Chemiluminescence and Phagocytic Responses of Rat Polymorphonuclear Neutrophils to Leptospires

Zbl. Bakt, 272, 36-46 (1989)

Chemiluminescence and Phagocytic Responses of Rat Polymorphonuclear Neutrophils to Leptospires EMIKO ISOGAlt, HIROSHI ISOGAI 2 , HITOMI WAKIZAKA 1, HIROKO MIURA 1, and YOICHI KUREBA YASHI3 Departments of Ipreventive Dentistry and 20 ral Anatomy, School of Dentistry, IshikariTobetsu 061-02, and 3New Product and Research Coordination Research Institute, Daiichi Seiyaku, Co., Ltd., 16-13, Kita-Kasai, Edogawa-Ku, Tokyo 134, Japan

With 5 Figures ' Received May 21, 1989 . Accepted in revised form July 7, 1989

Summary The interaction of leptospires with polymorphonuclear neutrophils (PMN) was examined by the luminol-dependent chemiluminescence (CL) test. Whole blood CL changed in relation to the stage of leptospiral infection both in susceptible (SUS) and resistant (RES) rats . The intensity of CL grew with an increasing number of leptospires in the blood. CL responses were observed in isolated PMN upon exposure to living leptospires. In contrast, the same bacteria, having been inactivated by formalin, did not stimulate PMN. A variation was found in the CL response by different living strains of Leptospira. The CL intensity was arranged as follows: L. illini > L. biflexa > L. interrogans avirulent strains> L. interrogans virulent strains . The CL response was markedly enhanced by an opsonization of leptospires. Specific opsonization was shown to increase the rate of phagocytosis of leptospires with relation to the CL response.

Zusammenfassung Die Wechselwirkung zwischen Leptospiren und polymophkernigen Granulozyten (PMN) wurde mit dem Luminol-Chemolumineszenztest (CL) untersucht. Vollblut-Chemolumineszenz zeigte Unterschiede in Abhangigkeit vom Stadium der Leptospiren-Infektion sowohl bei empfindlichen (sus) als auch bei resistenten (res) Ratten. Mit ansteigender Zahl der Leptospiren im Blut erhohte sich die Cl-Intensitat, An isolierten PMN wurde die CL-Reaktion auf lebende Leptospiren beobachtet. Mit Formalin inaktivierte Bakterien stimulierten dagegen die PMN nicht. Unterschiedliche CLIntensitat erlaubte eine Reihung der lebenden Starnme von Leptospira: L. illini > L. biflexa > L. interrogans avirulente Stamme > L. interrogans virulente Starnme. Die CL-Reaktion wurde durch Opsonisierung der Leptospiren deutlich verstarkt. Spezifische Opsonisierung erhohte die Phagozytoserate parallel zur CL-Reaktion.

PMN and Leptospir~s

37

Introduction Chemiluminescence (CL) is considered to be indicative of the generation of reactive species of oxygen, such as superoxide, hydrogen peroxide, hydroxy radical, and singlet oxygen (18). The generation of reactive oxygen species represents an important mechanism in the killing of bacteria (1, 2, 14, 18). Since CL response is one of the earliest phenomena of phagocytosis upon exposure to stimuli, the CL measurement is considered to be a useful aid for the investigation of early events in phagocytosis. PMN play an important role in defense system against leptospiral infection. Paine et al. demonstrated phagocytosis of leptospires from different animal species ranging from those susceptible to those highly resistant to leptospirosis (4). Specific opsonization increased the rate of phagocytosis by PMN (4, 19). It has been shown that differences in virulence exist between the various strains of Leptospira interrogans (15, 16). The degree of resistance to host defense mechanisms, such as phagocytosis is considered to be a factor which may contribute to the virulence of these organisms. McGrath et al. reported that a significant CL response was observed when guinea-pig PMN were incubated with virulent leptospires in the presence but not in the absence of specific immune serum and that the ability of leptospires to resist phagocytosis by PMN in the absence of immune serum did not appear to be a major determinant of virulence (13). Wang et al. showed that there was no ingestion or killing of non-pathogenic or pathogenic leptospires by human PMN without the presence of specific immune serum, but non-pathogenic leptospires were killed in a pellet of PMN (19). They also suggested that the virulence of leptospires appeared to be related to their ability to resist serum and to resist ingestion and killing by PMN. The aim of this study was to determine the CL of PMN stimulated by various strains of Leptospira and to investigate the influence of the opsonic factor on this CL. In most studies, purified and washed PMN have been used. It has been reported that PMN function in vivo, i.e. in blood or in the microenvironment of inflammatory sites, is greatly modified by the presence of other cells and serum factors (5, 6). In order to study cellular functions under leptospiral infection, an attempt was made to analyze CL in freshly drawn, unfractionated blood . For these studies, two strains of rats, susceptible (SUS) and resistant (RES) ones, were used.

Materials and Methods

Bacterial strains. Pathogenic Leptospira interrogans serovar copenhageni strain Shibaura (virulent: V and avirulent: AV), serovar canicola Moulton (V), serovar canicola Hond Utrecht IV (AV), serovar icterohaemorrhagiae strain CF-l, serovar hebdomadis strain Hebdomadis (AV), serovar perameles strain Bandicoot 343 (AV), non-pathogenic L. biflexa strain Patoc I and Leptospira (Leptonema) illini strain 3055 were used in this study (Table 1). The organisms were grown at 30 °C for 5 to 7 days in EMJH liquid medium (Difco) containing 10% EMJH enrichment (heat inactivated rabbit serum, Difco). The organisms were harvested by centrifugation at 5000 x g for 30 min, washed with phosphate-buffered saline (PBSS, pH 7.4), and resuspended in Hanks balanced salt solution (HBSS, pH 7.4). The organisms were counted in a counting chamber on EMJH solid agar medium with 10% EMJH enrichment and Noble agar (Difco). Inactivation of leptospires. Leptospires were killed by treating them with 1% (v/v) formalin which was removed by washing before their use.

38

E. Isogai et al.

Table 1. Leptospira used for experiments and its virulence Leptospira

% in mice experimentally infected with leptospires" Leptospiremia

Renal infection

100 100

100 100 10 0 0

Lethality in guinea-pigs or hamsters

Pathogenic leptospires

L. interrogans copenhageni Shibaura (V) canicola Moulton (V) copenhageni Shibaura (AV) canicola Hond Utrecht IV (AV) hebdomadis Hebdomadis (AV) perameles Bandiccot 343 (AV) icterohaemorrhagiae CF-1

Non-pathogenic leptospires L. bi(lexa patoc Patoc I

Leptonema illini3055

Ob

0 0 Nrc

NT

0

NT

NT NT

0

NT

+ +

NT

NT

Leptospiremia and renal infection were examined in ddY 10 mice after inravenous inoculation of 2 x 10 7 leptospires. b Complete clearance occured within 6 h. C Not tested . a

Animals. SUS and RES rats (5-7 weeks old, male) were used for the experiments. Both strains were derived from WistariKyoto (8). SUS rats were characterized by a lower potency of PMN functions (9) and lower immunoglobulin synthesis (11) than that of RES rats. Preparation of PMN. PMN (95% <, pure) were obta ined from SUS rats 15 h after stimulation with an intraperitoneal injection of 1 ml of 5% starch and 5% polypeptone, by double-Ficoll separation. The cells were counted in a hemocytometer and adjusted to a concentration of 1 x 10 6 to 1 x 10 7 per ml. Viability was more than 95% as determined by trypan blue exclusion. Wholeblood CL. Whole blood CL was performed according to the method of Kato et al. (12). After leptospiral infection, the heparinized blood was obtained from rats . In the whole blood CL, the reaction mixture consisted of 100 !tl of the blood, and 50 ul luminol solution (1 mM in HEPES-HBSS with 1 mM CaCl z) and 320 !tl of non-opsonized zymosan (final concentration, 2 mg/ml), Chemiluminescence was measured at 3rC in a luminometer (Aroka) . The experiment was started by the addition of luminol. When background counts became constant, zymosan was added, and counting was continued while stirring the mixture. Measurement of CL by PMN. Measurement of CL by PMN was performed according to the method of Allen et al. (1). In the experiment for measurement of CL response to living and killed leptospires, the reaction mixture consisted of 100 !tl of the 1 x 10 6/ml PMN solution and 100 !tl of 0.2 mM luminol solution, plus 100 ul of leptospires. HEPES-HBSS with 50 mM HEPES and 1 mM CaCh was used as buffer. CL was measured at 37°C in a luminometer (Aroca). The experiment was started by the addition of luminol. When the background count became constant, living and killed leptospires were added, and counting was continued. As positive standards, opsonized zymosan (4 mg/ml) and FMLP (3 x 10-4 mM) were used. To compare the CL intensity induced by various strains of Leptospira, the different system described above was used. The reaction mixture consisted of 100 ul of 1 x

PMN and Leptospires

39

106/ml PMN, 10 III of 1 mM luminol and 108/ml living leptospires in HEPES-HBSS. CL was measured at 3rC in a luminometer (Biolumat LB 9500T, Berthold) without stirring. Preopsonization procedure and CL assay. Leptospires (2 x 108) were centrifuged for 20 min at 1500 g. The pellets were resuspended in 0.5 ml pooled rat serum or 0.5 ml immune serum which had been diluted to a subagglutinating concentration, leptospires were again centrifuged and suspended in HEPES-HBSS. The mixture consisted of 100 III of 1 x 106 PMN cells, 10 III of 0.2 mM luminol and 10 III of opsonized leptospires. Phagocytosis in vitro. In the phagocytosis test, the reaction mixture consisted of 0.5 mlof the cell suspension containing 107 cells/ml (final concentration of 5 X 106 cells/ml), and 0.1 mlleptospires at a final concentration of 5 x 106Ieptospires/ml, and the total volume was adjusted to 1 ml by the addition of HBSS or by the addition of fresh rat normal serum (0.1 ml) as source of complement, diluted antiserum (0.1 ml, subagglutinating concentration) and HBSS. The mixtures were incubated at 3rc for 1 h. Samples (0.1 ml) were taken and serially diluted in cold sterile distilled water and plated on the agar plates for counting of surviving leptospires. Clearance test. The clearance test was performed by injection of 0.1 ml (2 X 107 leptospires) into the tail veins of the rats . At intervals, blood samples were taken, diluted and plated on EMJH agar medium. The clearance rate of the organisms per ml was calculated from the number of colonies which grew on agar. Results

CL of whole blood after leptospiral infection. The CL of whole blood after leptospiral infection was observed. Fig. 1 shows the typical CL pattern at 2 days after leptospiral infection. Two peaks (1 and 2) were recognized. At 6 hours after the infection, the CL intensity of peak 1 was the highest (Fig. 2). At 30 h after the infection, the CL intensity decreased in both rats . SUS rats showed an increasing CL intensity at 54 and 126 h after infection while RES rats did not. Table 2 shows the CL intensity of peak 2. The pattern change of peak 2 intensity was similar to that of peak 1. Clearance test. The clearance test was done at the same time as the CL assay. Fig. 3 shows the clearance of copenhageni Shibaura (V) from the blood of SUS and RES rats after intravenous inoculation. The number of leptospires in the blood of SUS rats was higher than in that of RES rats. At 126 h after the infection, leptospires were present in

PEAK 2

9

~ 8 ~ 7

z

~

~

6

5

::;j 4

g;

3 2 1

o

2

4

8

10 12 14 16 18 20 22 MINUTES AFTER STIMULATION

24

Fig. 1. Pattern of whole blood CL. This was obtained at 2 days after inoculation of L. copenhageni Shibaura (V).

40

E. Isogai et al.

o

6

30

5Il

126

Il:X.RS IfTER l£PIOSP(PJt IIffCTIOO

Fig. 2. Change of peak 1 intensity after L. copenhagen! Shibaura (V) inoculation . the blood of SUS rats. In contrast, no leptospires were present in the blood of RES rats. These results were related to the result s of the CL assay, because high CL was seen in the presentee of large number of leptospires in the blood. Difference of CL in various species of Leptospira. Fig. 4 shows the CL of PMN for var ious species of Leptospira. A different response to living leptospires was observed, when L mini, L. biflexa and L. interrogans were used. A high level of CL emission was observed upon exposure of PMN to L. mini and L. biflexa. Only a low level of CL Table 2. Change of peak 2 chemiluminescence intensity of whole blood after leptospiral infection Time after infection (h)

o

6 30 54 126 a

Mean ± SE

Peak 2 intensity of CLiI

x 105 PMN

SUS

RES

1.7 ± 0.5a 14.3±2.3 3.6±0.5

2.2±0.9 13.9± 1.8 1.3 ±0.5 2.9±0.2

7A± 1.0 8.1 ±0.2

2.7±OA

41

PMN and Leptospires 5

3'--

- - - - -............ Xl 5ll IOOlS N'TER lfPTOSPlfl..OJ.. llfECTIOO

Fig. 3. Comparison of clearance of leptospires from the blood between susceptible and resistant rats.

1.5

1. 2

'" 0.9

~

c

~

~

::> 0

u

0.6

canicola UT IV (AV)

.... -

..-l

u

-_

0 .3

o

-

•.._......

__

...,.

-..

copenhagen i

.

0

0

TIME (MIN) AFTER STIMULATION

Fig. 4 . Pattern of C L o f va rio us L eptospira species.

o

42

E.lsogai et aI.

Table 3. Chemiluminescence of polymorphonuclear neutrophil s induced by various leptospires

Leptospira

Peak 2 intensity of PMN

illini 3055

3.07±0.32" 2.07±OA1 1.00±0.24 1.10±0.23 0.58 ±0.35 0.35 ±0.14

patoc Patoc I canicola UT IV hebdomadis Hebdomadis copenhageni Shibaura (V) canicola Mouton (V)

a

X 104

counts, mean ± SD

emission was observed upon exposure of PMN to virulent strains of 1. interrogans serovars copenbageni and canicola. Table 3 presents a summary of the CL of PMN to various leptospiras. The peak intensity decreased in the following order; 1. illini > 1. biflexa> 1. interrogans (AV) > 1. interrogans (V). There were significant differences in CL among the species. Comparison of response of PMN to living and killedleptospires. The CL response of PMN to living and killed leptospires has been shown in Table 4. A different response to living and killed bacteria was observed. There were no peaks of CL upon exposure of killed leptospires. These results suggest that living, motile bacteria induce more CL generation from PMN than no nonmotile ones .

Table 4. Chemiluminescence of polymorphonuclear neutrophils induced by living and killed leptospires CL response of

Stimulation peak 1

Living leptospires copenhageni Shibaura (AV) icterohaemorrhagiae CF-l Killed leptospires copenhageni Shibaura

icterohaemorrhagiae CF-l hebdomadis Hebdomadis perameles Bandicoot 343

Opsonized zymosan" FMLp d a b C

d

X 103 counts,

Not detected

peak 2

Intensity

Time (min)

Intensity

Time (min)

4.2 ± 0.6" 5.2± 1.9

2.8 ±0.5 2.3 ± 1.2

9.2±OA 10.2±4.2

17.5±0.5 18.0±3.1

ND b ND ND ND

56± 7 57± 11

ND ND ND ND

3.2±0.5 3.0±0.5

126±4

ND

mean ± SE

Zymosan (12.5 mg/ml, opsonized with normal rat fresh sera) 3 x 10-7 M

12.5±0.6

PMN and Leptospires

43

hebdomad is (AV) copenhageni (v) canicola (AV) canicola (V)

6 5

4 ~3

M

o

N

0.1'"-_..-

-.-

PMN

_

PMN + COMPLEMENT + ANTISERUM

Fig. 5. Increased CL of PMN after incubation with opsonized Leptospira.

Table 5. Viable percentage after incubation with polymorphonuclear neutrophils Leptospora

Viable % of leptospires after incubation with PMN

illini3055 patoc Patoc I eanicola UT IV (AV) hebdomadis hebdomadis (A V) copenhageni Shibaura eanieola Moulton (V) a b

c

mean ± SO Not tested Not detected

16.7± 4.8" 17.0± 2.2 33.0±15.8 27.7± 5.2 56.3 ± 3.6 61.6± 15.9

PMN

+ complement + antiserum NTb NT

NT

NT NO' NO

44

E.Isogai et al.

The effectof opsonization with complement andantiserum. After opsonization of L. copenbageni Shibaura and the other 3 Leptospira species CL was significantly increased. Fig. 5 shows that the peak counts increased about 4 to 10 times. After incubation with PMN only, viable percentages were 16.7, 17.9,27.7,56.3,61.6 in L. illini, L. patoc, L. canicola Hond Utrecht IV, L. hebdomadis Hebdomadis, L. copenhageni Shibaura (V) and L. canicola Moulton (V), respectively (Table 5). After incubation with PMN, complement and immune serum, no leptospires were detected. Discussion In the experiments described, whole blood CL increased with active leptospiral infection. The intensity of CL was related to an increasing number of leptospires in the blood. In patients with bacterial infections, the measured CL of whole blood has been reported to be elevated background CL and a high peak of CL (3, 12). Thus , an increased stimulatory response ability of PMN could be demonstrated for man and animals with the development of an infection. Similar results were obtained in rats experimentally infected with leptospires. SUS rats were more sensitive in leptospiral infection than RES rats. This phenomenon is considered to contribute to its host's defense mechanisms, because SUS rats were characterized by a lower potency of PMN functions (9) and by a lower immunoglobulin synthesis (11) than that of RES rats. In the early phase of clearance, PMN functions may be related to the elimination of leptospires from the blood. In the later phase of clearance, additional immunoglobulin synthesis may be important for elimination of bacteria from the blood. A different CL response to various strains of living leptospires was observed with L. illini, L. biflexa, and L. interrogans (avirulent and virulent strains). A low level of CL emission was observed upon exposure of PMN to virulent strains. McGrath et al. have reported that no CL response of guinea-pig PMN was observed, when either normal serum or guinea-pig complement was exposed to virulent leptospires. Different results may be due to different assay systems of different laboratories. The following reasons were considered ; 1) Use of stirring (The chance of a contact of leptospires with PMN may increase under stirring condition), 2) Number of leptospires as a stimulant, 3) Motility of leptospires, 4) Number of PMN, 5) Source of PMN, 6) Concentration of luminol and other mixture condition . In the experiment, the CL response of PMN was much greater when the cells had been exposed to the living leptospires than when they had been exposed to the killed leptospires. It has been reported that a similar phenomenon was found in macrophages with Escherichia coli, Pseudomonas aeruginosa, Proteus morganii and Enterobacter aerogenes (17). It has been shown that simple attachment of particles to the phagocytic cell surface was not sufficient to trigger ingestion of the particle, and that ingestion requires the sequential circumferential interaction of particle-bound ligands with membrane receptors different from the initial sites (7). These findings may indicate that there is a requirement of more than 2 sites for phagocytic and CL responses. It may be suggested that living leptospires initially contact one site of the surface of PMN and then get a chance to attach to other receptor sites by moving. An increased CL response was observed when specific immune serum and complement were added. The addition of immune serum and complement also enhanced the killing activity of PMN. Opsonization was important to effectively eliminate leptos-

PMN and Leptospires

45

pires. It has been reported that CL response to leptospires is markedly enhanced by the addition of immune serum and complement (13). Specific antiserum enhanced the activities of not only PMN but also of macrophages. Macrophages have been found to be important as a defense against leptospiral infection (10) . SUS rats have shown lower PMN activities than RES rats (9). However, macrophage functions have been considered to be similar to those of RES rats (11 ). In the clearance of leptospires from the blood of SUS rats, macrophage may playa key role as a defense mechanism.

References

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16. Stahlheim, O. H. V.: Chemical aspects of leptospirosis. CRC Crit. Rev. Microbiol. (1973) 423-456

17. Tomita, T., E. Blumenstock, and S. Kanegasaki: Phagocytic and chemiluminescent responses of mouse peritoneal macrophages to living and killed Salmonella typhimurium and other bacteria. Infect. Immun. 32 (1981) 1242-1248 18. Trush, M. A., M. E. Wilson, and K. Van Dyke: The generation of chemiluminescence by phagocytic cells. Meth. Enzymol. 57 (1978) 462-494

19. Wang, B., J. Sullivan, C. W. Sullivan, and C.L. Mandell: Interaction of leptospires with human polymorphonuclear neutrophils . Infect. Immun. 44 (1984) 459-464

Dr. Emiko Isogai, Dept. of Preventive Dentistry, School of Dentistry, Higashi Nippon Gakuen University, Ishikari-Tobetsu 061-02, Japan