and methods
 Cases
 Results
 Discussion
 References
 
Detection of effector T cells and macrophages in urine from the two patients
On admission or at the start of consultation, before pulse therapy of methylprednisolone or cyclophosphamide, blood and fresh urine were sampled and analysed by flow cytometry as described in Subjects and methods. As shown in Table 1Go, the total number of T cells and macrophages in 1 ml of urine, corrected for urine osmolarity, were 9.6x102 and 26.3x102/ml, respectively. The main phenotype of the T cells in the urine was of the immediate effector type (CD62L-CD45RO+RA-), while peripheral blood T cells were mainly of the naive (CD62L+CD45RO-RA+) or resting memory types (CD62L+CD45RO+RA-).


View this table:
[in this window]
[in a new window]
 
Table 1.  Flow cytometric analysis of urine and peripheral blood

 
These results suggested that cellular immunity-mediated inflammatory renal lesions such as fresh cellular crescent formations and diffuse tubulointerstitial cell infiltrations might be present.

Renal histopathology of autopsy specimens
The first patient (Case 1) had candidiasis in multiple organs such as lungs, kidneys, heart and alimentary tract. Diffuse fibrous crescentic formation was observed in the kidneys (Figure 1AGo). Although the elastic fibres of arterioles were ruptured in some fields of the renal specimens, specific pathological features of arteriolitis or arteritis were not found at all in the autopsy specimens.



View larger version (101K):
[in this window]
[in a new window]
 
Fig. 1.  Crescent formation in autopsy kidney specimens. The diffuse formation of fibrous or fibrocellular crescents was observed in the kidneys of (A) case 1 and (B) case 2. Tissues were fixed by formaldehyde and stained by periodic acid–Schiff (PAS) (original magnification x100).

 
The second patient (Case 2) had disseminated microembolisms of aspergillus in multiple organs including the brain, which may have been the cause of the cerebral infarction and bleeding. In the kidneys there were diffuse fibrous or fibrocellular crescent formations in the glomeruli, and interstitial fibrosis with scattered small, round cell infiltration (Figure 1BGo). Arteritis or arteriolitis was not observed in the autopsy specimens, although the cutaneous leukocytoclastic vasculitis seen in the skin biopsy gave support to the diagnosis of systemic vasculitis.

These findings revealed that systemic infection was the cause of death in these two patients. Furthermore, there were diffusely formed crescents, although almost fibrous, which might have resulted from suppression of the diseases by a series of treatments with prednisolone and cyclophosphamide.



   Discussion
 Top
 Introduction
 Subjects and methods
 Cases
 Results
 Discussion
 References
 
In the urine of patients with GN accompanied by active cellular infiltration, including IgA nephropathy, Henoch-Schönlein purpura nephritis, and ANCA-associated GN, T cells appear together with macrophages. The urine T cells are mainly CD62L(L-selectin)-, CD45RO+ and CD45RA-, which are the phenotypic manifestations of effector T cells [1,4]. These T cells are thought to have been participants in the development of the diseases appearing in the urinary space while functioning as effector cells in renal inflammatory lesions. In agreement with this presumption, T cells infiltrating glomeruli, crescents, and tubulointerstitial lesions are also of the effector type [1,5]. The cells found in urine express the mRNA of the Th1 cytokines, interleukin-2 and interferon-{gamma}. The appearance of effector T cells and macrophages in urine can be considered to reflect the cellular immune reaction that occurs in the kidneys of patients with GN accompanied by active cell infiltration [1]. As shown in our current case reports, the detection of immune effectors in urine of a patient is very informative with respect to the diagnosis of inflammatory renal involvement, especially when the patient's condition is too severe to allow renal biopsy.

However, one patient who was diagnosed to have classical periarteritis nodosa and developed acute renal failure during treatment (which included prednisolone) did not show any T cells or macrophages in the urine (case not presented). The renal biopsy showed arteritis accompanied by fibrinoid necrosis at the arcuate artery level, although neither crescent formation nor diffuse tubulointerstitial nephritis were observed. Thus, the detection of effector T cells and macrophages in urine indicates that attack by the immune effector cells upon kidney tissue may be orientated to glomerular capillary walls or tubules, which separate the inner space from the outer urinary space.



   Acknowledgments
 
The authors thank Noriko Ikeda for her skilful technical assistance. This work was supported by the Ministry of Education, Science, Sports and Culture (Grant-in-Aid for Scientific Research [C] 12670420).



   Notes
 
Correspondence and offprint requests to: Minoru Sakatsume MD PhD, 1–757 Asahimachi-dori, Niigata 951-8510, Japan. Email: sakatsum{at}med.niigata-u.ac.jp Back



   References
 Top
 Introduction
 Subjects and methods
 Cases
 Results
 Discussion
 References
 

  1. Sakatsume M, Xie Y, Ueno M et al. Human glomerulonephritis accompanied by active cellular infiltrates shows effector T cells in urine. J Am Soc Nephrol2001; 12:2636–2644[Abstract/Free Full Text]
  2. Thompson JM, Gralow JR, Levy R, Miller RA. The optimal application of forward and ninety-degree light scatter in flow cytometry for the gating of mononuclear cells. Cytometry1985; 6:401–406[ISI][Medline]
  3. Salzman GC, Crowell JM, Martin JC et al. Cell classification by laser light scattering: identification and separation of unstained leukocytes. Acta Cytol1975; 19:374–377[ISI][Medline]
  4. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature1999; 401:708–712[CrossRef][ISI][Medline]
  5. Cunningham MA, Huang XR, Dowling JP, Tipping PG, Holdsworth SR. Prominence of cell-mediated immunity effectors in ‘pauci-immune’ glomerulonephritis. J Am Soc Nephrol1999; 10:499–506[Abstract/Free Full Text]
Received for publication: 30. 3.02
Accepted in revised form: 19. 9.02





This Article
Extract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Sakatsume, M.
Articles by Gejyo, F.
PubMed
PubMed Citation
Articles by Sakatsume, M.
Articles by Gejyo, F.