Defective chemotactic response of human alveolar and colostral macrophages

hnmunology Letters, 12 (1986) 271-276 Elsevier lmlet 740

DEFECTIVE

CHEMOTACTIC RESPONSE OF HUMAN COLOSTRAL MACROPHAGES

ALVEOLAR

AND

J. CLEMENTE, N. CLERICI, M. A. ESPINOSA and E LEYVA-COBIAN* Department of Immunology, Centro "'Rarn6n y Cajal", 28034 Madrid, Spain (Received 30 September 1985) (Modified versions received 18 November 1985 and 17 January 1986) (Accepted 18 January 1986)

1. Summary

2. Introduction

This report describes the chemotactic response of human alveolar macrophages (AM~) and milk macrophages (MM~) to a panel of chemotactic agents: endotoxin (EAS) and zymosan (ZAS) activated serum, lymphocyte derived chemotactic factor (LDCF) and formylated synthetic peptides. The locomotion studies were compared with the responses of peripheral blood monocytes. Both AM4~ and MMq~ exhibited an extremely poor chemotactic response to all agents in comparison with the monocytic response. When monocytes were cultured for long periods, a defective response was likewise demonstrated. The chemotactic response was significantly higher in AM4~ from smokers. The stimulated locomotion was not increased by the addition of a surfactant lipoprotein to the AM4~ suspension. Moreover, monocytes incubated with fat- and cell-free human milk exhibited lower chemotactic responses than normal monocytes and practically in the same range as that obtained with MM~.

Several human M¢ functions have been poorly studied due, in part, to the difficulties in obtaining pure populations [1]. In the last few years, the introduction of bronchofiberscopy has facilitated the collection of large numbers of AM¢ (reviewed in [2]). Human breast milk provides an easily obtainable M¢ population (MM~) although, surprisingly, it has received little attention. Thus, for instance, recent reviews or books dealing with M~ biology do not mention MM¢ [3-5]. This could be one of the reasons why the M~ chemotactic response has rarely been studied and is far from being completely understood, as is neutrophil locomotion. Moreover, although about 80% of the cells obtained from colostrum and early human are M~ [6, 7], there are no reports on the chemotactic response of MMO. Considering that M~b represent the larger cell population from both lung washing fluid and breast milk, the chemotactic response is likely to be an early and important event in the response against infection. Because it has not been established how chemotactic responsiveness is related to M¢ heterogeneity, we have studied the chemotactic responses of both AM~ and MM~ populations and compared them with the response of monocytes to chemotactic stimuli.

Key words." chemotaxis - human colostral macrophages milk - human alveolar macrophages - lung

*Correspondence address." Department of Pathology Washington University School of Medicine, Box 8118, 660 South Euclid Avenue, St. Louis, MO 63110, U.S.A.

0165-2478 /' 86 / $ 3.30 ~c~ 1986 Elsevier Science Publishers B.V. (Biomedical Division)

271

3. Materials and Methods 3.1. Preparation and characterization of cells AMq~ were o b t a i n e d f r o m n o r m a l c o n t r o l s by b r o n c h o a l v e o l a r washing a c c o r d i n g to a m e t h o d previously d e s c r i b e d [8]. S a m p l e s were o b t a i n e d f r o m five s m o k e r s a n d eight n o n s m o k e r s . C o l o s t r u m s a m p l e s were o b t a i n e d f r o m a selected g r o u p o f 10 n o n s m o k i n g w o m e n between the first a n d fifth d a y p o s t p a r t u m . M M ~ were o b t a i n e d a n d h a n d l e d as d e s c r i b e d elsewhere [7]. In a d d i t i o n , p e r i p h e r a l b l o o d f r o m 10 s m o k e r a n d from 10 n o n s m o k e r h e a l t h y volunteers was d r a w n in sterile Vacutainer tubes c o n t a i n i n g h e p a r i n as d e s c r i b e d [101. 3.2. Preparation of cell suspension Cell s u s p e n s i o n s were p r e p a r e d as previously d e s c r i b e d [7, 8]. Briefly, b r o n c h o a l v e o l a r cells were w a s h e d in H a n k s ' b a l a n c e d salt s o l u t i o n ( H B S S ) a n d total a n d differential cell c o u n t s were p e r f o r m e d . B r o n c h o a l v e o l a r cells were adj u s t e d to 1 x 10 6 A M O / m l in Gey's m e d i u m . A f t e r r e m o v i n g fat f r o m m i l k samples, m i l k cells were washed in H B S S a n d layered o n t o FicollH y p a q u e (FH). T h e interface was removed a n d the cells washed a n d r e s u s p e n d e d in R P M I 1640

at 1 x 10 6 M M 4 , / m l . M o n o n u c l e a r p e r i p h e r a l cells were s e p a r a t e d by F H g r a d i e n t c e n t r i f u g a t i o n . A f t e r washing twice, m o n o n u c l e a r p e r i p h e r a l cells were a d j u s t e d to 1 x 10 6 m o n o c y t e s / m l . In all cases viability was d e t e r m i n e d by t r y p a n blue exclusion. Differential c o u n t s for b r o n c h o a l v e o l a r cells, milk cells a n d m o n o n u c l e a r p e r i p h e r a l cells were d o n e using a non-specific esterase stain (NSE) [9]. O n l y b r o n c h o a l v e o l a r a n d milk samples with m o r e t h a n 80°7o o f Mq5 were selected for o u r e x p e r i m e n t s (Table 1). 3.3. Chemotaxis C h e m o t a x i s was p e r f o r m e d as previously d e s c r i b e d [10] a n d a d a p t e d to A M O a n d M M O with slight m o d i f i c a t i o n s . Briefly, the u p p e r c o m p a r t m e n t s o f blind well c h a m b e r s ( B i o - R a d L a b o r a t o r i e s , R i c h m o n d , C A , U.S.A.) were filled with 200 #1 o f a cell s u s p e n s i o n at 1 x 10 6 cells/ml. Lower c o m p a r t m e n t s c o n t a i n e d the c h e m o t a c t i c factor. Both c o m p a r t m e n t s were s e p a r a t e d by p o l y c a r b o n a t e m e m b r a n e discs (5 a n d 8 tzm pore size for m o n o c y t e s a n d M~,, respectively) a n d i n c u b a t i o n p e r f o r m e d for 90 min (monocytes) [I0] or 3 h ( M ~ ) at 37 °C in a h u m i d a t m o s p h e r e with (monocytes, M M ~ ) or w i t h o u t (AM¢~) 5o70 CO2 [13]. T h e c h e m o t a c t i c factors used were: n o r m a l hu-

Table 1 Cellular composition of the samples Samples

Cell populations M¢/Mo a

Bronchoalveolar cells Smokers (5)~ Nonsmokers (8)

88-+6 85_*8

Colostrum cellso Nonsmokers (10)

86 -+5

Mononuclear peripheral cells Smokers (10) Nonsmokers (10)

16+5 13 +_2

Lymphocytes b 5-+4 12_+7 < 10 80_+4 78 -+ 1

PMN b

Others b

4_* 1 - 1

- 1 - 1

<5

- 2

<2 <2

<2 <2

Determined by a non-specific esterase stain. b Established by a Wright's stain. Number of samples in parentheses. a Percentages from these samples are calculated after Ficoll- Hypaque separation. a

272

man serum activated with Salmonella typhosa endotoxin (EAS) or zymosan (ZAS), the chemotactic peptides N-formyl-methionylphenylalanine (NfMP), N-formyl-methionylleucyl-phenylalanine (NfMLP) and a lymphocyte derived chemotactic factor (LDCF). All were prepared as previously described [10, 11] and used in a wide range of concentrations. Surfactant lipoprotein (SL) was obtained from cell-free supernatant fluid of pooled bronchoalveolar washings by centrifugation at 1000xg for 60 rain. The SL pelleted fraction was concentrated 100x and stored at - 8 0 ° C until use. In some experiments, SL was added to the upper compartment containing AMq~. In other studies, monocytes were incubated with different dilutions of fat- and cell-free colostral samples, the monocytes having been washed twice and assayed for chemotaxis as described above. Results are expressed as the number of cells migrated to the lower surface of the filter per high power immersion oil field (HPF). 3.4. Statistical analysis The standard error of the mean (SEM) was used as an estimation of variance and the onetailed Student's t-test was used to compare means. 4. Results

The cellular composition of our samples is shown in Table 1. Increased numbers of total recovered bronchoalveolar cells and neutrophils and decreased lymphocyte count were observed in smokers compared with nonsmokers. However, routinely > 8 0 % of cells in both smokers and nonsmokers were AMq5 as established by a NSE stain. The macrophage percentage in our 10 highly selected women was 86_+5% after FH separation. These samples contained less than 10°70 lymphocytes and less than 5°70 PMN. The percentage of monocytes in both smokers and nonsmokers was around 15°70 after FH centrifugation. AMq~ chemotactic response was studied under variable conditions, i.e., different cell concentra-

I

1:8

L4e

N|MP

10.5 M

6

NIMLP

10 .8 M

LDCF

1:4

ZAS

I

p < 0.01

I

I

p < 0.05

I

I

p<0005

I14 17 .-..i , I O

}

,4 p -c 0.01

I 4

i 12

L 16

i 20

HAMI~ / 10 hpf

Fig. 1. Chemotactic response of human alveolar macrophages. The striped (smokers) and white (nonsmokers) bars indicate the mean and lines indicate the SEM. The number of experiments is indicated in the bars. Results are expressed as the number of migrated cells per 10 hpf. tions and incubation time periods, and in the presence or absence of C O 2. AS no significant differences were found with the various protocols, the procedures employed were those described in Materials and Methods. As shown in Fig. 1, the number of cells migrating through the polycarbonate filters was extremely poor in all cases. Chemotaxis of AM4) from smokers and nonsmokers was compared. When ZAS or LDCF were used as chemotactic agents, significant differences were observed between smokers and nonsmokers (p<0.01). Chemotactic activity generated by formylated peptides was also significantly lower in nonsmokers than in smokers ( p < 0.05 for NfMP and p < 0.005 for NfMLP). Chemotactic response of MM4) to different concentrations of chemotactic agents is shown in Fig. 2A. As with AMqh the number of migrated cells was lower than for monocyte migration (Fig. 2B). This response was low at all the concentrations of the chemotactic factors tested. At a given concentration of any one chemotactic factor, migration of MM4) was significantly higher than that of AMq~. For instance, 45.7+_ 5.6 migrated MM~ per 10 hpf (Fig. 2A) vs 9.7+5.2 migrated AMq~ per 10 hpf (Fig. 1) with 12.5% EAS as chemoattractant. Milk M~ from smoking women was not available and thus the possible influence of smoking 273

I

I

ZASi dilution

1:4 1:9

EAS, dilution

1:2 1:4 1:8

NIMLP, molar

10 .6 10 .7 10-9

LDCF, dilution

1:2 I :4

~{.

B

I

1."2 . _..k-~ - 7 ]

~'

6

[ 5 ~iiiiiiiiiii i iiiiii iiTii_--~

L

t

I

O

20

40 HMMO/10

66

[12

..

L--. 0

1

[

1

20

40

60

hpf

. ] ]]].71

I

PlVlo / hpf

Fig. 2. Chemotactic response of human milk macrophages and peripheral blood monocytes. Chemotactic response ot" MN'IO(AI and monocytes (B) to different concentrations of the indicated chemotactic agents is shown. Results are expressed as the number of migrated cells per 10 hpf for MMO or per hfp for monocytes. See also footnote to Fig. 1. on MM4~ c h e m o t a x i s c o u l d not be evaluated. C h e m o t a c t i c responses o f m o n o c y t e s f r o m smokers an d n o n s m o k e r s were c o m p a r e d ; no significant differences were observed a n d the results were p o o l ed . T h e results described above for AM4~ a n d M M 0 were relatively low when c o m p a r e d with monocytes. Using the same c h a m b e r assay but with a smaller pore filter (5/~m) in place o f the 8-/,m pore filters a n d a s h o r te r i n c u b a t i o n time (90 min instead o f 3 h for MO), a higher c h e m o t a c t i c response was observed for m o n o cytes with o p t i m a l c o n c e n t r a t i o n s o f the c h e m o t a c t i c agents (Fig. 2B). In physiological c o n d i t i o n s milk cells are in a

m e d i u m rich in protein and lipids. In order to ascertain w h et h er milk contains s o m e factors able to i m p a i r M M 0 m i g r a t i o n , n o r m a l m o n o cytes were incubated with a cell- and fat-free milk supernatant. Table 2 shows that preincubation o f m o n o c y t e s with this material caused a striking fall in their l o c o m o t i v e behavior to E A S an d L D C E T h e c h e m o t a c t i c response o f A M O was not increased when a d d i n g SL to the upper c o m p a r t ment o f the c h e m o t a c t i c chamber, nor when A M 0 were p r e i n c u b a t e d with SL, washed and then tested for the c h e m o t a c t i c assay (not shown).

Table 2 Chemotactic response of peripheral blood monocytes incubated ~ith human milk Cells

Monocytes incubated with: milk supernatant b RPMI 1640 medium Milk macrophages

Chemotactic responsea to EAS

LDCF

10.3+_ 5.8 (3) 44.6_+ 11.2 (14)

15.1_+ 6.3 (3) 59.3_+ 11.8 (18)

4.5+_ 0.5 (6)

2.1 _+ 0.8 (4)

Results are expressed as the mean ,]umber of migrated cells per hpf. Nuntber of experiments in parentheses. b One million monocytes were incubated with 1 ml cell-free milk supernatant for 60 mitt, then washed twice and assayed for chemotaxis. 274

5. Discussion There is little information available on chemotactic response in human MO. Human peritoneal M~b (PMO) and AMO chemotaxis has rarely been studied and with contradictory results [12-15]. Moreover, to our knowledge MMO chemotaxis has not been reported. A defective chemotactic response by AM0 has been reported [12, 13] although this impaired response was not mentioned by others [15]. The chemotactic behavior of PMO has been studied too and shown to be significantly reduced in comparison with monocytic chemotaxis [14]. A priori this impaired chemotactic response can be interpreted in several ways. H u m a n MO are terminal mature cells which may lose their chemotactic receptors. In this paper a defective chemotactic response of PMO cultured for long periods has been demonstrated, as previously described in mice [16]. It is worth mentioning that a defective response by lung MO occurs in other mammalian species, such as the guinea pig [17, 18], hamster [19], rabbit [20] and monkey [21]. It is also well known that the local environment of AMO plays an important role in altering expression of plasmatic membrane components. H u m a n alveolar fluid has been shown to possess nonspecific neutral protease activity [22] and surface changes in AMq~ might occur by proteolytic loss of chemotactic receptors. An interesting effect is that provoked by the smoking habit on the locomotion of AM4,. It has been reported that AM~ from smokers show higher stimulated and unstimulated locomotion than those from nonsmokers [12, 15]. Others found that monocyte chemotaxis was the same in both these groups [23]. In this study, no differences were observed in the chemotactic response of monocyte from smokers and nonsmokers. Nevertheless, it was reported that in burned subjects, acute inhalation of smoke depressed neutrophil locomotion [24]. We have found the chemotactic response of AM0 from smokers to be higher with all the chemotactic agents tested. In another study, the chemotactic response of AM¢b from smokers to C5a also was significantly higher [15]. These findings indicate that the

smoking habit could be associated with an AM4~ population capable of an enhanced response. We have recently studied the distribution of class II antigens on human Mq~ [7, 8, 25]. DR antigen expression was higher in AMO from nonsmoker subjects [8]. In addition, AMq~ from nonsmokers seem to be as effective as monocytes in their cooperative role with Y cells [26], whereas AMO from smokers are not able to provide an optimal response [27]. It remains to be explained clearly in what way smoking habit influences the AM4~ functions. The chemotactic response to MM4~ has not previously been investigated specifically. In our study, these cells displayed impaired chemotaxis for all the chemotactic agents tested. Impaired chemotaxis of milk neutrophils has been reported, too [28]. In our study, when monocytes were preincubated with milk supernatants and then assayed for chemotaxis, a depressed chemotactic response was observed. Monocytes, as well as MMq~ [29, 30], ingest lipids and proteins and these laden cells could have certain defective functions. This situation may be analogous to that observed in pulmonary alveolar proteinosis, in which the overloaded AM4~ show depressed chemotaxis among other impairments [31]. Moreover, phagocytosis may lead to an inhibition of leukocyte locomotion [32]. Another explanation for the impaired chemotactic response of human M~b could be the lack of a hypothetical factor necessary for optimal chemotaxis. For instance, when mouse resident peritoneal cells are suspended in RPMI 1640 medium without serum, virtually none of the cells respond to chemotactic agents. Restoration of the response was achieved when a serum protein, M~b stimulating protein (MSP), was added to the M~ [33]. In addition, when rabbit alveolar and peritoneal MO were supplemented with SE, the migration of both populations was stimulated [20]. In the present study, however, no significant increase in the chemotactic response was obtained when SL was added to the HAM4,. It remains to be elucidated how chemotactic responsiveness is related to Mq~ heterogeneity. For instance, are chemotaxis and antigen presentation performed equally efficiently by the same 275

MqS? O n e c o u l d h y p o t h e s i z e t h a t c h e m o t a c t i c f a c t o r s , like L D C F , m i g h t act o n D R - p o s i t i v e M ~ , w h e r e a s o t h e r c h e m o a t t r a c t a n t s , s u c h as C 5 a , o p e r a t e o n D R - n e g a t i v e M~b. T h e sign i f i c a n c e o f t h i s h y p o t h e t i c a l r e l a t i o n s h i p is being investigated.

Acknowledgements T h i s w o r k was s u p p o r t e d by g r a n t no. TR-182015 f r o m I n s t i t u t o N a c i o n a l d e la S a l u d and by a grant from Heinz-Koch Foundation. The authors wish to thank the patients, nurses and staff of the Department of Pulmonary Medicine (Centro "Ramon y Cajal", Madrid) and of the Department of Obstetrics (Hospital " L a P a z " , M a d r i d ) for t h e i r c o l l a b o r a t i o n .

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