FEMS MicrobiologyImmunology105 (1992) 211-218 © 1992 Federation of European MicrobiologicalSocieties 0920-8534/92/$05.00 Published by Elsevier
Phagocytosis of polymorphonuclear leukocytes by guinea pig peritoneal macrophages: effects of serum and temperature in vitro Chiyuki Y a m a m o t o , Shin-ichi Y o s h i d a and Y a s u o M i z u g u c h i Department of Microbiology, School of Medicine, Unicersity of Occupational and Ent:ironmental Health, Kitakyushu, Japan
Received 3 February 1992 Revision received 25 June 1992 Accepted 26 June 1992 Key words: Polymorphonuclear neutrophil (PMN); Macrophage; Autophagocytosis; PMN-phagocytosing macrophage; Hyperthermia; Pyknosis
1. S U M M A R Y The regulation of phagocytosis of neutrophils by peritoneal macrophages was studied in Vitro. Peritoneal exudate cells (PECs) of guinea pigs were lavaged 15 h after the i.p. injection of thioglycollate medium and were cultured in chamberslides. When PECs were cultured in RPM! 1640 medium in the absence of serum, approximately 20% of the macrophages phagocytized autologous neutrophils during 48-72 h of culture. Addition of guinea pig serum to the culture (2.5-20% v / v ) suppressed the extent of the phagocytosis. The suppression was induced by globulin-rich ammonium sulfate fractions of the serum. Sera from rat, mouse, hamster, horse or calf aIso suppressed the phagocytosis, but fetal bovine serum (FBS) supported the phagocytosis, which was inhibited
Correspondence to: S. Yoshida, Department of Microbiology,
School of Medicine, Universityof Occupational and Environmental Health, Kitakyushu, 807 Japan.
by globulin-rich Cohn fractions of bovine serum. The rate of neutrophil-phagocytosing macrophages was proportional to the rate of the pyknotic change of neutrophils. At a high temperature (42°C), the autophagocytosis took place at 12 h of culture when fresh, but not heat-inactivated, autologous serum was added, implying that complement components may play a role in the hyperthermia-induced phagocytosis of neutrophils by macrophages. At 42°C, ingested neutrophils did not show the pyknotic changes, indicating that intact neutrophils were ingested by macrophages.
2. I N T R O D U C T I O N The life span of polymorphonuclear neutrophils (PMNs) is thought to be very short, especially after they have migrated into the inflammatory sites. To limit the expansion and to overcome the inflammation, it is necessary to scavenge PMNs before their autolysis takes place. It
212 has been known that mature inflammatory macrophages phagocytize aged autologous PMNs [1,2]. We reported previously that, when guinea pigs were injected i.p. with irritants or microorganisms, P M N - p h a g o c y t o s i n g m a c r o p h a g e s (PPMs) appeared in the peritoneal exudate cells (PECs) . Since our data suggested that almost all of the infiltrating neutrophils were phagocytosed by the macrophages, we discussed in the report that macrophages infiltrating into inflammatory lesions play an important role in scavenging aged neutrophils. Although the phagocytosis of neutrophils by macrophages is considered to be very important in the regulation of inflammation , the mechanism of the phagocytosis is not yet fully understood. To analyze this phenomenon more precisely, we have developed an in vitro experimental system in which PMN-phagocytosing macrophages can be clearly demonstrated. In the inflammatory lesion, exudation of serum components and elevation of temperature take place. These factors could affect the removal of infiltrating neutrophils by macrophages from the lesion. We examined the effects of these factors on the regulation of the appearance of PMN-phagocytosing macrophages in relation to the pyknotic change of nuclei of PMNs.
3. M A T E R I A L S AND M E T H O D S 3.1. Animals Male and female guinea pigs of outbred Hartley strain weighing 450-550 g were purchased from Seiwa Experimental Animal Co. (Oita, Japan) and used in this study. 3.2. Preparation of peritoneal exudate cells Guinea pigs were injected i.p. with 10 ml of 3% thioglycollate medium (Eiken Chemical Co., Tokyo). 15 h after the injection, the guinea pigs were bled by cardiac puncture and peritoneal exudate cells (PECs) were collected with RPMI 1640 medium (Nissui Pharmaceutical Co., Tokyo) containing 10 U / m l of heparin. The number of viable cells was determined by the Trypan blue
dye exclusion method in a hemocytometer. Differential cell counts were done on Wright-Giemsa stained smears of PECs. After washing twice with RPMI 1640 medium by centrifugation, PECs were suspended in the same medium supplemented with 20 mM of H E P E S (Nacalai Tesque, Kyoto), 100 U / m l of penicillin G and 100 g g / m l of streptomycin, and adjusted to a concentration of 1 × 107 cells/ml. 3.3. In t,itro assay ]or PMN-phagocytosing macrophages The PEC suspension was mixed with an equal amount of serum-free RPMI 1640 medium or RPMI 1640 medium containing various amounts of serum samples. Each 0.25 ml of the mixture was transferred to 8-well chamber slides (Lab-Tek 4808, Nunc Inc., Naperville, IL), and incubated in a CO2-incubator for up to 3 days. The culture was usually carried out at 37°C but in some experiments it was done at 42°C. At intervals during incubation, the culture media were discarded and wells were washed gently twice with Dulbecco's phosphate buffered saline (PBS). After drying, the slides were stained with WrightGiemsa's solution (Muto Pure Chemicals Ltd., Tokyo). The stained slides were observed under a light microscope and the percentage (%) of PMN-phagocytosing macrophages among 200 randomly observed macrophages was determined. The neutrophils in which nuclei shrink in size and the chromatin condense to solid, structureless masses (that is pyknotic change) were also examined simultaneously. The rate (%) of neutrophils showing the pyknotic change was calculated after examining 200 neutrophils. 3.4. Sera and serum components The blood samples were collected by cardiac puncture from Hartley guinea pigs, ddY mice, Wister rats and Golden hamsters and they were kept at room temperature for clotting. The sera were immediately used or stored at - 2 0 ° C until use. Horse serum (lot 94989) and calf serum (lot 120677) were purchased from Microbiological Associates (Walkersville, MD) and Chiba Serum Institute (Ichikawa, Chiba), respectively. Fetal bovine serum (FBS, lot 32K7145) was obtained
213 from G1BCO Laboratories (Grand Island, NY). Guinea pig albumin (lot 75F-9405), and Cohn fractions of bovine serum, that is, Fraction II (lot 56F-9320, predominantly y-globulins), Fraction III (lot 95F-9300, predominantly y- and /3-globulins), Fractions II, III (lot 85F-9375, primarily/3-, and y-globulins), Fraction I V - 1 (lot 76F-9306, predominantly a-globulins) were from Sigma (St. Louis, MO).
3.5. Ammonium sulfate fractionation and other treatment of sera Pooled guinea pig serum (50 ml) was subjected to a m m o n i u m sulfate fractionation. Precipitation of proteins was accomplished by stepwise addition of (NH4)2SO 4 crystals (Nacalai Tesque, Kyoto) to achieve 33, 50 and 80% saturation at 4°C. The precipitates were collected by centrifugation at 15 000 × g for 20 min, and a m m o n i u m sulfate in precipitates was removed by extensive dialysis in Dulbecco's PBS. After the dialysis, the final volume of each fraction was adjusted to 50 ml. The amounts of proteins were measured by Lowry's method and it was found that each fraction contained 0.8 g ( 0 - 3 3 % saturation), 0.9 g ( 3 3 - 5 0 % saturation) and 2.5 g ( 5 0 - 8 0 % saturation) of proteins. Electrophoretic analysis showed that the major components of these fractions were y-globulin ( 0 - 3 3 % saturation), oq-globulin ( 3 3 - 5 0 % saturation) and albumin ( 5 0 - 8 0 % saturation). Heat inactivation was carried out by incubating the serum at 56°C for 30 min. Acidification at p H 2.0 was carried out by adding 37% HC1 to the serum, set at room t e m p e r a t u r e for 30 min, and then neutralized to p H 7 with N a O H .
0.8% (mean + standard deviation of seven experiments). At this stage, polymorphonuclear neutrophil ( P M N ) - p h a g o c y t o s i n g m a c r o p h a g e s (PPMs) were detected in only 1 - 2 % of the infiltrating macrophages. When the PECs were cultured in the absence of serum, the rate of PMNphagocytosing macrophages to total macrophages reached nearly 20% after 2 to 3 days of cultivation (Fig. 1). Microphotographs of PPMs at day 2 of the culture are shown in Fig. 2 (a and b). Most of the ingested neutrophils showed pyknotic change in nuclei and sometimes only neutrophil granules were observed inside the macrophages. When more than 2.5% of autologous guinea pig serum was added to the culture medium, the
4. R E S U L T S
4.1. Effects of serum on the proportion of neutrophil-phagocytosing macrophages in relation to pyknotic changes of neutrophil nuclei Usually, 4 - 6 × 10 s PECs were lavaged from a guinea pig which was injected with 10 ml of 3% thioglycollate medium 15 h previously. The percentages of differential cell types were as follows: neutrophils, 74.6 + 4.1%; macrophages, 23.2 + 3.7%; lymphocytes, 0.6 + 0.6%; eosinophils, 1.6 +_
DOyS of In v l t r o culture
Fig. 1. Effects of autologons serum concentrations on the proportion of PPMs, Guinea pigs were injected i.p. with 10 ml of 3% thioglycollate medium and PECs were lavaged 15 h after the injection. Cells were adjusted to 5 × 106 cells/ml in RPMI 1640 medium and cultured on chamber-slides. Autologous guinea pig serum was added at the concentrations of 0 (©), 1 (zx), 2.5 (el, 5 (11), 10 (A), and 20 (O) % (v/v) in RPMI 1640 medium. After 1, 2 and 3 days, slides were stained with Wright-Giemsa and PPMs were examined under a light microscope. 200 macrophages were counted and the ratio of PPMs were plotted. Data represent the mean % of PPMs_+ S.D. from four independent experiments.
Fig. 2. Light micrograpbs of neutrophil-ingesting macrophages in vitro. (a) and (b): in serum-free cultt~re at 4g h; (e-f): itx the presence of fetal bovine serum at 48 h; (g) and (h): at 42°C in the presence of fresh guinea pig serum at 12 h. Scale bar shows 10 /xm. Wright-Giemsa staining.
215 e x t e n t o f t h e p h a g o c y t o s i s was s u p p r e s s e d (Fig. 1). T h e s u p p r e s s i v e activity o f t h e s e r u m was n o t a b r o g a t e d by i n c u b a t i n g at 56°C for 30 min o r by the t r e a t m e n t at p H 2.0 ( d a t a not shown). A m m o n i u m sulfate fractions of 0 - 3 3 a n d 3 3 50% s a t u r a t i o n s h o w e d s u p p r e s s i v e effects similar to t h o s e o f w h o l e g u i n e a pig s e r u m ( T a b l e 1). H o w e v e r , a f r a c t i o n of 5 0 - 8 0 % s a t u r a t i o n (alb u m i n - r i c h f r a c t i o n ) s h o w e d no s u p p r e s s i v e effect on t h e phagocytosis. Next, s e r a f r o m a l l o g e n e i c g u i n e a pigs a n d o t h e r a n i m a l species w e r e a s s a y e d to d e t e r m i n e w h e t h e r they have s u p p r e s s i v e effects on t h e p r o p o r t i o n o f P P M s . T h e sera, e x c e p t for f e t a l b o v i n e s e r u m (FBS), s u p p r e s s e d t h e level of p h a g o c y t o s i s ( T a b l e 1), w h e r e a s F B S s o m e w h a t a u g m e n t e d t h e phagocytosis. M i c r o p h o t o g r a p h s o f P P M s at day 2 in t h e p r e s e n c e o f F B S a r e shown in Fig. 2 c - f . M o s t o f t h e m a c r o p h a g e s i n g e s t e d m o r e t h a n two
10 "6 2o
Days of in v i t r o culture
Fig. 3. Comparison of the kinetics of neutrophil pyknosis and
PPMs in reference to the effects of serum. (A): rate (%) of pyknotic change of neutrophils; (B): rate (%) of PPMs. No serum ( • ), FBS (o), calf serum ( a ) and guinea pig serum (e) were added at a concentration of 10%. Each serum was incubated at 56°C for 30 min before use. Data represent the mean +_S.D. from five independent experiments.
Table 1 Effects of allogeneic, and xenogeneic sera and ammonium sulfate fractions on the proportion of PMN-phagocytosing macrophages in vitro
Source of serum *
Rate (%) of PPMs on day 2 +
No serum Autologous serum Allogeneic serum Ammonium sulfate fraction 0-33% saturation 33-50% saturation 50-80% saturation Xenogeneic sera Rat serum Mouse serum
30.6_+ 13.0 5.8+ 1.9 7.1_+ 4.8
Hamster serum Horse serum Calf serum Fetal bovine serum (FBS) FBS + Cohn Fraction I1 FBS+Cohn Fraction III FBS+Cohn Fraction II, III FBS+Cohn Fraction IV-1 Guinea pig serum albumin (5 mg/ml)
2.8_+ 5.5_+ 5.0_+ 44.3_+ 16.5 + 9.0+ 7.7+ 4.3_+ 24.5 +
2.7_+ 1.6 2.9+ 1.7 23.5_+ 6.7 1.0+ 0.7 1.9_+ 1.1
0.0 4.4 1.4 4.2 6.3 4.6 3.6
* All sera were used at a concentration of 10% after heat-inactivation at 56°C for 30 min. + Ratio of PPMs on day 0 was 1.1+0.6%. Data represent the m e a n + S.D. from four individual experiments.
n e u t r o p h i l s . T h e high level o f p h a g o c y t o s i s in t h e p r e s e n c e o f F B S was i n h i b i t e d w h e n g l o b u l i n - r i c h C o h n f r a c t i o n s o f b o v i n e s e r u m (2 m g / m l ) w e r e a d d e d to the c u l t u r e ( T a b l e 1).
4.2. Relation between pyknotic change of neutrophils and the proportion PMN-phagocytosing macrophages It is a s s u m e d t h a t e l i c i t e d m a c r o p h a g e s p h a g o cytize only s e n e s c e n t o r d e g e n e r a t i n g n e u t r o p h i l s . T o test this possibility, effects o f s e r u m s a m p l e s on the p y k n o t i c c h a n g e o f n e u t r o p h i l nuclei w e r e e x a m i n e d (Fig. 3 A ) a n d c o m p a r e d to t h e e x t e n t o f p h a g o c y t o s i s (Fig. 3B). In s e r u m - f r e e R P M I 1640 m e d i u m (control), n e a r l y 80% o f t h e n e u t r o p h i l s s h o w e d pyknosis a f t e r 2 days o f cultivation. T h e r a t e o f m a c r o p h a g e s p h a g o c y t i z i n g P M N in t h e s e r u m - f r e e m e d i a was a p p r o x i m a t e l y 24% on d a y 2. A d d i t i o n o f g u i n e a pig or calf s e r u m to t h e c u l t u r e (10%) s u p p r e s s e d b o t h p y k n o t i c c h a n g e s a n d t h e level o f P M N - p h a g o c y t o s i n g m a c r o p h a g e s . W h e n fetal b o v i n e s e r u m was a d d e d , the p r o p o r t i o n o f n e u t r o p h i l s with pyknotic c h a n g e was 35% at day 2 a n d the p r o p o r -
216 Table 2 Effects of hyperthermia on the proportion of pyknosis of neutrophils and PMN-phagocytosing macrophages (PPMs) Supplement
Appearance (%) of
18.0+ 1.2 18.7+2.2
Fresh guinea pig serum (10%)
2.8 _+0.3 3.2_+0.6
1.4 + 0.2 13.0+0.9
Heat-inactivated guinea pig serum (10%)
4.9 _+0.9 6.7+0.6
0.5 + 0.2 2.4_+0.3
Data represent the mean +_S.D. from 15 independent experiments.
tion of PMN-phagocytosing macrophages was more than 30%.
4.3. Effects of hyperthermia on PMN-phagocytosing macrophages When the culture temperature was elevated to 42°C in the presence of 10% fresh autologous serum, the proportion of PPMs after 12 h of culture reached 13.0% in comparison to 1.4% at 37°C (Table 2). When the serum was absent or it was previously heat-inactivated, the extent of PMN-phagocytosing macrophages stayed at the low level of 2.3 or 2.4% respectively, at 42°C. Pyknosis was only 3.2 + 0.6% at 12 h of culture in the presence of guinea pig serum where ingested neutrophils looked intact (Fig. 2 g and h).
5. DISCUSSION We observed that the appearance of PMNphagocytosing macrophages (PPMs) was suppressed by adding autologous guinea pig serum into the in vitro assay system (Fig. 1). Furthermore, globulin-rich fractions of ammonium sulfate precipitate of guinea pig serum suppressed the appearance of PPMs (Table 1). A high level of phagocytosis in FBS-added culture was also suppressed by adding globulin-rich Cohn fractions of bovine serum (Table 1). Although the mechanism of suppression by globulin is not obvious, one
possible mechanism is the prevention of the ageing of guinea pig neutrophils. Kay [4,5] reported that lgG autoantibody is required for phagocytosis of aged autologous erythrocytes by human macrophages. Our results imply that IgG autoantibody, even globulins, may not mediate the phagocytosis of neutrophils by macrophages. Among sera, only FBS supported the phagocytosis of PMN by macrophages. In the presence of FBS, neutrophil pyknosis was observed at a relatively low rate (35%) at 48 h of culture, but about 31% of the macrophages phagocytosed autologous neutrophils (Fig. 3A and B). FBS is known to have a different composition from adult bovine serum including a lack of y-globulin fraction (we also confirmed this by electrophoresis) which suppressed the phagocytosis of neutrophils by macrophages in our experiments. The phagocytosis was suppressed by adding the Cohn fractions of bovine serum which contained globulins. Furthermore, an accelerating factor may be present in FBS, because macrophages usually phagocytized more than two neutrophils (Fig. 2 c-f). It has been reported that FBS was required for optimal phagocytosis of old red blood cells by mouse macrophages . When the culture temperature was elevated to 42°C, the autophagocytosis of the neutrophils by macrophages was accelerated. When the serum was absent or it was previously heat-inactivated at 56°C for 30 min, the phagocytosis did not accelerate (Table 2). This suggests that the recognition process of neutrophils by macrophages at 42°C may be mediated by complements. Although we have no information on whether the hyperthermic condition activates the alternative complement pathway, it may have occurred in the autophagocytosis in our system. On the contrary, Peterson et al.  reported that opsonization of streptococcus by the complement system was depressed at 41°C. From the proportion of PPMs to neutrophil pyknosis, at least two mechanisms can be considered for the autophagocytosis of neutrophils by macrophages. Firstly, a senescence-dependent mechanism was observed in the culture without any serum added. In that condition, pyknosis of the neutrophils occurred in 80% at 48 h of cul-
ture (Fig. 3) and macrophages phagocytosed mainly pyknotic neutrophils (Fig. 2 a and b). Some changes of neutrophil membrane, such as diminution in sialic acid contents , followed by decreased negative charge  may be responsible for this phagocytosis. Secondly, a senescence-independent mechanism was observed when PECs were cultured at 42°C in the presence of fresh autologous serum. In this case, the rate of the pyknosis of neutrophils was only 3.2% at 12 h, but macrophages phagocytosed neutrophils at a rate of 13.0%. The neutrophils ingested under this condition appeared non-pyknotic (Fig. 2 g and h). The phagocytosis at 42°C may be mediated by the complement, because the rate of PPMs decreased after the serum was heat-inactivated (Table 2). It is important to elucidate whether macrophages ingest only senescent or dead neutrophils, though it is very difficult to judge definitely whether the ingested neutrophils are alive or dead. Newman et al.  mentioned that the death of the neutrophils did not appear to be required for the phagocytosis. Brewer  reported that phagocytosed neutrophils preserved their morphology fairly well in macrophages. Dresch et al.  showed the phagocytosis of neutrophils by macrophages in human bone marrow and presented micrographs indicating that ingested neutrophils were nearly intact within a macrophage. We also previously observed that when oyster glycogen was reinjected i.p. 4 days after the first i.p. injection in guinea pigs, peritoneal macrophages could phagocytose fresh neutrophils immediately after the neutrophils infiltrated into the peritoneal cavity . In our in vitro study, we observed that neutrophils were nearly intact within the macrophages (Fig. 2 g and h). The above observations imply that macrophages ingest not only senescent but intact neutrophils. To examine whether the cells are alive or dead, the Trypan blue dye exclusion method is commonly used. In our system, the method was not suitable for that purpose because inflammatory nentrophils adhered to the glass. Instead, we used pyknosis of nuclei as the pathological sign of degeneration of the cells.
Newman et al.  cultured human neutrophils in the presence of serum to obtain 'aged' neutrophils. Although the details of the change of 'aged' neutrophils is not described, they showed that the serum accelerated the phagocytosis of neutrophils by human peripheral blood monocytes. In our system, however, not only autologous but also allogeneic and xenogeneic sera, except for FBS, suppressed the appearance of PPMs. The reason for this discrepancy is not known, but it may be due to the difference in the source and the method of collecting neutrophils and monocytes, and the time course observed. Whether PPMs have any role in the protection against microorganisms is an interesting subject. Recently Silva et al.  reported the neutrophils ingesting Mycobacterium aurum are present inside macrophages during the mycobacterial infection in the peritoneal cavity of the mouse. Lactoferrin, which is a neutrophil marker, was also present inside the macrophages. Heifets et al.  reported that mouse peritoneal macrophages ingesting human neutrophils use the neutrophil myeloperoxidase for iodination. It is estimated that guinea pig macrophages also utilize neutrophil myeloperoxidase or lactoferrin [11,13]. Clearance of neutrophils from an inflammatory site seems to be essential for the healing of tissue injury [14,15]. In this sense, PPMs play an important role in the repair or resolution of the inflammatory response. The mode of phagocytosis of neutrophils by macrophages observed in vitro could work in the inflammatory lesion. The in vitro experimental system presented here would be a meaningful model for the analysis of the phenomenon.
ACKNOWLEDGEMENTS The authors are Nishimura and Dr. helpful discussions. Midori Ogawa and acknowledged.
grateful to Dr. Yoshihisa Hideyuki Hirano for their The technical assistance of Kumiko Mizue is gratefully
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