Nucleolar silver staining patterns of lymphocytes in sarcoidosis

Path. Res. Pract. 188, 131-134 (1992)

Nuc1eolar Silver Staining Patterns of Lymphocytes in Sarcoidosis W. Popp, H. Zwick and T. Wanke Lungenabteilung, KH der Stadt Wien-Lainz, Vienna, Austria

O. Braun and J. H. Holzner Pathologisch-Anatomisches Institut, Universität Wien, Vienna, Austria

F. Wachtier Histologisch-Embryologisches Institut, Universität Wien, Vienna, Austria

SUMMARY

Bronchoalveolar lavage lymphocytes from 15 patients with pulmonary sarcoidosis and 8 healthy controls were investigated for nudeolar silver staining patterns and lymphocyte subpopulations. Patients with sarcoidosis had increased numbers of silver stained dots versus controls (2.20 ± 0.24 versus 1.78 ± 0.07; P < 0.001). The number of silver stained dots showed the strongest positive correlation to helper cells (OKT 4+) (r = 0.781; P < 0.0001). These results may be interpreted as further evidence of lymphocytic activation, especially of helper cells (OKT 4+) in pulmonary sarcoidosis.

Introduction

Silver staining of nucleoli is a phenomenon first described by Ruzicka in 1899 22 • A correlation between certain chromosomes and nucleoli was recognized by Heitz 8 and these regions were later called "nucleolar organizing" by McClintock ll . The general concept of nucleolar organizer region (NOR) has not changed essentially since that time. The NOR is a region of acrocentric chromosomes containing the main r-RNA genes which can be stained by silver staining of NOR-associated proteins if the NOR has been transcriptionally active beforehand3 ,6,24,25. The amount and distribution of NOR associated, silver stained proteins have been correlated with cellular activity both in vitro and in viv0 2,4,5, 10, 12, 15, 17, 18,26,28. In all these studies, silver staining has been found to be a very sensitive indicator of cellular activity. Lymphocytes from patients with sarcoidosis were described as highly activated at the site of disease 9, 13, 14,27. © 1992 by Gustav Fischer Verlag, Stuttgart

The aim of this investigation was to study cellular activation of lymphocytes from bronchoalveolar lavage (BAL) of patients with sarcoidosis and healthy controls by silver staining of NOR associated proteins.

Material and Methods The sampIe comprised 15 patients with pulmonary sarcoidosis and 8 healthy voluntary controls. All probands underwent BAL and the diagnosis of sarcoidosis was confirmed by transbronchial lung biopsy. After local anaesthesia with 2% xylocaine, a fiberoptic bronchoscope (Olympus BF 10) was used for BAL in the middle lobe20 • One hundred ml 3rc isotonic saline was placed in 20 ml aliquots and recovered by gentle aspiration. BAL fluid 'Yas collected into siliconized glass tubes and processed immediately at 4°C. Viability of more than 90% of cells was shown with Trypan Blue exclusion in all cases. Cells were counted in a haemocytometer and cell count was given per milliliter. Cytocentrifuge ceII preparations were made for May-Grünwald-Giemsa staining and 0344-0338/92/0188-0131$3.50/0

132 . W. Popp et al. silver staining. Differential ceIl counts were made on 400 ceIls. Lymphocytes were identified by their light scattering properties and the subpopulations were investigated after immunofluorescent staining with f1uorescein-isothiocyanate (FITC) or phycoerythrin (PE) conjugated monoclonal antibodies on at least 400 lymphocytes in a cytofluorometer (Ortho-Spectrum III) in conneetion with a Data;Handler 2140; for this procedure aliquots of 0.5 ml of BAL fluid were incubated with 25 fll of the foIlowing antibodies: OKT 3(CD 3)-FITC, OKT 4(CD 4)-FITC with OKT 8(CD 8)-PE, OKT 4-FITC with OKDR-PE, OKNK (CD 16)-FITC (Ortho Diagnostic)16. Silver staining was performed after fixation in phosphate buffered formalin with freshly prepared solution: 2 g of gelatin were dissolved in a 1 % aqueous solution of formic acid and this was mixed with a 50% aqueous solution of silver nitrate in a proportion of 1 : 2 5 • Cytocentrifuge ceIl preparations were incubated in this reagent at room temperature in the dark for 30 minutes. Afterwards, slides were washed thoroughly with deionized water, dehydrated in xylene, and mounted in EnteIan. For each proband the number of silver-stained dots per nucleus were counted in 200 lymphocytes without foreknowledge of the dia gnosis. Reproducibility of counts of silver stained dots was

Table 1. Background data on patients and controls Sarcoidosis n = 15 Age (years) Sex (maleIfemale) Chest X-ray (stage I1IfIIII) ACE increased

38.9 ± 13.6 619 915/1

7

Controls

n = 8

43.5 ± 15.4

4/4

01010

o

Chest X-ray stage I = bilateral hilar lymphadenopathy, II = I + infiltrates, III = lung fibrosis; ACE (Serum angiotensin converting enzyme, normal range 7-26 Vlml).

shown by an interobserver coefficient of variation less than 10% and an intraobserver coefficient of variation less than 5%. Statistical analysis was done using the Mann-Whimey V test, and correIation analysis 23 . A p value smaIler than 0.05 was considered statisticaIly significant.

Results The data from study subjects are listed in Table 1, and BAL results are given in Table 2. Patients with sarcoidosis showed increased total cell count and increased lymphocytes in the BAL. Lymphocyte subpopulations showed highly increased helper cells (OKT 4+) and slightly decreased suppressor cells (OKT 8+), resulting in an increased helper/suppressor cell index (OKT 4+/0KT 8+) as weil as an increase of D R + helper cells (0 KT 4 + OKDR +) when compared to controls. The silver staining in lymphocytes both in healthy controls and in patients with sarcoidosis consisted of round stained silver dots. The lymphocytes from healthy controls showed one or two silver stained dots, whereas those from patients with sarcoidosis showed several, up to four silver stained dots (see Fig. 1). The me an number of silver stained dots was significantly increased in pulmonary sarcoidosis versus controls (Table 2). The increase of silver stained dots showed the strongest positive correlation to helper cells (OKT 4+) but also in a lesser degree to activated helper cells (OKT 4+ OKDR +) (Table 3). Positive correlations to the percentage of T-lymphocytes (OKT 3+), and negative correlation to suppressor cells (OKT 8+) appear secondary due to the strong correlations to helper cells (OKT 4+). No correlation was found to OKDR + lymphocytes or natural killer cells (0 KNK +) .

Table 2. Rcsults from BAL and silver stained dots of lymphocytes Sarcoidosis n = 15

n

Recovery (%) Total ceIl count CeIls per ml

72.7 ± 8.4% 16.6 ± 10.3 X 10 6 2.2 ± 1.2 x 10 5

62.5 ± 13.6% 7.8 ± 1.7 x 10 6 1.3 ± 0.4 x 10 5

n.s. p < 0.01 P < 0.05

Alveolar macrophages Lymphocytes Neutrophil granulocytes Eosinophil granulocytes

56.7 ± 37.1 ± 5.6 ± 0.7±

92.4 ± 7.9 ± 0.9 ± 0.1±

P < 0.001 P < 0.01 n.s. n.s.

OKT3+ OKT4+ OKT8+ OKT 4+IOKT 8+ OKDR+ OKT4+ OKDR+ OKNK+

81.3 ± 15.5% 70.4 ± 19.8% 13.1 ± 6.8% 8.29 ± 7.1 15.7 ± 10.2% 10.0 ± 7.9% 13.2 ± 14.0%

63.3 ± 21.4% 40.0 ± 13.7% 22.7± 11.1% 1.99 ± 0.99 11.6 ± 10.1 2.8 ± 2.3% 16.8 ± 15.0%

p< p< p< p< n.s. p< n.s.

Number of silvcr stained dots (range)

2.20 ± 0.24 (1.78--2.48)

1.78 ± 0.07 (1.67-1.87)

p < 0.001

22.3% 18.4% 15.4% 0.8%

Controls = 8

5.2% 5.7% 0.9% 0.4%

0.05 0.01 0.05 0.01 0.05

Differential ceIl counts are given in perccntage and lymphocyte subpopulations as percent of lymphocytes, aIl me an ± standard deviation; for the silver stained dots, the lower and the upper value of the range are given additionaIly in brackets. Statistical analysis was performcd using the Mann Whitney V test (n.s. = not significant).

Nucleolar Silver Staining . 133

Fig. 1. On the left a Iymphocyte from a healthy control subject can be seen with one silver stained dot (nucleolus); the Iymphocytes from patients with sarcoidosis show rwo (center) and three silver stained dots (right), approx, 1800x. Table 3. Correlations berween Iymphocyte subpopulations and number of silver stained dots Lymphocyte subpopulations

Correlation coefficient to Significance of silver stained dots correlation

OKT3+ 0.590 OKT4+ 0.781 OKT 8+ -0.678 OKT 4+/0KT 8+ 0.717 OKDR+ 0.319 OKT4+ OKDR+ 0.622 OKNK+ -0.301

p< p< p< p< n.s. p< n.s.

0.01 0.0001 0.001 0.001 0.01

Discussion Nucleolar organizer regions have become of increasing interest in recent years, not only in basic research but also in clinical investigations 1,4,5,24. Transcriptionally active NORs are associated with highly argentaphilic proteins (AgNOR associated proteins) in their neighbourhood. These proteins are located predominantly ifl the fibrillar centers and in the dense fibrillar component of nucleoli 24, 25. Many factors, e.g. cellular activation and inactivation, satellite associations, and polyploidy may contribute to different results of silver staining on nucleoli5 , 12, 17,28. Increased amounts of AgNOR associated pro teins are generally considered reflecting activated cellular metabolism24 . The small round, silver stained dots correspond to individual ring-shaped nucleoli 24 . These ring-shaped nucleoli may contain one or more transcriptionally active NORs29,31. Lymphocytes from patients with pulmonary sarcoidosis showed statistically significant increased numbers of silver positive dots, i.e., nucleoli as compared to healthy controls. Cell preparations from BAL fluid gave optimal results not influenced by the pretreatment procedures of embedding, dewaxing, or sectioning and with low background deposits. The results of BAL cellular distribution and lymphocyte subpopulation counts are similar to those from recent investigations and may be interpreted in the light of other observations of increased metabolie activity of lymphocytes in patients with sarcoidosis at the site of disease 9, 13, 14,21,27. The increase in number of silver stained dots evidently corresponds to a previous activation of

NORs7,29-31. Furthermore the increase in the illimber of silver stained dots correlated well with the increase of T-Iymphocytes (OKT 3+), helper cells (OKT 4+), activated DR + helper cells (OKT 4+ OKDR +), and the helperlsuppressor cell index (OKT 4+/0KT 8+). The strong positive correlation of increase of silver stained dots to helper cells (OKT 4+) underlines the importance of the helper cell population in pulmonary sarcoidosis not only as an inflammatory reaction but also as an activation of cells which may play itself a role in immunomodulation 19 . The silver staining of lymphocytes proved to be an easy and rapid technique for investigating lymphocytic activation in sarcoidosis. If pulmonary sarcoidosis is suspected, silver staining should be performed for assessing of lymphocytic activation, especially ofhelper cells (OKT 4+) in pulmonary sarcoidosis. Acknowledgement We thank E. Handler (MTA) for technical assistance.

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Received January 21, 1991 . Accepted in revised form April 16, 1991

Key words: Nucleoli - Silver staining - Lymphocyte subpopulations - Sarcoidosis Dr. Wolfgang Popp, Lungenabteilung, KH der Stadt Wien-Lainz, Wolkersbergenstraße 1, 1130 Vienna, Austria