659

Macrophages in the Urine in Acute Bacterial Cystitis

Taiji NISHIMURA, Yasunori TERASHIMA, Jun WATANABE, Kenji AMAGAI,Mitsuhiro SATOH, Qiang SHAO and Masao AKIMOTO

Department of Urology, Nippon Medical School(Received: March 8, 1993)(Accepted: April 12, 1993)

Key words: macrophages, acute bacterial cystitis, urinary leukocyte

Abstract

In the previous study in this series of studies concerning the role of macrophages in urinary tract

infection, we attempted to detect macrophages in the urine of acute bacterial cystitis patients by non-

specific esterase staining of urinary sediment, however none of the leukocytes stained, probably because of

cell damage caused by the urine and by centrifugation. In the present study, detection of macrophages in

urine was again attempted, this time by prompt transfer of urinary leukocytes to culture medium after

minimum centrifugation, 1 hr culture in a glass bottom chamber and non-specific staining of leukocytes

adhering to glass. Macrophages in urine were detected by this method, and they comprised 5 .9% of theadherent leukocytes, although macrophage spreading, which implies macrophage activation and is often

seen in the early stage of nonbacterial prostatitis, was hardly ever observed.

The percentages of adherent leukocytes were not correlated with urine osmolarity, probably because

the effect of urine was minimized by prompt transfer of urinary leukocytes to culture medium after the

urine samples had been collected. There have been quite few studies involving culture of urinary

leukocytes in the past. Our simple techniques, such as prompt transfer of urinary leukocytes to culture

medium after centrifuging with minimum gravity and for a minimum period of time, appear to be useful in

the study of urinary leukocytes using other cells which appear in urinary tract infection, as well as

cytokines and antibiotics, to clarify cellular mechanisms of defense in urinary tract infection.

Introduction

Although there has been a debate concerning the phagocytic ability of leukocytes in urine1,2), mostrecent reports3,4,5,6,7), including our own study8 , have confirmed phagocytosis by leukocytes in urine .Nevertheless, macrophages in urine have not been well studied, and most urologists still have the

traditional idea that macrophages are just scavengers to clean up the field of infection or inflammation, and

they are not very concerned about macrophages. However, over the past several years there has been an

increasing number of papers on new concepts of the role of macrophages, such as tissue destruction by

superoxide produced by macrophages9), tissue reconstruction by macrophages10), regulation of the survival

or growth of tissue macrophages by phospholipids11, production of various cytokines, such as tumornecrosis factor and interleukin-1, by macrophages, which is important not only for tumor immunology but

infection and inflammation12), and about new ideas on precursors and subpopulations of macrophages13,14).Therefore, macrophages in urine deserve to be studied from the standpoint of these new concepts. In this

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西村 泰司

平成5年7月20日

660 Taiji NISHIMURA et al

preliminary study we investigated whether macrophages exist in the urine of acute bacterial cystitis

patients using fresh urine samples and transferring urinary leukocytes to culture medium as soon as

possible after minimum centrifuging15) to minimize cell damage caused by the urine, such as by the effects

of its osmolarity, and by centrifugation. This was followed by nonspecific esterase staining after 1-hr

culture, because no macrophages were detected by non-specific staining of sediment from oridinary

specimens of urine from acute bacterial cystitis patients in a previous experiment"). The percentage of

activated leukocytes, detected among urinary leukocytes by their adherence to the glass bottom of culture

chamber, among urinary leukocytes and its correlation with urine osmolarity and time since the onset of

symptoms of cystitis were also studied.

Materials and Methods

Urine specimens obtained within a week of the onset of symptoms from 20 female patients with

untreated, culture-confirmed acute bacterial cystitis with more than 104/ml bacteria were used for this

study.

Urine specimens were collected by catheterization within 2 hours after the previous urination and

were immediately centrifuged at low speed (75 g) for 5 minutes to minimize damage by centrifugation.

Then, 1 ml of leukocyte suspension (2.5 X 105 cells/ml) in eagle's minimal essential medium (Gibco, USA)

containing 10% fetal calf serum (Gibco) (MEM/10% FCS) was introduced into a each chamber of a Lab-Tek

4 chamber (Miles Laboratories, Naperville, III, USA) (Fig. 1) and was incubated at 37•Ž in a CO2 incubator

for 60 minutes to allow the leukocytes to attach to the glass. Leukocytes in the original urine were counted

using a hemocytometer. Osmolarity was measured with an osmometer (Advanced Instruments Inc,

Needham Heights, Mass., USA).

For the observation of adherence of leukocytes on glass, the chamber was washed three times with

MEM to discard the unadherent leukocytes after 1 hr culture. The percentage of adherent leukocytes was

determined by examining the same area of the glass plate of the chamber before and after the washing14).

The percentage of macrophages among the adherent leukocytes was obtained after nonspecific

esterase staining and counterstaining15> (Fig. 2). Student's t test was used for statistical analysis of the

results.

Results

Urine osmolarity, urine leukocyte counts, percentages of adherent urinary leukocytes, percentages of

macrophages among adherent urinary leukocytes and the results of bacterial culture are shown in Table 1.

Fig. 1 Lab-Tek 4 chamber to culture urinary

leukocytes.

Fig. 2 Macrophage (center) among urinary leuko-

cytes after nonspecific esterase staining (•~400).

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Macrophages in the Urine in Acute Bacterial Cystitis 661

Table 1 Urine osmolarity, urinary leukocyte counts, percentages of adherent leukocytes, percent-

ages of macrophages among adherent leukocytes and bacterial culture in 20 acute bacterial cystitis

patients

Fig. 3 Relation between percentages of adherent

urinary leukocytes and urine osmolarity.

Fig. 4 Relation between percentages of adherent

urinary leukocytes and duration after onset of

symptoms of cystitis.

The mean percentages of adherent leukocytes was 8.19% (standard deviation=9.969). There were no

correlations between percentages of adherent leukocytes and urine osmolarity or number of days after theonset of symptoms (Figs. 3 and 4).

The mean percentages of macrophages among adherent leukocytes was 5.885% (standard devia-tion=9.826). There was no correlation between the percentages of macrophages and duration after theonset of symptoms (Fig. 5).

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662 Taiji NISHIMURA et al

Fig. 5 Relation between percentages of macro-

phages among adherent urinary leukocytes anddurations after onset of svmntoms of cystitis.

Fig. 6 Spreading of macrophages in prostatic fluid

from a nonbacterial prostatitis patient (•~400).

Discussion

The presence of live macrophages was confirmed by prompt transfer of urinary leukocytes to culture

medium and the nonspecific esterase staining of adherent urinary leukocytes which attached to the glass of

the culture chamber during 1-hr culture. Macrophages comprised an average of 5.9% of adherent

leukocytes. These macropahges and leukocytes appearing in urine probably retain their ability to cope with

urinary tract infection, as indicated in previous reports5,6), including our own study8), since they are stillalive and have the ability to adhere to glass.

The percentage of macrophages among urinary leukocytes tended to be higher up to 3 days after the

onset of acute bacterial cystitis than at 7 days, but this tendency was not statistically significant in the

present study. Macrophages in prostatic fluid were greater in number and activated, as revealed byspreading, in the early stage of nonbacterial prostatitis15)(Fig. 6), but urinary macrophages in acute

bacterial cystitis were smaller in number and spreading was hardly ever observed , even a few days afterthe onset of symptoms of cystitis. Therefore it seems that activation and the role of macrophages and

subpopulations of macrophages13,14) in the two may be different.

There was no correlation between the percentages of adherent urinary leukocytes and urine

osmolarity in present study, while Maeda et al.4) and Suzuki et al.51 reported higher rates of phagocytosis by

urinary leukocytes in 300-500 mOsm/kg and 300-600 mOsm/1 urine than in less than 300 mOsm/1 urine or

greater than 500 or 600 mOsm/1 urine. The reason for the different results may be less effect of urinaryosmolarity because of quicker transfer of urinary leukocytes to culture medium and centrifugation with

less gravity and duration in our study than in other studies. There have been quite a few studies of urinary

tract infection using urinary leukocytes4,5,6) in the past. Our simple techniques , such as prompt transfer ofurinary leukocytes to culture medium after centrifuging at minimum gravity for a minimum amount of

time appear to be useful for the study of urinary leukocytes using other cells appearing in urinary tractinfection and cytokines and antibiotics to identify cellular mechanisms in the body's defense against

urinary tract infection.

References

1) Knoll, B. F., Johnson, A. J. & Pearce, C. W.: The effect of autogeneous urine on leukocytic defenses in man. Invest.Urol. 6: 406-411, 1969.

2) Orikasa, S. & Hinman, F. Jr.: Reaction of the vesical wall to bacterial penetration. Resistance to attachment ,

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Macrophages in the Urine in Acute Bacterial Cystitis 663

desquamation and leukocytic activity. Invest. Urol. 15: 185-193, 1977.3) Fukushi, Y., Orikasa, S. & Kagayama, M.: An electrom microscopic study of the interaction between vesical

epithelium and E. coli. Invest. Urol. 17: 61-68,1979.4) Maeda, S., Deguchi, T., Kanimoto, Y., Kuriyama, M., Kawada, Y. & Nishiura, T.: Studies on the phagocytic

function of urinary leukocytes. J. Urol. 129: 427-429, 1983.5) Suzuki, Y., Toyoda, S., Magima, K., Fukushi, Y. & Orikasa, S.: Studies of anti-bacterial defense mechanism of

urinary bladder: 1) Activity of phagocytosis of PMN in urine. 2) Viability, cell population and activity of

phagocytosis of urinary PMN. Jap. J. Urol. 77: 574-580,1986.6) Kataoka, S. & Fujita, Y.: Basal experiment of active oxygen generation in urinary polymorphonuclear leukocytes.

Jap. J. Urol. 82: 16-23,1991.7) Goto, H. & Kiyota, H.: Superoxide anion generation of human polymorphonuclear leukocytes in urine. J. J. A. Inf. D.

65: 718-724,1991.8) Nishimura, T., Kanamori, S., Akimoto, M. & Kawai, H.: Study of macrophages in prostatic fluid. 1. Methods for

detection of macrophages. Jap. J. Urol. 72: 785-789, 1981.9) Ando, M., Sugimoto, M., Nishi, R., Suga, M., Horio, S., Kohrogi, H., Shimazu, K. & Araki, S.: Surface morphology

and function of human pulmonary alveolar macrophages from smokers and non-smoker. Thorax 39: 850-856,1984.10) Miyahara, S., Yokomuro, K., Takahashi, H. & Kimura, Y.: Regeneration and the immune system. I. In vitro and in

vivo activation of lymphocytes by liver regeneration and the role of Kupffer cells in stimulation. Eur. J. Immunol. 13:878-883, 1983.

11) Yui, S. & Yamazaki, M.: Induction of macrophage growth by negatively charged phospholipids. J. Leukocyte Biol.39: 713-716, 1986.

12) Yui, S. & Yamazaki, M.: Augmentation and suppression of release of tumor necrosis factor from macrophages bynegatively charged phospholipids. Jpn. J. Cancer Res. 82: 1028-1034,1991.

13) Naitoh, K., Ichiki, Y., Tokushima, M., Haraoka. S., Muraguchi, A. & Kimoto, M.: Development of cells withdendric/macrophage morphology from human lymphoid progenitors. Proc. Jpn. Soc. Immunol. 21: 289, 1991.

14) Komuro, T., Mabuchi, A., Yokomuro, K. & Asano, G.: Liver regeneration and immune system. Morphological andcytochemical studies of activated spleen cells during the liver regeneration after partial hepatectomy. Jap. J.Allergol. 39: 1529-1540, 1990.

15) Nishimura, T., Terashima, Y., Hattori, T., Satoh, M., Yoshida, K. & Akimoto, M.: Study of macrophages in prostaticfluid from nonbacterial prostatitis patients. V. Relation between activation of macrophages and stage of prostatitis.Urol. Int. 46: 15-17, 1991.

16) Nishimura, T., Kanamori, S., Akomoto, M. & Kawai, H.: Macrophages in prostatic fluid. Br. J. Urol. 52: 381-385,1980.

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664 Taiji NISHIMURA et al

急性細菌性膀胱炎患老の尿中に出現す るマク ロファージについて

日本医科大学泌尿器科学教室

西村 泰司 寺島 保典 渡辺 潤 天谷 健二

佐藤 三洋 郡 強 秋元 成太

(平成5年3月8日 受付)

(平成5年4月12日 受理)

要 旨

尿路感染症において下部尿路に出現するマクロ

ファージの役割に関す る著者 らの一連の研究 とし

て,以 前急性細菌性膀胱炎患者の尿沈査を非特異

的エステラーゼ染色 し,尿 中に出現す るマクロ

ファージの検出を試みたがマクロファージは全 く

検 出でず,そ の原因として尿の影響および尿沈査

をつ くる過程 における細胞傷害が考 えられた.今

回は急性細菌性膀胱炎発症後1週 間以内の患者か

ら得 られた尿を直ちに最小限の遠心後,培 養液に

尿中白血球を移 し,1時 間ガラス板底の培養チャ

ンバーで培養後,ガ ラス板に付着 した白血球を非

特異的エラステーゼ染色しマクロファージを検 出

する方法を試みた.そ の結果,付 着した白血球の

5.9%に マクロファージが認められた.し かし,非

細菌性前立腺炎の早期 に見 られるよ うなマクロ

ファージの活性化を意味す るマク ロファージの

spreadingは 認め られなかった.付 着性白血球の

割合は尿浸透圧と相関 しなかったが,そ の原因と

して尿採取後,可 及的に早 く尿中白血球を培養液

に移した ことが考えられた.過 去に尿中白血球を

用い培養 した研究は比較的少なく,最 小限の遠心

後直ちに培養液に尿中白血球を移すわれわれの簡

便 な方法は,尿 路感染症に出現す る他の細胞,サ

イ トカイン,抗 菌剤 と尿中白血球を培養す るなど

の尿路感染防御機構における細胞の役割の研究に

有用 と思われた.

感染症学雑誌 第67巻 第7号