Respirology (2002) 7, 273–279
CASE REPORT
Increased circulating CD16 + CD14dim monocytes in a patient with pulmonary alveolar proteinosis Y YOSHIOKA,1 A OHWADA,1 N HARADA,1 N SATOH,2 S SAKURABA,1 T DAMBARA1 and Y FUKUCHI1 Departments of 1Respiratory Medicine and 2Clinical Pathology, Juntendo University, School of Medicine, Tokyo, Japan
Increased circulating CD16+ CD14dim monocytes in a patient with pulmonary alveolar proteinosis YOSHIOKA Y, OHWADA A, HARADA N, SATOH N, SAKURABA S, DAMBARA T, FUKUCHI Y. Respirology 2002; 7: 273–279 Abstract: Pulmonary alveolar proteinosis (PAP) is characterized by filling of the alveoli with a periodic acid-Schiff-positive proteinaceous material. Although the pathogenesis of primary or idiopathic PAP remains unknown, it has been proposed that a deficiency or loss of responsiveness of the monocyte/macrophage lineage to granulocyte–macrophage colony stimulating factor (GM-CSF) is involved in PAP. Secondary PAP is associated with haematological malignancies, especially in myeloid disorders. Herein, we report on an adult with PAP associated with myelodysplastic syndrome (MDS). The CD16+ CD14dim monocytes comprise 5–10% of circulating monocytes in healthy volunteers. Flow cytometric analysis of the patient in the present study revealed increased CD16+ CD14dim monocytes in the peripheral blood. It has been demonstrated that the expression of CD16 and CD14 is regulated by macrophage colony stimulating factor (M-CSF) and GM-CSF. Hence, serum cytokines were analysed in our patient and the concentration of serum GM-CSF was found to be less than the lower limit of the assay. In addition, serum M-CSF and granulocyte colony stimulating factor levels were only slightly increased above the normal range. These results suggest that the increase in the CD16+ CD14dim subpopulation in the circulation of our patient indicates another pathogenetic mechanism for secondary PAP, such as hyperresponsiveness of the monocyte/ macrophage lineage to these cytokines. Key words: CD14, CD16, myelodysplastic syndrome, pulmonary alveolar proteinosis.
INTRODUCTION Pulmonary alveolar proteinosis (PAP) is characterized by filling of the alveoli with a periodic acid-Schiff (PAS)-positive proteinaceous material rich in phospholipid.1 Recently, the importance of granulocyte– macrophage colony stimulating factor (GM-CSF) in pulmonary proteinosis has been recognized since Dranoff et al. demonstrated the appearance of
Correspondence: Dr Yasuko Yoshioka, Department of Respiratory Medicine, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Email:
[email protected] Received 24 September 2001; revised 14 December 2001; accepted for publication 18 January 2002.
PAP-like pulmonary lesions in the GM-CSF knockout mouse without any deficit in basal haematopoiesis.2 Primary or idiopathic forms of PAP occur spontaneously in normal patients. The association of PAP with haematological disorders such as leukaemia or lymphoma, is well established and considered as secondary PAP. Cordonnier et al. demonstrated that the incidence of secondary PAP in patients with pneumonia and haematological disorders was estimated to be 5.3% among all haematological patients examined and 10% in patients with myeloid disorders, such as acute or chronic myeloid leukaemia.3 Pulmonary alveolar proteinosis in myelodysplastic syndrome (MDS) has also been reported.4,5 It is now accepted that there is a CD16+ CD14dim or CD16+ CD14+ subpopulation representing 5–10% of total circulating monocytes in healthy individuals.6,7 The remaining 90% of monocytes (conventional monocytes) are represented as CD16- CD14bright or CD16- CD14++ cells. Nearly 70% of alveolar
274 macrophages are estimated to be derived from circulating monocytes that highly express the CD16 molecule but express the CD14 molecule at low levels.6–8 In vitro studies have revealed that macrophage colony stimulating factor (M-CSF) is capable of inducing CD16 expression on monocytes, whereas GM-CSF downregulates CD14 expression.9,10 Therefore, it is possible that these cytokines regulate the expression of CD16 and CD14 and the differentiation of CD16+ CD14dim monocytes in the circulation. Herein, we report on a patient with PAP associated with MDS, with a high percentage of circulating CD16+ CD14dim monocytes. We measured serum cytokines and discuss the association of increased CD16+ CD14dim monocytes with cytokine levels in peripheral blood.
CASE REPORT A 66-year-old male patient diagnosed as having hypoplastic MDS has been under medical care in the haematology department of our hospital since 1988. The white blood cell count (WBC) at diagnosis was 3.3 ¥ 109 /L (3% band, 33% segmented neutrophils, 59% lymphocytes, 2% monocytes and 3% eosinophils), the red blood cell (RBC) count was 2.92 ¥ 1012 /L, haemoglobin (Hb) was 11.1 g/dL and the platelet count was 103 ¥ 109 /L. The bone marrow was hypocellular with myeloid hypoplasia (2.8 ¥ 1010 /L nucleated cell count, ratio of myeloid cells to erythroid cells 0.55, 0.6% blasts). Cytogenetic analysis revealed(47,XY,add(1)(p11),+add(1),t(1;19)(p10;q10), –14,+mar1) in all 20 metaphases examined. The patient had smoked one packet of cigarettes every 3 days for 45 years. He complained of a fever and skin rash on his trunk in 1999 that waxed and waned. After 3 months of these episodes, he was referred to our department because he had developed pulmonary infiltrates in the left upper lobe as evidenced on chest radiographic examination and thoracic computed tomography (CT) scan. The patient did not complain of dyspnoea at rest or on exertion. Bronchoscopic biopsy examination was aborted due to massive bleeding. The patient’s spike fever returned and the degree of pulmonary infiltrate progressed without any improvement following the administration of antibiotics. The presence of focal vasculitis was diagnosed clinically and steroid therapy was commenced, initially with methylprednisolone pulse therapy (1000 mg/day) for 3 days and then with prednisolone (PSL) at a dose of 1 mg/kg bodyweight. Consequently, the patient’s fever and the infiltrates on chest radiography improved dramatically. The dose of PSL was gradually tapered and the patient was maintained on 5 mg PSL thereafter. One and a half years later, the patient was readmitted with infiltrates in the left upper lung despite an increased dose of PSL up to 35 mg/day. Chest radiographic examination revealed bilateral multifocal pulmonary infiltrates (Fig. 1a). A thoracic CT scan confirmed the presence of pulmonary infiltrates and air bronchogram in the left upper lobe (Fig. 1b; upper panel) and increased opacities in both the right middle lobe and bilateral lower lobes (Fig. 1b;
Y Yoshioka et al.
lower panel). Laboratory tests revealed anaemia (Hb 10.9 g/dL) and elevated serum lactate dehydrogenase (1763 IU/L, with dominance of the type II isozyme). The WBC count was 5.2 ¥ 109 /L with mild eosinophilia and basophilia (0.5% myeloblasts, 2% bands, 65.5% segmented neutrophils, 0.5% monocytes, 14% lymphocytes, 14.5% eosinophils, 2.5% basophils). Arterial blood gas analysis on room air revealed no deterioration in the arterial oxygen level (pH 7.466, PaC O 2 36.3 mmHg, PaO 2 92 mmHg). Left upper lobe bronchoalveolar lavage (BAL) was performed. The lavage fluid was opaque and contained amorphous debris and cholesterol crystals. The amorphous material was PAS positive and electron microscopy examination revealed multilamellated tubular myelin structures in the cytoplasm of alveolar macrophages and in the extracellular space (Fig. 1c,d). The number of BAL cells was 6.6 ¥ 105 /mL. Cell differentiation revealed 1.7% alveolar macrophages, 28.7% lymphocytes and 59.7% neutrophils. Lipid analysis of the BAL fluid revealed that the fluid was rich in phosphatidylcholine. All cultures from the lavage fluid were negative. Autoimmune antibody against GM-CSF, usually detectable in patients with idiopathic PAP, was not detected either in the serum or the BAL fluid. We diagnosed the patient as having PAP associated with MDS. After diagnosis of PAP, the dose of PSL was tapered rapidly from 35 to 5 mg/day. After administration of amobroxol (Mucosolvan; Teijin, Tokyo, Japan), gradual improvement of pulmonary infiltrates was noted. Determination of serum cytokines was undertaken during the administration of PSL at 35 mg/day. The serum GM-CSF concentration was immeasurable due to the fact that the concentration was less than the lower limit of the assay (8 pg/mL), but the level detected in BAL fluid (120 pg/mL) suggested an ability to produce GM-CSF in this patient. The serum concentration of M-CSF was slightly elevated (7660 pg/mL). The concentrations of granulocyte colony stimulating factor (G-CSF) in the serum and BAL fluid were 41 and 333 pg/mL, respectively, and the serum concentration was elevated compared with published accepted values;11 there is no standard value for BAL. The presence of CD16+ CD14dim monocytes among peripheral blood leucocytes was determined by flow cytometric analysis (FACSvantage; Becton-Dickinson, Rutherford, NJ, USA). Heparinized whole blood was obtained when the dose of PSL was tapered to 5 mg/day and stained directly for three-colour immunofluorescence with allophycocyanin (APC)conjugated CD14 (LPS receptor; Beckman Coulter, Fullerton, CA, USA), phycoerythrin (PE)-conjugated CD16 (FcgRIII; Nichi-rei, Tokyo, Japan) and fluorescein isothiocyanate (FITC)-conjugated CD64 (FcgR1; Beckman Coulter), along with the respective isotypematched controls. To confirm the CD16+ CD14dim subpopulation among the circulating monocytes, gated CD64+ cells were analyzed for CD16 and CD14 expression. The findings revealed that CD16+ CD14dim monocytes were increased in the peripheral blood of our patient compared with healthy control subjects (88.6 vs 7.6% of gated CD64+ monocytes; Fig. 2a,b).
Increased CD16 + CD14dim monocytes in PAP
275
Figure 1 (a) Chest radiograph revealing bilateral multifocal pulmonary infiltrates predominantly involving the left upper lung field. (b) A thoracic computed tomography scan revealed dense pulmonary infiltration in the upper lobe (upper panel) and increased opacity in both the right middle lobe and bilateral lower lobes (lower panel). (c) Electron micrograph of the cytoplasm of an alveolar macrophage. Note the various morphological forms of lamellar bodies and dense granules (original magnification ¥4000). (d) Electron micrograph of bronchoalveolar lavage fluid. Note the myeloid bodies and numerous myelin structures (original magnification ¥100 000).
276
Y Yoshioka et al.
CD64
0
50
100
150
200
250
50
100
150
200
250
100
101
102
103
104
101
102
103
104
100
100
101
101
102
102
103
103
104
104
(b)
CD14
100
100
101
101
102
102
103
103
104
104
(a)
0
FSC
100
CD16
Figure 2 Flow cytometric analysis by direct three-colour immunofluorescence staining of the circulating monocytes. (a) Peripheral blood from the patient in the present study with pulmonary alveolar proteinosis. (b) Peripheral blood from a healthy control subject. Left panels, CD64-positive cells were gated as monocytes. Right panels, CD14 and CD16 expression were then analyzed. The CD16+ CD14dim subpopulation comprised 88.6% of CD64-positive monocytes in the patient compared with 7.6% of CD64-positive monocytes in the healthy control subject.
Even in the CD16+ cell fraction, the tendency to decreased expression of CD14 among these cells was obvious. The BAL cells in cytospin preparations were examined by immunohistochemical staining. Cells double stained with PE-conjugated CD16 and FITCconjugated CD64 were examined by fluorescence microscopy (Nikon ECLIPSE TE300; Nikon, Tokyo, Japan; and ZEISS Axioplan; Carl Zeiss Japan, Tokyo, Japan). The CD16+ monocyte/macrophage cells were detected among the BAL cells from our patient (Fig. 3). It was difficult to calculate the ratio of CD16+ cells because the majority of BAL cells on the cytospin slides were disfigured.
DISCUSSION The genetic predisposition for PAP has been investigated. Four patients with congenital PAP were
confirmed to have a bc-chain defect in the GM-CSF receptor on peripheral blood mononuclear cells.12 Another patient with a mutation at position 382 of GM-CSF cDNA has also been reported.13 However, these mutations may not be common among adult patients with PAP.13 In addition, it has been reported that some PAP patients have a functional deficiency of GM-CSF caused by the inhibitory cytokine interleukin (IL)-1014 and by a blunted responsiveness of the GM-CSF receptor to GM-CSF and IL-3.15 Recently, Kitamura et al. demonstrated that a neutralizing antibody against GM-CSF was detectable in the BAL fluid and serum of all patients with idiopathic PAP.16 Interestingly, this autoimmune antibody against GM-CSF was not detected in patients with secondary PAP, including the present case. The observations of Kitamura et al. indicate that the aetiologies of deficiency or loss in responsiveness of monocyte–macrophage cells to GM-CSF seen in PAP are divergent between primary and secondary PAP.
Increased CD16 + CD14dim monocytes in PAP
277
Figure 3 Immunohistochemical staining of a bronchoalveolar lavage cell. (a) A representative green-coloured CD64-positive cell observed through the filter for fluorescein isothiocyanate immunofluorescence (original magnification ¥400). (b) The same cell shown in (a) was stained positively with CD16 (red coloured) and photographed through the filter for phycoerythrin (PE; original magnification ¥400). (c) Double staining for CD64 and CD16 (yellow coloured) in the same cell (original magnification ¥400). (d) Phase-contrast microscopic view of the same cell (original magnification ¥400).
The serum concentration of G-CSF in our patient was slightly higher than that in healthy control subjects.11 Janowska-Wieczorek et al. reported that the serum M-CSF concentration in a normal control group was 4464 ± 1332 pg/mL, whereas among patients with MDS the mean serum M-CSF concentration was 11 496 ± 4080 pg/mL.17 Based on these findings, the M-CSF values in patients with MDS are significantly elevated compared with healthy control subjects. However, the serum M-CSF concentration in our patient was only slightly higher than the normal range. In a clinical study with recombinant human M-CSF administration, the dose-related effects of combined M-CSF and g-interferon therapy on the increase of CD16+ CD14dim monocytes among circulating monocytes was recognized, occurring with maximal induction of these monocytes, in the 80–100 mg/kg per day M-CSF dose range.18 In a pharmacokinetic study with consecutive 7 day infusion of recombinant M-CSF in patients with cancer, serum concentrations of M-CSF were greater than 1 mg/mL with M-CSF administration over 100 mg/kg per day.19 These findings suggest that a serum M-CSF concentration of approximately 1 mg/mL increases CD16+ CD14dim monocytes in the circulation. Hence, it is postulated that there was a certain pathophysiological abnormality that increased CD16+ CD14dim monocytes in the peripheral blood of the present patient and this was inconsistent with the serum level of MCSF. The CD16 and CD14 phenotype of peripheral blood monocytes in MDS has not been reported previously. We have made a preliminary assessment of CD16+ CD14dim monocytes in the peripheral blood of MDS patients. The mean (±SD) percentage of circulating CD16+ CD14dim monocytes was 44.3 ± 9.5% in three MDS patients other than the present case compared with 8.3 ± 4.8% in four normal volunteers. This analysis is premature because we have not yet evalu-
ated the cytokine profiles or chest radiographs in these patients. However, increased circulating CD16+ CD14dim monocytes in MDS patients may be a predisposing factor for the appearance of PAP lesions. Because the percentage of circulating CD16+ CD14dim monocytes in our patient was much higher than in other MDS patients examined, there may exist some possibility of clonal expansion of CD16+ CD14dim monocytes in the present case. Although several bone marrow aspiration examinations were conducted during the clinical course, no increase in monocytes or monoclonal expansion of monocytes was recognized. It is well known that circulating CD16+ CD14dim monocytes are increased in many pathological situations, such as malignancy,7 sepsis20 or human immunodeficiency virus infection.21 In this particular case, we were unable to detect or identify any coexisting disorder. Rather than the presence of a concomitant disorder, hyperresponsiveness of the monocyte/macrophage lineage to M-CSF may exist in a cooperative manner with other cytokines, such as GM-CSF, even though the levels of GM-CSF were below the measurable lower limit in our patient’s serum. In the present patient, pulmonary infiltrates were initially identified and resolved rapidly with steroid therapy. Bronchiolitis obliterans with organizing pneumonia (BOOP)-like pulmonary disease responsive to steroid therapy in MDS patients has been reported.22 However, the pulmonary infiltrates in our case were later diagnosed as being due to PAP by BAL. It is not clear whether circulating CD16+ CD14dim monocytes can move into the alveoli and contribute to the pulmonary disease, but the appearance of CD16+ monocytes in BAL cells suggests the possibility that CD16+ CD14dim monocytes are involved in the pathogenesis of PAP pulmonary lesions. In addition, the responsiveness of pulmonary infiltrates to steroid
278 therapy concurs with a report that high-dose glucocorticoid therapy selectively depletes CD16+ CD14dim monocytes in the peripheral blood, possibly by blocking the influx of new CD16+ CD14dim monocytes into the circulation.23 Although the most effective treatment for PAP is alveolar lavage, we postponed this procedure until clinical deterioration was evident. Because there were no respiratory symptoms and signs, a surfactant activator (Ambroxol) was chosen for this patient.24,25 We have reported on a patient with PAP associated with MDS in whom the CD16+ CD14dim subpopulation of circulating monocytes was increased. The pathophysiological meaning of this increase is unknown, but further evaluation to determine whether CD16+ CD14dim monocytes are increased in other PAP and MDS patients would be of interest.
ACKNOWLEDGEMENTS The authors thank Dr K. Nakata (Department of Pulmonary Medicine, International Medical Center of Japan, Tokyo, Japan) for measurement of the autoimmune antibody against GM-CSF in the serum and BAL fluid. The authors also thank Dr H. Miyajima for expert technical assistance in FACS and Dr A. Ohsaka (Division of Blood Transfusion, Juntendo University) for his valuable knowledge and suggestions.
REFERENCES 1 Mani S, Eugene J, Sullivan EJ, Piccin R, Thomassen MJ, Stoller JK. Exogenous granulocyte–macrophage colony stimulating factor administration for pulmonary alveolar proteinosis. Am. J. Respir. Crit. Care Med. 2000; 161: 1143–8. 2 Dranoff G, Crawford AD, Sadelain M et al. Involvement of granulocyte–macrophage colony-stimulating factor in pulmonary homeostasis. Science 1994; 264: 713–16. 3 Cordonnier C, Fleury-Feith J, Escudier E, Atassi K, Bernaudin JF. Secondary alveolar proteinosis is a reversible cause of respiratory failure in leukemic patients. Am. J. Respir. Crit. Care Med. 1994; 149: 788–94. 4 Terashima T, Nakamura H, Meguro S et al. Pulmonary alveolar proteinosis associated with myelodysplastic syndrome. Nihon Kyobu Shikkan Gakkai Zasshi 1995; 33: 645–51 (in Japanese with an English abstract). 5 Tamaki Y, Seyama K, Takahashi H et al. Progressive interstitial pneumonia associated with myelodysplastic syndrome: Implication of superoxide hyperproduction by neutrophils. Respirology 1997; 2: 295–8. 6 Ziegler-Heitbrock HWL. Definition of human blood monocytes. J. Leukoc. Biol. 2000; 67: 603–6. 7 Saleh MN, Goldman SL, LoBuglio AF et al. CD16+ monocytes in patients with cancer: Spontaneous elevation and pharmacologic induction by recombinant human macrophage colony-stimulating factor. Blood 1995; 85: 2910–17. 8 Prakash UBS, Barham SS, Carpenter HA, Marsh HM. Pulmonary alveolar phospholipoproteinosis: Experience with 34 cases and a review. Mayo Clin. Proc. 1987; 62: 499–518.
Y Yoshioka et al. 9 Geissler K, Harrington M, Srivastava C, Leemhuis T, Tricot G, Broxmeyer HE. Effects of recombinant human colony stimulating factors (CSF) (granulocyte–macrophage CSF, granulocyte CSF, and CSF-1) on human monocyte/macrophage differentiation. J. Immunol. 1989; 143: 140–6. 10 Young DA, Lowe LD, Clark SC. Comparison of the effects of IL-3, granulocyte–macrophage colony-stimulating factor, and macrophage colony-stimulating factor in supporting monocyte differentiation in culture. Analysis of macrophage antibody-dependent cellular cytotoxicity. J. Immunol. 1990; 145: 607–15. 11 Watari K, Asano S, Shirafuji N et al. Serum granulocyte colony-stimulating factor levels in healthy volunteers and patients with various disorders as estimated by enzyme immunoassay. Blood 1989; 73: 117–22. 12 Dirksen U, Nishinakamura R, Groneck P et al. Human pulmonary alveolar proteinosis associated with a defect in GM-CSF/IL-3/IL-5 receptor common b chain expression. J. Clin. Invest. 1997; 100: 2211–17. 13 Bewig B, Wang X-D, Kirsten D, Dalhoff K, Schäfer H. GM-CSF and GM-CSF bc receptor in adult patients with pulmonary alveolar proteinosis. Eur. Respir. J. 2000; 15: 350–7. 14 Tchou-Wong K-M, Harkin TJ, Chi C, Bodkin M, Rom WN. GM-CSF gene expression is normal but protein release is absent in a patient with pulmonary alveolar proteinosis. Am. J. Respir. Crit. Care Med. 1997; 156: 1999–2002. 15 Seymour JF, Begley CG, Dirksen U et al. Attenuated hematopoietic response to granulocyte–macrophage colony-stimulating factor in patients with acquired pulmonary alveolar proteinosis. Blood 1998; 92: 2657– 67. 16 Kitamura T, Tanaka N, Watanabe J et al. Idiopathic pulmonary alveolar proteinosis as an autoimmune disease with neutralizing antibody against granulocyte/ macrophage colony-stimulating factor. J. Exp. Med. 1999; 190: 875–80. 17 Janowska-Wieczorek A, Belch AR, Jacobs A et al. Increased circulating colony-stimulating factor-1 in patients with preleukemia, leukemia, and lymphoid malignancies. Blood 1991; 77: 1796–803. 18 Weiner LM, Li W, Holmes M et al. Phase I trial of recombinant macrophage colony-stimulating factor and recombinant g-interferon: Toxicity, monocytosis, and clinical effects. Cancer Res. 1994; 54: 4084–90. 19 Jakubowski AA, Bajorin DF, Templeton MA et al. Phase I study of continuous-infusion recombinant macrophage colony-stimulating factor in patients with metastatic melanoma. Clin. Cancer Res. 1996; 2: 295–302. 20 Fingerle G, Pforte A, Passlick B, Blumenstein M, Strobel M, Ziegler-Heitbrock HW. The novel subset of CD14+/ CD16+ blood monocytes is expanded in sepsis patients. Blood 1993; 82: 3170–6. 21 Allen JB, Wong HL, Guyre PM, Simon GL, Wahl SM. Association of circulating receptor Fc gamma RIII-positive monocytes in AIDS patients with elevated levels of transforming growth factor-beta. J. Clin. Invest. 1991; 87: 1773–9. 22 Matsushima T, Murakami H, Kim K et al. Steroidresponsive pulmonary disorders associated with myelodysplastic syndromes with der (1q; 7p) chromosomal abnormality. Am. J. Hematol. 1995; 50: 110–15.
Increased CD16 + CD14dim monocytes in PAP 23 Fingerle-Rowson G, Angstwurm M, Andreesen R, Ziegler-Heitbrock HW. Selective depletion of CD14+ CD16+ monocytes by glucocorticoid therapy. Clin. Exp. Immunol. 1998; 112: 501–6. 24 Diaz JP, Manresa Presas F, Benasco C, Guardiola J, Munoz L, Clariana A. Response to surfactant activator
279 (ambroxol) in alveolar proteinosis. Lancet 1984; i: 1023. 25 Naito M, Kunieda T, Yoshioka T, Okubo S, Yutani C. A case of pulmonary alveolar proteinosis treated with oral administration of ambroxol. Nihon Kyobu Shikkan Gakkai Zasshi 1985; 23: 912–20 (in Japanese).
