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Leukokinesis-enhancing factor in human serum: Partial characterization and relationship to disorders of leukocyte migration
(:LINI(:AL
IMMUNOLOGY
AND
I.MMMUNOPATHOLOCY
12, 382- 395 ( 1979)
Leukokinesis-Enhancing Factor in Human Serum: Partial Characterization and Relationship to Disorders of Leukocyte Migration’ EUFRONIO Mrdiccrl Medicine, Pathology,
CHARLES L. WORONICK, PETER A. WARD
G. MADERAZO,’
Research Hartford University
AND
Lnhorator~ und Irzjrt~tious Disewc Di~~ision. Depcwtnret~: Hospital, Hartford, Connecticut 06115, and Department of Connecticut Health Center, Farmirqton, Connecticut
I$ qf 06032
Received October 25. I978 In the presence (as contrasted to the absence) of serum. human polymorphonuclear leukocytes respond with enhanced movement in modified Boyden chambers. This observation is largely related to the presence in serum of a factor which enhances random migration of leukocytes (leukokinesis). This substance, termed the leukokinesisenhancing factor (LEF), can be fractionated from normal human serum by precipitation with ammonium sulfate. LEF has a sedimentation coefficient of approximately 2.9 S and has an isoelectric point between 4.2 and 5.6. It antagonizes the effects of the cell-directed inhibitor of leukotaxis, thus providing evidence for a complex control of leukocyte movement by serum factors. LEF also has a stimulatory effect on the in vitro movement of human monocytes. A small effect of LEF in phagocytic function of human leukocytes was shown. Three patients with persistent and recurrent bacterial infections have been discussed. In each case, the major in Isitro defect of leukotaxis has been a relative deficiency of LEF in their serum. This would appear to represent a previously undescribed defect of leukocyte motility.
INTRODUCTION
Studies of leukotactic abnormalities in humans have demonstrated two general types of defects, namely, abnormal regulation of chemotactic factors and cellassociated defects of locomotion. The former abnormality can be due to deficiencies of complement-derived chemotactic factors (1, 2). or abnormal elevations of the chemotactic factor inactivator (CFI) in serums of patients with Hodgkin’s disease (3), cirrhosis (4), sarcoidosis (5), and lepromatous leprosy (6). Cellassociated defects of leukocyte movement have been reported in patients with a variety of diseases such as rheumatoid arthritis (7), diabetes meilitus (g), hyperimmunoglobulinemia E syndrome (9), cancer (lo), periodontitis (11). and others. With the exception of a single patient with dysfunctional leukocytic actin (12) and the microtubule defect in Chediak-Higashi leukocytes (13), the only other well-defined mechanism in these abnormalities is the presence of a celldirected inhibitor of leukotaxis (CDI) which was found in the serum of a patient with listeria meningitis (14). Similar inhibitors have also been demonstrated in ’ Supported by grants from the Hartford Hospital Research Free Funds and National Institutes of Health Grant HL-22257. 2 To whom requests for reprints should be sent at the Medical Research Laboratory and Division of Infectious Disease, Department of Medicine. Hartford Hospital. Hartford. Conn. 06115. 382 0090-1229/79/040382-14$01.00/O Copyrtghf c:: 1979 by Academic Press. Inc. AlI rights of reproduction in any form rtservrd
LEUKOKINESIS-ENHANCING
FACTOR
IN
HUMAN
SERUM
383
patients with other diseases such as patients with anergy (15), IgA myeloma (16), and various neoplasms (17). Recent studies have provided us with evidence for the presence of a factor in normal serum that stimulates polymorphonuclear leukocyte locomotion and antagonizes the effect of the cell-directed inhibitor. Because of its functional behavior, we have called it the leukokinesis-enhancing factor (LEF). This evidence has also lead to the recognition of a different type of disorder of leukocyte motility which is due to an apparent deficiency of this factor. MATERIALS
AND METHODS
Subjects. Normal leukocytes and sera were obtained from healthy laboratory personnel. Patients included an 18-month-old male child (Pl) who suffered from recurrent Staphylococcus aUYeUS skin infections since birth, a 53-year-old woman (P2) with recurrent skin and pulmonary infections due to the same organism, and a 35year-old man (P3) with recalcitrant periodontitis but no history of recurrent infections elsewhere. A healthy age-matched cousin of the 18-month-old patient provided the control blood samples for his leukotactic studies. Leukocyte locomotion assay. Polymorphonuclear leukocytes (PMN) were prepared from heparinized (50 units heparin/ml) whole blood by sedimentation of the red blood cells at lg for 30 to 90 min at room temperature. The resulting leukocyte-rich plasma was centrifuged at 5OOg for 10 min, and the cell pellet obtained was washed once, resuspended in Medium 199, and adjusted to a concentration of 5 x lo6 PMN/ml. Isolation of monocytes was achieved using the method described by Boyiim (18). Sera were prepared from clotted whole blood. For the leukocyte locomotion assay, modified Boyden chambers made from clear acrylic (Ahlco Corporation, Southington, Conn.) and micropore filters of 5-pm porosity (Millipore Corporation, Bedford, Mass.) were used. The modifications of the Bbyden technique, as well as methods for calculating the locomotion index (LI), have been described in earlier reports (19, 20). In brief, the prepared chambers containing the test PMN in the upper compartment and medium or chemotactic factor in the lower compartment were placed in an incubator at 37°C for 90 min. This short incubation period prevented complete penetration of the filter by the migrating cells, thus the problem of cell detachment from the distal filter surface was avoided. After incubation, the filters were removed, fixed, stained, cleared, and mounted as described previously (21). The locomotion index was then determined as follows: (i) PMN were counted at every IO-pm interval beneath the proximal filter surface and progressing to the distal surface; (ii) the number of cells counted at every IO-pm level was multiplied by the distance of that level from the proximal surface; (iii) the products calculated from each lo-pm level were added (to obtain the total distance migrated by the migrating cell population); and (iv) the sum was divided by the total number of cells counted. The quotient is the locomotion index (LI), which is the average distance in micrometers migrated by those PMN which have entered the filter. All determinations were performed in duplicate, and at least three fields in each filter were counted. To test for LEF activity in whole sera, aliquots of normal cells (0.1 ml of 5 X lo6 PMN/ml) were first preincubated at 37°C for 30 min in a shaking water bath with
384
MADERAZO
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50 ~1 of the serum in question, or with 50 ,ul of normal serum which served as a control and which was tested simultaneously. In partially purified serum fractions, LEF activity was determined by treating the PMN with 50 ~1 (unless otherwise specified) of the fraction; in this case, controls consisted of normal PMN treated with the same volume of medium in which the fractions were dissolved. After incubation, the mixture was diluted to 0.7 ml and added to the upper compartment. Subsequently, it was found that chemotactic factor, either as zymosanactivated normal serum or bacterial chemotactic factor, was not necessary to elicit the stimulatory effect of serum on PMN locomotion. Thus, in most latter experiments Medium 199 alone in the lower compartment was used. Cell-directed inhibitor activity of serum fractions were determined by experiments similar to those previously described ( 14). The bacterial chemotactic factor was prepared from overnight cultures of Escherichia coli in Medium 199 at 37°C. After incubation the culture was centrifuged at 45,OOOg for 30 min, and the supernatant fluid filtered through a 0.2-pm pore size filter and adjusted to pH 7.4 with sodium hydroxide. A 3% concentration of this filtrate in Medium 199 was used in experiments requiring its use. In monocyte experiments, zymosan-activated serum prepared by incubation of 5 mg zymosan with 1 ml of serum at 37°C for 30 min was used. After incubation, the zymosan was removed by centrifugation, and 0.1 ml of the activated serum diluted to 1.5 ml with Medium 199 was used in the lower compartment. Nitroblue tetrazolium (NBT) reduction assay. Phagocytosis of opsonized zymosan particles by PMN using NBT as indicator dye was used to determine the effects of partially purified LEF on engulfment and NBT reduction. The quantitative spectrophotometric method of Baehner and Nathan was used (22). To test for LEF activity on phagocytosis, aliquots of cells (2 x IO6 PMN in 0.4 ml Medium 199itube) were incubated at 37°C for 30 min with 0.2 ml of LEF-rich fractions obtained by DEAE chromatography of whole serum. NBT reduction by the treated PMN was then assayed and compared with controls using PMN treated with whole serum and PMN incubated with Medium 199 alone. Ammonium sulfate fractionation. Salt fractionation of serum was performed by sequential precipitation using increasing concentrations of ammonium sulfate at 4°C. The various precipitated fractions obtained were redissolved in sodium phosphate buffer at pH 7.4 and dialyzed in cellulose tubing against the buffer (MW cutoff of 12,000, Arthur H. Thomas Co., Philadelphia, Pa.) overnight at 4°C. The fractions were diluted to the original serum volume before testing for LEF activity. Ultracentrifugation. Whole normal serum was ultracentrifuged in a linear 5 -20% sucrose density gradient (23) using human immunoglobulin G and albumin as reference proteins. The fractions were dialyzed, as above, prior to testing for LEF activity. DEAE-cellulose chromatography. The precipitated fraction of serum treated with ammonium sulfate at 60% of saturation, dialyzed against 0.01 M potassium phosphate buffer at pH 6.8, was applied to a DEAE column which had been equilibrated with the same buffer (24). The retained protein was then eluted by 0.5 M potassium phosphate buffer, pH 6.8. Preparative isoelectric focusing. Electrofocusing of whole serum was performed at 7°C with pH 3.5 to 10 ampholyte, using the LKB 2117 Multiphore
LEUKOKINESIS-ENHANCING
FACTOR
IN
HUMAN
385
SERUM
electrofocusing apparatus (LKB Instruments, Inc., Rockville, Md.) (25) which uses a flat-bed granulated Sephadex G-75 (Ultrodex) gel as the supporting and stabilizing medium. The positions of the protein bands in the gel were determined by placing a sheet of filter paper on the surface of the gel for 2 min, futing with 12.5% trichloroacetic acid, and staining with 0.2% Coomassie brilliant blue R-250. The pH range was determined by extracting each l-cm wide section of the gel with deionized water, and measuring the pH of each extract at 7°C. The fractions were then passed through a column of Sephadex G-25 to remove the ampholyte prior to testing for LEF activity. Preparation of chemotactic factor inactivator. The (Y- and @globulin forms of the chemotactic factor inactivator were prepared from human serum by a combination of fractionation techniques which included precipitation with ammonium sulfate, anionic exchange chromatography, gel filtration, and preparative acrylamide gel electrophoresis (26). RESULTS
Increased Migration of Leukocytes Treated with Normal Serum As shown in Table 1, the leukotactic response was clearly greater for PMN treated with normal serum than for PMN treated with medium alone. This leukotactic-enhancing property of normal serum was highly consistent and was detected in each of more than 100 consecutive experiments, three of which are reported in Table 1. These results are similar to those reported by Maderazo and Woronick (20) concerning the influence of normal serum on the migration of cells from normal donors under similar experimental conditions. They found that in the absence of serum the cells had a LI of 28.6 ? 0.8 pm (mean rf: SE) (7 normals, 42 fields counted), whereas in the presence of serum the LI was 41.3 + 0.3 Frn (36 normals, 282 fields), which represents a 44% increase (P < 0.001). As an alternate explanation, this phenomenon could be produced if a potent chemotaxin was produced by bacterial contamination of the culture medium, and which could be destroyed by the chemotactic factor inactivator normally present
TABLE ENHANCING
EFFECT
OF SERUM
1
ON GRANULOCYTE
MIGRATION
Locomotion Cells treated with: No serum With 7% autologous serum P value
Experiment 16.2 k 1.P 49.3 k 1.3
1
IN VITRO”
index (pm)
Experiment 2
Experiment 3
29.7 2 1.3 46.7 2 2.5
25.6 k 1.4 50.4 f 3.4
a Normal eranulocvtes susuended in Medium 199 were incubated with or without serum in the upper compartment and with 3% chemotactic factor derived from E. coli in the lower compartment. Three typical experiments using cells and sera from three different normal donors are shown. Each experiment was performed in duplicate, and three fields per filter were counted. The probabilities were calculated by Student’s I test. @Values are mean 2 SD.
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MADERAZO
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in serum, thus producing a concentration gradient across the filter. However, the medium was found to be sterile. The stimulatory effect of serum was also found to occur when a variety of tissue culture media were used in place of Medium 199, thus proving the essential role of serum. Furthermore, purified cy-and P-globulin forms of human chemotactic factor inactivators failed to produce stimulation when used in place of serum in the upper compartment. These findings indicate that the presence of chemotactic factor in the medium does not explain the migration-enhancing effect of normal serum, and that serum contains a naturally occurring stimulator. Chemokinetic
Versus
Chemotactic
Effects
of Partially
Purified
LEF
The relative effects of absolute concentration of partially purified LEF (obtained by preparative isoelectric focusing of whole serum) above and below the filter was investigated, using experiments similar to those used by Zigmond and Hirsch (27). Various concentrations of LEF in Medium 199 were placed in the upper and lower compartments of the chamber, and the PMN responses were assessed using no other attractant. The results of this experiment are shown in Table 2. In view of objections to some of the assumptions used by Zigmond and Hirsch in predicting random migration (19, 20) gross evaluation of kinetic and tactic effects was made using Wilkinson’s method (28). The numbers along the diagonal from upper left to lower right record PMN responses to increasing absolute concentrations of LEF (chemokinetic responses). The numbers above and to the right of this diagonal demonstrate the response to an increasing concentration gradient of LEF (positive chemotactic response), while the numbers below and to the left of the diagonal demonstrate the response to a decreasing concentration gradient (negative chemotactic response). Chemotactic response is roughly assessed by examining the numbers along the same horizontal from left to right or along the opposite diagonal from lower left to upper right. As shown by the results along the diagonal in Table 2, PMN migration increased as the absolute concentration of LEF increased. This finding resembles the chemokinetic effect of whole serum in the horse (27) and in humans (Maderazo and Woronick, unpublished observation). Unlike whole serum which has chemotactic activity, partially purified LEF showed no such response. Physical
Characteristics
of LEF
To test the heat stability of LEF, serum was preincubated at 56°C for 1 hr prior to testing with cells. The results of this experiment, shown in Table 3 (Experiment A), indicate marked reduction of LEF activity, which was almost completely abolished, after heat treatment. Dialysis of normal serum against sodium phosphate-buffered saline (pH 7.4, ionic strength 0.15) for 24 hr at 4°C did not change the LEF activity of the serum (Table 3, Experiment B). The various fractions obtained by ultracentrifugation of whole serum in 5 -20% sucrose density gradient were tested for their effects on PMN. Each fraction was dialyzed to remove sucrose and tested for LEF activity. As shown in Fig. 1, most of the LEF activity was found in a position above albumin. Cell-directed inhibitor activity was noted in fractions in the region of immunoglobulin G and a region
LEUKOKINESIS-ENHANCING
FACTOR
TABLE EFFECT
OF CONCENTRATION
IN
HUMAN
387
SERUM
2
GRADIENTS AND ABSOLUTE CONCENTRATIONS OF PARTIALLY ON POLYMORPHONUCLEAR LEIJKOCVTE MIGRATIONS
LEF
Percentage LEF preparation Percentage LEF preparation in upper compartment
0
1
in lower compartment
5 Locomotion
PURIFIED
10
25
indexb
0 1 5 10 25 a The LEF used in this experiment was prepared by isoelectic focusing of normal serum and had an absorbance at 280 nm of 1.66. b Mean + SD in pm.
below it, with sedimentation coefficients of 7 S and 10.5 S, respectively. This finding is consistent with the previously reported sedimentation characteristics of the cell-directed inhibitor (14). The sedimentation coefficient of LEF was found to be 3.0 S when calculated from the position of human albumin and 2.8 S when calculated from the position of human IgG, using the method of Martin and Ames (23). The molecular weight was estimated to be approximately 37,000 + 600 (mean + SE). TABLE EFFECT
OF HEAT
Experiments
TREATMENT
AND
DIALYSIS
3
ON THE LEIJKOKINESIS-ENHANCING
Cells treated with:
ACTIVITY
OF SERUM
Locomotion index (km)”
P value@
A
1 No serum 2 Unheated serum (7%) 3 Heated sentme (7%)
19.3 + 0.9 37.2 e 2.4 20.8 f 1.2
.4-l vs A-2
B
1 Nondialyzed serum (7%) 2 Dialvzed serumd (7%)
31.1 2 1.4 32.8 -+ 1.2
B-l vs B-2 >0.0.5
a Mean * SD. b Probabilities were calculated by the Student’s t test. c Sera from four normal individuals were tested for leukokinesis-enhancing activity both before and after treatment at 56°C for 1 hr. Each assay was performed in triplicate, and three fields in each of the three filters were counted. d Serum was dialyzed against sodium phosphate-buffered saline (pH 7.4, ~=0.15) for 24 hr at 4°C. The control nondialyzed serum was diluted to compensate for the volume change of dialyzed serum.
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% MIGRATION ALBUMIN
12
3
4
IgG
5
6
.---.
7
8
A
9
280
. 1.0
i
. 0.8
8 2
10
FRACTIONS FIG. 1. Sucrose density gradient characteristics of LEF. Whole serum was ultracentrifuged in a 5-20% sucrose density gradient and the fractions obtained were tested for LEF activity. Response of PMN treated in medium alone was used as the control and the percentage change from this value [( +) for stimulation or LEF activity, and C-) for inhibition] was calculated from response of cells treated with 50 ~1 of the various fractions in the upper compartment.
Separation of LEF Activity from CDI Activity Examination of the various fractions obtained by ammonium sulfate fractionation showed that most of the LEF activity, which is found in the nonsoluble fractions between 40-60% saturation, is distinctly separable from the celldirected inhibitor (CDI) activity, which is found in the precipitate obtained at 20-40% of saturation (Fig. 2). DEAE-cellulose chromatography was also found to be useful in separating LEF from the CD1 of serum. When either whole serum or the O-60% ammonium sulfate precipitate of whole serum was dialyzed against 0.01 M potassium phosphate buffer, pH 6.8, and applied to a DEAE-cellulose column equilibrated against the same buffer, the CD1 passed directly through the column, whereas the LEF was retained. The LEF activity was recovered when the column was eluted with a 0.5 M phosphate buffer which was applied to elute all the protein from the column in a single peak. This simple experiment demonstrates that DEAE-cellulose can be used to separate CD1 and LEF (which have opposite effects on leukocyte migration) from each other at low ionic strength, and that the LEF activity can be recovered at high ionic strength (Fig. 3). Isoelectric focusing of whole serum revealed the presence of LEF activity in fractions with isoelectric points of 4.2 to 5.6 (Fig. 4). More than 11 distinct protein bands were detected within this zone by staining with Coomassie brilliant blue. Eluted fractions in the higher pH range (pH 7-9) contained CD1 activity. These preliminary investigations demonstrated that standard protein purification procedures can be used to separate LEF from its CD1 antagonist normally
LEUKOKINESIS-ENHANCING
FACTOR
IN
HUMAN
SERUM
389
-30 1
FRACTIONS (% SATURATION
OF APMONIUM
SULFATE)
2. LEF activity in ammonium sulfate fractions of human serum. Serum was fractionated seqwntially by increasing ammonium sulfate concentrations. The percentage change from the control value was determined as described in the legend to Fig. 1. FIG.
present in human serum. The CDI-free LEF preparations were found to be suitable for further studies of LEF functional activity on PMN as described below. Work is now in progress to refine the application of these techniques so that they may be used as part of a practical purification procedure for LEF. Stimulation of Monocyte Migration
by Serum and by Partially PuriJied LEF
To determine whether LEF has activity on the migration of cells other than PMN, normal human monocytes were tested in the presence of normal human serum and in the presence of partially purified LEF obtained by DEAE chromatography of normal serum. Each chamber contained 5 x lo5 monocytes. Techniques for assessment of monocyte activity were similar to those performed with PMN, except that B-pm pore-size filters were used and chamber incubation was continued for 2 to 3 hr. When the effect of normal serum was tested in the absence of chemotactic attractant in the lower compartment, the locomotion indexes obtained at 2 hr were 10.4 ? 0.9 pm (mean * SD) without normal serum in the upper chamber, and 13.2 -c 1.8 pm with 10% normal serum in the upper chamber [26% increase, Student’s t = 3.425, degrees of freedom (df)= 10, P < 0.011. On the other hand, when the chemotactic response to zymosan-activated normal serum in the lower compartment was measured, the locomotion indexes obtained were 11.8 + 0.6 pm without normal serum in the upper compartment, and 15.7 + 1.0 pm with 10% normal serum in the upper compartment (33% increase, t = 10.032, df = 16, P < 0.001). Thus, 10% normal serum in the cell compartment increases both the non-
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ENiiANCFMENT
_L
INBIBITION
10
20
30
90
100
110
120
FRACTIONS
FIG. 3. DEAE-cellulose chromatography of LEF. The nonsoluble fraction of normal serum at 60% ammonium sulfate saturation was applied to a DEAE column. LEF activity was found in fractions with 0.5 h4 phosphate buffer (pH 6.8) (arrow). The initial peak (fractions 12-20) was rich in IgG and contained inhibitor activity.
chemotactic and the chemotactic responses of normal human monocytes. In separate experiments using a 3-hr incubation time, an increased response was also observed when 0.1 ml of an LEF fraction (Azso = 0.77) prepared by DEAE chromatography was used with monocytes in the cell compartment, with activated normal serum in the lower compartment. In this case the LI was 22.7 + 0.8 pm without LEF and 31.5 t 1.7 pm with LEF (39% increase, f = 14.768, df= 15;P < 0.001).
LEUKOKINESIS-ENHANCING
FACTOR
IN
HUMAN
SERUM
392
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ET AL.
Effect of LEF on Phagocytosis and NBT Reduction by PMN Pooled partially purified LEF fractions (A 28,,= 2.25) obtained by DEAE chromatography of whole serum enhanced phagocytosis of opsonized zymosan by PMN as determined by NBT reduction, when compared with PMN treated with Medium 199 alone (AA,,, = 0.52 + 0.02 without LEF versus 0.55 -+: 0.01 with LEF, t = 3.171, df = 4, P < 0.05). Phagocytic activity was also augmented by treatment of the cells with normal serum (AA,,, = 0.58 t 0.01 with serum, P < 0.01). These results indicate that LEF is not only a specific stimulator of leukocyte migration, but also a weak stimulator of phagocytosis. Dysfunction of Leukocyte Migration Due to a Deficiency of Serum LEF Activity Three patients, one child (PI) and two adults (P2 and P3), were evaluated because of recurrent Staphylococcus aureus infections (PI and P2) or recalcitrant periodontitis (P3). The association between leukotactic dysfunction and periodontal disease was shown previously (11). Initial studies of granulocyte migration showed no intrinsic abnormality of the cells (Table 4, Experiment A), but a serum-associated defect resembling cell-directed inhibitor activity was noted (Table 4, Experiment B). Further studies of their sera, however, demonstrated some differences from the previously described dysfunctions caused by increased inhibitor activity (14). For instance, unlike patients with high serum cell-directed inhibitor activity, where washed cells continue to be abnormal until reincubated with normal serum (14), washed cells of these patients showed normal nonstimulated
TABLE 4 PRESENCE OF A SERUM-ASSOCIATED DEFECI OF LEUKOCYTE MICRAIION IYX PATIENTS WITH RECURRENT OR CHRONIC INFECTIONS" Materials Upper compartment cells + serum
Experiment A
B
added to:
Locomotion Lower compartment
1 2
Normal Patient’s
+ none + none
Medium Medium
199 199
3 4
Normal Patient’s
+ none + none
Bacterial Bacterial
factor factor
I 2
Normal Normal
+ normaId + patient’s
Medium Medium
199’ 199
3 4
Patient‘s Patient’s
+ normal + patient’sd
Medium Medium
199 199
Patient 1
index (pm)*
Patient 2
30.1 t 8.0 28.1 -r 4.9 (>0.5) Not done Not done
29.6 _f 27.4 + (>O.l) 43.1 * 42.1 k (BO.7)
1.3 2.1
52.9 2 2.1 36.2 2 5.4 (
49.0 k 1.9 35.1 2 4.6 (
4.3 1.4
Patient 3 21.4 ? 22.5 A (>0.5) 23.1 ? 23.5 r (>0.5)
1.5 1.8 2.0’ 2.0c
40.4 + 3.9 25.8 + 4.5 (
@Normal controls were tested simultaneously with each experiment to correct for day-to-day variability of the leukotactic assay. The control and patient’s values were compared and the significance of the difference was determined using Student’s f test. Numbers in parentheses are P values. b Mean + SD.
LEUKOKINESIS-ENHANCING
FACTOR
IN
HUMAN
393
SERUM
and chemotactic responses (Table 4, Experiment A). Secondly, LEF-deficient sera from all three patients lost their abnormality and became indistinguishable from normal serum after storage at -90, -10, and 4°C for 1 to 2 weeks. This reversal towards normal after storage was confirmed in all three patients by simultaneous testing of aged and fresh sera. The most convincing evidence that these sera were deficient in LEF rather than possessing high inhibitor activity was obtained by heat treatment of sera at 56°C for 1 hr. As shown in Table 5, heat treatment reduced the serum leukokinetic activity of all serum samples (both normal and abnormal). Activity of heated normal sera and LEF-deficient set-a were reduced to approximately similar levels, whereas that of inhibitor-rich sera were reduced to significantly lower levels. The explanation of this observation is that because LEF is heat labile, heat treatment of the serum permits one to measure the activity of unopposed heat-stable cell-directed inhibitor. DISCUSSION
The data presented above demonstrate the presence in normal serum of a stimulator of leukocyte migration. This factor, the leukokinesis-enhancing factor (LEF) is heat labile and nondialyzable. It can be fractionated from whole serum using a variety of techniques including ammonium sulfate fractionation, DEAEcellulose chromatography, and isoelectric focusing in granulated G-75 gel. The sedimentation coefficient of LEF is estimated to be about 2.9. The spectrum of activity of LEF appears to be wide and includes stimulatory effects on PMN and monocyte locomotion and on PMN phagocytosis. Every known activity of the cell-directed inhibitor appears to have a corresponding antagonistic LEF activity. The stimulatory effect of LEF on monocytes suggests its importance as a regulator for both acute and chronic inflammatory reactions. In TABLE HEAT
TREATMENT
OF SERA
CELL-DIRECTED
Diagnosis
Serum Donor
5
T O DIFFERENTIATE
LEF
INHIBITION
DEFICIENCY
FROM
INCREASED
ACTIVITY
Locomotion
index (prn)O
Unheated
Heatedb
Normal:
Normal 1 Normal 2
52.4 2 0.8 50.7 + 1.6
33.4 k 1.4 29.4 f 1.0
4 LEF:
Patient 1 Patient 2 Patient 3
49.9 2 1.4’ 42.8 2 1.7’ 53.0 2 0.9’
30.0 zt 0.6’ 30.0 2 1.4’ 27.7 k 0.8’
T CDI:
Patient 4d Patient 5 Patient 6
31.6 ? 1.7 24.2 ” 0.6 25.9 k 0.8
23.6 k 0.8 15.6 k 0.9 19.8 _’ 0.8
a Three fields on each of two filters were counted. Values are mean 2 SD. b Sera were incubated at 56°C for 1 hr. c Stored frozen sera were used. These results indicate complete (patients 1 and 3) or partial (patient 2) reversal of LEF-deficient serum to normal after storage in a freezer for 1 week or longer. See Table 4, Experiment B-2 for the LI values obtained under the same conditions on fresh serum from these patients. d This patient was previously reported [Ref. (14)].
394
MADERAZO
ET
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viva studies with experimental animals should further clarify its role in inflammation. The recognition of the existence of LEF naturally implied the possible existence of abnormalities of leukocyte migration owing to a deficiency of this factor in serum. Such an abnormality has now been identified, as described in the preceding sections, in humans with recurrent or chronic infections. In vitro laboratory observations which were found useful for the differentiation of LEF deficiency from increased inhibitor activity include: (i) heat treatment of sera at 56°C for 1 hr, where there was reduction of stimulatory activity such that the difference between abnormal and normal sera was lost, unlike serum with high cell-directed inhibitor activity where heat treatment produced reduction of PMN locomotion greater than that produced in similarly heat-treated normal serum: (ii) nonstimulated locomotion of PMN treated with LEF-deficient serum and washed was similar to the response of PMN pretreated with normal serum, whereas, nonstimulated response of cells treated with inhibitor and washed (PMN preincubated in serum with high cell-directed inhibitor activity and subsequently washed) was lower than the corresponding control (PMN preincubated in normal serum and washed); and (iii) reversal to normal of LEF-deficient sera upon storage at -90, -10, and 4”C, whereas this was not observed in sera containing high celldirected inhibitor activity. It should be noted that normal serum also showed increased enhancing activity upon storage, indicating that LEF may have an inactive (or less active) precursor from which it is derived in serum, and that apparent deficiencies of LEF can result either from a lack of conversion of this precursor or from a rapid metabolism or destruction of LEF once formed from this precursor. Presumably, there is a net gain of LEF in serum upon storage and this net gain is higher in the three patients with apparent LEF deficiency studied so far. A substance that functionally resembles LEF was described previously by Jacobs and Goetzl in the serum of a patient with recurrent sterile pyoderma gangrenosum (29). Their studies revealed the presence of a migration-enhancing factor found in the serum of the patient, which increased both PMN and monocyte migration. The effect on monocytes was minor compared with the effect on PMN. Based upon results of studies using Sephadex G-200. they estimated the molecular weight of their stimulator to be 160,000. This is different from the LEF we have found in normal serum which has a sedimentation coefficient of approximately 2.9 S and an estimated molecular weight of approximately 37,000. Nevertheless, it is possible that this higher molecular weight LEF of Jacobs and Goetzl does occur normally. The latter LEF preparation and CD1 have molecular weights that are similar and can therefore be expected to be present in the same serum fractions prepared by Sephadex G-200. Because the two preparations have antagonistic activities, inhibitory or stimulatory activity will then be observed in these fractions depending upon the relative concentrations of each of these opposing factors. In normal serum this zone, which is easily detected because of its immunoglobulin G content, is inhibitory. In the patient described by Jacob and Goetzl it is likely that the abnormal elevation of the heavier LEF was sufficient to overcome the activity of normal CDI, and thus, LEF activity became more apparent. This possible contamination of CDI-rich serum fractions with heat-labile higher molecular weight LEF can explain the augmentation of CD1 activity by heat treatment as observed previously by two groups of investigators (17,30).
LEUKOKINESIS-ENHANCING
FACTOR
IN
HUMAN
SERUM
395
Recently, Wilkinson and Allan (31) have shown the chemokinetic effect of bovine serum albumin. This is of interest because LEF activity is frequently found in albumin-rich fractions. However, as shown by the results of the ultracentrifugation studies (Fig. l), maximum LEF activity was not found in the fraction containing the highest concentration of albumin.Additionally, some bovine serum albumin preparations tested in our laboratory do not contain appreciable chemokinetic activity. These findings suggest that LEF is distinct from albumin. The effects of removing albumin from our LEF-rich fractions on LEF activity is presently being studied. The results of these studies should prove or disprove our suspicion that LEF and albumin are not identical. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
Ballow, M., Shira, J. E., Harden, L., Yang, S. Y., and Day, N. K.,J. C/in. Z~zvest.56,703, 1975. Rosenfeld, S. I., Baum, J., Steigbigel, R. T., and Leddy, J. P., J. Clin. Invest. 57, 1635, 1976. Ward, P. A., and Berenberg. J. L., N. Engl. J. Med. 290, 76, 1974. Maderazo, E. G., Ward, P. A., and Quintiliani, R., J. Lab. C/in. Med. 85, 621, 1975. Maderazo, E. G., Ward, P. A. Woronick, C. L., Kubik, J., and DeGraff, A. C., Jr., Ann. Intern. Med. 84, 414, 1976. Ward, P. A., Goralnick, S., and Bullock, W. E., J. Lab. C/in. Med. 87, 1025, 1976. Mowat, A. G., and Baum, J., J. Clin. Invest., 50, 2541, 1971. Mowat, A. G., and Baum, J., N. Engl. J. Med. 284, 621, 1971. Hill, H. R., and Quie, P. G., In “The Phagocytic Cell in Host Resistance” (J. A. Bellanti and D. H. Dayton, Eds.), p. 249, Raven Press, New York, 1975. Baum, J., In “The Phagocytic Cell in Host Resistance” (J. A. Bellanti and D. H. Dayton, Eds.) p. 267, Raven Press, New York, 1975. Maderazo, E. G., Lavine, W., Stolman, J., Ward, P. A., and Cogen, R., “16th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Illinois. 1976” (abstract). Boxer, L. A., Hedley-Whyte, E. T., and Stossel, T. P., N. Engl. J. Med. 291, 1093, 1974. Oliver, J. M., Amer. J. Pathol. 85, 395, 1976. Maderazo, E. G., Ward, P. A., Woronick, C. L., and Quintiliani, R., J. Lab. Clin. Med. 80, 190, 1977. Van Epps, D. E., Palmer, D. L., and Williams, R. C., J. Zmmunol. 113, 189, 1974. Van Epps, D. E., and Williams, R. C. Jr., J. Exp. Med. 144, 1227, 1976. Maderazo, E. G., Anton, R., and Ward, P. A., Clin. Zmmunol Zmmunopathol. 9, 166, 1978. Boyilm, A., Stand. J. C&z. Lab. Invest. 21, 77, 1%8. Maderazo, E. G., and Woronick, C. L., In “Leukocyte Chemotaxis: Methods, Physiology and Clinical Implications” (J. I. Gallin and P. G. Quie, Eds.), p. 43, Raven Press, New York, 1978. Maderazo, E. G., and Woronick, C. L., Clin. Zmmunol. Zmmunopathol. 11, 196, 1978. Ward, P. A., and Schlegel, R. A., Lancer 11, 344, 1969. Baehner, R. L., and Nathan, D. G., N. Engl. J. Med. 278, 971, 1968. Martin, R. G., and Ames, B. N., J. Biol. Chem. 236, 1372, 1961. Livingston, D. M., In “Methods in Enzymology” (A. S. Pietro, Ed.), Vol. 24, p. 723, Academic Press, New York, 1974. Winter, A., Perlmutter, H., and Davies, H., LKB Application Note 198, LKB Instruments, Inc., Rockville, Md., 1975. Till, G., and Ward, P. A., J. Zmmunol. 114, 843, 1975. Zigmond, S. H., and Hirsch, J. G., /. Exp. Med. 137, 387, 1973. Wilkinson, P. C., In “Primitive Sensory and Communication Systems. The Taxes and Tropisms of Micro-organisms and Cells” (M. J. Carlile, Ed.), p. 205, Academic Press, New York. 1975. Jacobs, J. C., and Goetzl, E. J., Pediatrics 56, 570, 1975. Van Epps, D. E., and Williams, R. C. Jr., Znfec. Zmmun. 13, 741, 1976. Wilkinson, P. C., and Allan, R. B., In “Leukocyte Chemotaxis: Methods, Physiology and Clinical Implications” (J. I. Gallin and P. G. Quie, Eds.), p. 1, Raven Press, New York, 1978.
IMMUNOLOGY
AND
I.MMMUNOPATHOLOCY
12, 382- 395 ( 1979)
Leukokinesis-Enhancing Factor in Human Serum: Partial Characterization and Relationship to Disorders of Leukocyte Migration’ EUFRONIO Mrdiccrl Medicine, Pathology,
CHARLES L. WORONICK, PETER A. WARD
G. MADERAZO,’
Research Hartford University
AND
Lnhorator~ und Irzjrt~tious Disewc Di~~ision. Depcwtnret~: Hospital, Hartford, Connecticut 06115, and Department of Connecticut Health Center, Farmirqton, Connecticut
I$ qf 06032
Received October 25. I978 In the presence (as contrasted to the absence) of serum. human polymorphonuclear leukocytes respond with enhanced movement in modified Boyden chambers. This observation is largely related to the presence in serum of a factor which enhances random migration of leukocytes (leukokinesis). This substance, termed the leukokinesisenhancing factor (LEF), can be fractionated from normal human serum by precipitation with ammonium sulfate. LEF has a sedimentation coefficient of approximately 2.9 S and has an isoelectric point between 4.2 and 5.6. It antagonizes the effects of the cell-directed inhibitor of leukotaxis, thus providing evidence for a complex control of leukocyte movement by serum factors. LEF also has a stimulatory effect on the in vitro movement of human monocytes. A small effect of LEF in phagocytic function of human leukocytes was shown. Three patients with persistent and recurrent bacterial infections have been discussed. In each case, the major in Isitro defect of leukotaxis has been a relative deficiency of LEF in their serum. This would appear to represent a previously undescribed defect of leukocyte motility.
INTRODUCTION
Studies of leukotactic abnormalities in humans have demonstrated two general types of defects, namely, abnormal regulation of chemotactic factors and cellassociated defects of locomotion. The former abnormality can be due to deficiencies of complement-derived chemotactic factors (1, 2). or abnormal elevations of the chemotactic factor inactivator (CFI) in serums of patients with Hodgkin’s disease (3), cirrhosis (4), sarcoidosis (5), and lepromatous leprosy (6). Cellassociated defects of leukocyte movement have been reported in patients with a variety of diseases such as rheumatoid arthritis (7), diabetes meilitus (g), hyperimmunoglobulinemia E syndrome (9), cancer (lo), periodontitis (11). and others. With the exception of a single patient with dysfunctional leukocytic actin (12) and the microtubule defect in Chediak-Higashi leukocytes (13), the only other well-defined mechanism in these abnormalities is the presence of a celldirected inhibitor of leukotaxis (CDI) which was found in the serum of a patient with listeria meningitis (14). Similar inhibitors have also been demonstrated in ’ Supported by grants from the Hartford Hospital Research Free Funds and National Institutes of Health Grant HL-22257. 2 To whom requests for reprints should be sent at the Medical Research Laboratory and Division of Infectious Disease, Department of Medicine. Hartford Hospital. Hartford. Conn. 06115. 382 0090-1229/79/040382-14$01.00/O Copyrtghf c:: 1979 by Academic Press. Inc. AlI rights of reproduction in any form rtservrd
LEUKOKINESIS-ENHANCING
FACTOR
IN
HUMAN
SERUM
383
patients with other diseases such as patients with anergy (15), IgA myeloma (16), and various neoplasms (17). Recent studies have provided us with evidence for the presence of a factor in normal serum that stimulates polymorphonuclear leukocyte locomotion and antagonizes the effect of the cell-directed inhibitor. Because of its functional behavior, we have called it the leukokinesis-enhancing factor (LEF). This evidence has also lead to the recognition of a different type of disorder of leukocyte motility which is due to an apparent deficiency of this factor. MATERIALS
AND METHODS
Subjects. Normal leukocytes and sera were obtained from healthy laboratory personnel. Patients included an 18-month-old male child (Pl) who suffered from recurrent Staphylococcus aUYeUS skin infections since birth, a 53-year-old woman (P2) with recurrent skin and pulmonary infections due to the same organism, and a 35year-old man (P3) with recalcitrant periodontitis but no history of recurrent infections elsewhere. A healthy age-matched cousin of the 18-month-old patient provided the control blood samples for his leukotactic studies. Leukocyte locomotion assay. Polymorphonuclear leukocytes (PMN) were prepared from heparinized (50 units heparin/ml) whole blood by sedimentation of the red blood cells at lg for 30 to 90 min at room temperature. The resulting leukocyte-rich plasma was centrifuged at 5OOg for 10 min, and the cell pellet obtained was washed once, resuspended in Medium 199, and adjusted to a concentration of 5 x lo6 PMN/ml. Isolation of monocytes was achieved using the method described by Boyiim (18). Sera were prepared from clotted whole blood. For the leukocyte locomotion assay, modified Boyden chambers made from clear acrylic (Ahlco Corporation, Southington, Conn.) and micropore filters of 5-pm porosity (Millipore Corporation, Bedford, Mass.) were used. The modifications of the Bbyden technique, as well as methods for calculating the locomotion index (LI), have been described in earlier reports (19, 20). In brief, the prepared chambers containing the test PMN in the upper compartment and medium or chemotactic factor in the lower compartment were placed in an incubator at 37°C for 90 min. This short incubation period prevented complete penetration of the filter by the migrating cells, thus the problem of cell detachment from the distal filter surface was avoided. After incubation, the filters were removed, fixed, stained, cleared, and mounted as described previously (21). The locomotion index was then determined as follows: (i) PMN were counted at every IO-pm interval beneath the proximal filter surface and progressing to the distal surface; (ii) the number of cells counted at every IO-pm level was multiplied by the distance of that level from the proximal surface; (iii) the products calculated from each lo-pm level were added (to obtain the total distance migrated by the migrating cell population); and (iv) the sum was divided by the total number of cells counted. The quotient is the locomotion index (LI), which is the average distance in micrometers migrated by those PMN which have entered the filter. All determinations were performed in duplicate, and at least three fields in each filter were counted. To test for LEF activity in whole sera, aliquots of normal cells (0.1 ml of 5 X lo6 PMN/ml) were first preincubated at 37°C for 30 min in a shaking water bath with
384
MADERAZO
ET
AL.
50 ~1 of the serum in question, or with 50 ,ul of normal serum which served as a control and which was tested simultaneously. In partially purified serum fractions, LEF activity was determined by treating the PMN with 50 ~1 (unless otherwise specified) of the fraction; in this case, controls consisted of normal PMN treated with the same volume of medium in which the fractions were dissolved. After incubation, the mixture was diluted to 0.7 ml and added to the upper compartment. Subsequently, it was found that chemotactic factor, either as zymosanactivated normal serum or bacterial chemotactic factor, was not necessary to elicit the stimulatory effect of serum on PMN locomotion. Thus, in most latter experiments Medium 199 alone in the lower compartment was used. Cell-directed inhibitor activity of serum fractions were determined by experiments similar to those previously described ( 14). The bacterial chemotactic factor was prepared from overnight cultures of Escherichia coli in Medium 199 at 37°C. After incubation the culture was centrifuged at 45,OOOg for 30 min, and the supernatant fluid filtered through a 0.2-pm pore size filter and adjusted to pH 7.4 with sodium hydroxide. A 3% concentration of this filtrate in Medium 199 was used in experiments requiring its use. In monocyte experiments, zymosan-activated serum prepared by incubation of 5 mg zymosan with 1 ml of serum at 37°C for 30 min was used. After incubation, the zymosan was removed by centrifugation, and 0.1 ml of the activated serum diluted to 1.5 ml with Medium 199 was used in the lower compartment. Nitroblue tetrazolium (NBT) reduction assay. Phagocytosis of opsonized zymosan particles by PMN using NBT as indicator dye was used to determine the effects of partially purified LEF on engulfment and NBT reduction. The quantitative spectrophotometric method of Baehner and Nathan was used (22). To test for LEF activity on phagocytosis, aliquots of cells (2 x IO6 PMN in 0.4 ml Medium 199itube) were incubated at 37°C for 30 min with 0.2 ml of LEF-rich fractions obtained by DEAE chromatography of whole serum. NBT reduction by the treated PMN was then assayed and compared with controls using PMN treated with whole serum and PMN incubated with Medium 199 alone. Ammonium sulfate fractionation. Salt fractionation of serum was performed by sequential precipitation using increasing concentrations of ammonium sulfate at 4°C. The various precipitated fractions obtained were redissolved in sodium phosphate buffer at pH 7.4 and dialyzed in cellulose tubing against the buffer (MW cutoff of 12,000, Arthur H. Thomas Co., Philadelphia, Pa.) overnight at 4°C. The fractions were diluted to the original serum volume before testing for LEF activity. Ultracentrifugation. Whole normal serum was ultracentrifuged in a linear 5 -20% sucrose density gradient (23) using human immunoglobulin G and albumin as reference proteins. The fractions were dialyzed, as above, prior to testing for LEF activity. DEAE-cellulose chromatography. The precipitated fraction of serum treated with ammonium sulfate at 60% of saturation, dialyzed against 0.01 M potassium phosphate buffer at pH 6.8, was applied to a DEAE column which had been equilibrated with the same buffer (24). The retained protein was then eluted by 0.5 M potassium phosphate buffer, pH 6.8. Preparative isoelectric focusing. Electrofocusing of whole serum was performed at 7°C with pH 3.5 to 10 ampholyte, using the LKB 2117 Multiphore
LEUKOKINESIS-ENHANCING
FACTOR
IN
HUMAN
385
SERUM
electrofocusing apparatus (LKB Instruments, Inc., Rockville, Md.) (25) which uses a flat-bed granulated Sephadex G-75 (Ultrodex) gel as the supporting and stabilizing medium. The positions of the protein bands in the gel were determined by placing a sheet of filter paper on the surface of the gel for 2 min, futing with 12.5% trichloroacetic acid, and staining with 0.2% Coomassie brilliant blue R-250. The pH range was determined by extracting each l-cm wide section of the gel with deionized water, and measuring the pH of each extract at 7°C. The fractions were then passed through a column of Sephadex G-25 to remove the ampholyte prior to testing for LEF activity. Preparation of chemotactic factor inactivator. The (Y- and @globulin forms of the chemotactic factor inactivator were prepared from human serum by a combination of fractionation techniques which included precipitation with ammonium sulfate, anionic exchange chromatography, gel filtration, and preparative acrylamide gel electrophoresis (26). RESULTS
Increased Migration of Leukocytes Treated with Normal Serum As shown in Table 1, the leukotactic response was clearly greater for PMN treated with normal serum than for PMN treated with medium alone. This leukotactic-enhancing property of normal serum was highly consistent and was detected in each of more than 100 consecutive experiments, three of which are reported in Table 1. These results are similar to those reported by Maderazo and Woronick (20) concerning the influence of normal serum on the migration of cells from normal donors under similar experimental conditions. They found that in the absence of serum the cells had a LI of 28.6 ? 0.8 pm (mean rf: SE) (7 normals, 42 fields counted), whereas in the presence of serum the LI was 41.3 + 0.3 Frn (36 normals, 282 fields), which represents a 44% increase (P < 0.001). As an alternate explanation, this phenomenon could be produced if a potent chemotaxin was produced by bacterial contamination of the culture medium, and which could be destroyed by the chemotactic factor inactivator normally present
TABLE ENHANCING
EFFECT
OF SERUM
1
ON GRANULOCYTE
MIGRATION
Locomotion Cells treated with: No serum With 7% autologous serum P value
Experiment 16.2 k 1.P 49.3 k 1.3
1
IN VITRO”
index (pm)
Experiment 2
Experiment 3
29.7 2 1.3 46.7 2 2.5
25.6 k 1.4 50.4 f 3.4
a Normal eranulocvtes susuended in Medium 199 were incubated with or without serum in the upper compartment and with 3% chemotactic factor derived from E. coli in the lower compartment. Three typical experiments using cells and sera from three different normal donors are shown. Each experiment was performed in duplicate, and three fields per filter were counted. The probabilities were calculated by Student’s I test. @Values are mean 2 SD.
386
MADERAZO
ET
AL.
in serum, thus producing a concentration gradient across the filter. However, the medium was found to be sterile. The stimulatory effect of serum was also found to occur when a variety of tissue culture media were used in place of Medium 199, thus proving the essential role of serum. Furthermore, purified cy-and P-globulin forms of human chemotactic factor inactivators failed to produce stimulation when used in place of serum in the upper compartment. These findings indicate that the presence of chemotactic factor in the medium does not explain the migration-enhancing effect of normal serum, and that serum contains a naturally occurring stimulator. Chemokinetic
Versus
Chemotactic
Effects
of Partially
Purified
LEF
The relative effects of absolute concentration of partially purified LEF (obtained by preparative isoelectric focusing of whole serum) above and below the filter was investigated, using experiments similar to those used by Zigmond and Hirsch (27). Various concentrations of LEF in Medium 199 were placed in the upper and lower compartments of the chamber, and the PMN responses were assessed using no other attractant. The results of this experiment are shown in Table 2. In view of objections to some of the assumptions used by Zigmond and Hirsch in predicting random migration (19, 20) gross evaluation of kinetic and tactic effects was made using Wilkinson’s method (28). The numbers along the diagonal from upper left to lower right record PMN responses to increasing absolute concentrations of LEF (chemokinetic responses). The numbers above and to the right of this diagonal demonstrate the response to an increasing concentration gradient of LEF (positive chemotactic response), while the numbers below and to the left of the diagonal demonstrate the response to a decreasing concentration gradient (negative chemotactic response). Chemotactic response is roughly assessed by examining the numbers along the same horizontal from left to right or along the opposite diagonal from lower left to upper right. As shown by the results along the diagonal in Table 2, PMN migration increased as the absolute concentration of LEF increased. This finding resembles the chemokinetic effect of whole serum in the horse (27) and in humans (Maderazo and Woronick, unpublished observation). Unlike whole serum which has chemotactic activity, partially purified LEF showed no such response. Physical
Characteristics
of LEF
To test the heat stability of LEF, serum was preincubated at 56°C for 1 hr prior to testing with cells. The results of this experiment, shown in Table 3 (Experiment A), indicate marked reduction of LEF activity, which was almost completely abolished, after heat treatment. Dialysis of normal serum against sodium phosphate-buffered saline (pH 7.4, ionic strength 0.15) for 24 hr at 4°C did not change the LEF activity of the serum (Table 3, Experiment B). The various fractions obtained by ultracentrifugation of whole serum in 5 -20% sucrose density gradient were tested for their effects on PMN. Each fraction was dialyzed to remove sucrose and tested for LEF activity. As shown in Fig. 1, most of the LEF activity was found in a position above albumin. Cell-directed inhibitor activity was noted in fractions in the region of immunoglobulin G and a region
LEUKOKINESIS-ENHANCING
FACTOR
TABLE EFFECT
OF CONCENTRATION
IN
HUMAN
387
SERUM
2
GRADIENTS AND ABSOLUTE CONCENTRATIONS OF PARTIALLY ON POLYMORPHONUCLEAR LEIJKOCVTE MIGRATIONS
LEF
Percentage LEF preparation Percentage LEF preparation in upper compartment
0
1
in lower compartment
5 Locomotion
PURIFIED
10
25
indexb
0 1 5 10 25 a The LEF used in this experiment was prepared by isoelectic focusing of normal serum and had an absorbance at 280 nm of 1.66. b Mean + SD in pm.
below it, with sedimentation coefficients of 7 S and 10.5 S, respectively. This finding is consistent with the previously reported sedimentation characteristics of the cell-directed inhibitor (14). The sedimentation coefficient of LEF was found to be 3.0 S when calculated from the position of human albumin and 2.8 S when calculated from the position of human IgG, using the method of Martin and Ames (23). The molecular weight was estimated to be approximately 37,000 + 600 (mean + SE). TABLE EFFECT
OF HEAT
Experiments
TREATMENT
AND
DIALYSIS
3
ON THE LEIJKOKINESIS-ENHANCING
Cells treated with:
ACTIVITY
OF SERUM
Locomotion index (km)”
P value@
A
1 No serum 2 Unheated serum (7%) 3 Heated sentme (7%)
19.3 + 0.9 37.2 e 2.4 20.8 f 1.2
.4-l vs A-2
B
1 Nondialyzed serum (7%) 2 Dialvzed serumd (7%)
31.1 2 1.4 32.8 -+ 1.2
B-l vs B-2 >0.0.5
a Mean * SD. b Probabilities were calculated by the Student’s t test. c Sera from four normal individuals were tested for leukokinesis-enhancing activity both before and after treatment at 56°C for 1 hr. Each assay was performed in triplicate, and three fields in each of the three filters were counted. d Serum was dialyzed against sodium phosphate-buffered saline (pH 7.4, ~=0.15) for 24 hr at 4°C. The control nondialyzed serum was diluted to compensate for the volume change of dialyzed serum.
388
MADERAZO
ET
AL.
% MIGRATION ALBUMIN
12
3
4
IgG
5
6
.---.
7
8
A
9
280
. 1.0
i
. 0.8
8 2
10
FRACTIONS FIG. 1. Sucrose density gradient characteristics of LEF. Whole serum was ultracentrifuged in a 5-20% sucrose density gradient and the fractions obtained were tested for LEF activity. Response of PMN treated in medium alone was used as the control and the percentage change from this value [( +) for stimulation or LEF activity, and C-) for inhibition] was calculated from response of cells treated with 50 ~1 of the various fractions in the upper compartment.
Separation of LEF Activity from CDI Activity Examination of the various fractions obtained by ammonium sulfate fractionation showed that most of the LEF activity, which is found in the nonsoluble fractions between 40-60% saturation, is distinctly separable from the celldirected inhibitor (CDI) activity, which is found in the precipitate obtained at 20-40% of saturation (Fig. 2). DEAE-cellulose chromatography was also found to be useful in separating LEF from the CD1 of serum. When either whole serum or the O-60% ammonium sulfate precipitate of whole serum was dialyzed against 0.01 M potassium phosphate buffer, pH 6.8, and applied to a DEAE-cellulose column equilibrated against the same buffer, the CD1 passed directly through the column, whereas the LEF was retained. The LEF activity was recovered when the column was eluted with a 0.5 M phosphate buffer which was applied to elute all the protein from the column in a single peak. This simple experiment demonstrates that DEAE-cellulose can be used to separate CD1 and LEF (which have opposite effects on leukocyte migration) from each other at low ionic strength, and that the LEF activity can be recovered at high ionic strength (Fig. 3). Isoelectric focusing of whole serum revealed the presence of LEF activity in fractions with isoelectric points of 4.2 to 5.6 (Fig. 4). More than 11 distinct protein bands were detected within this zone by staining with Coomassie brilliant blue. Eluted fractions in the higher pH range (pH 7-9) contained CD1 activity. These preliminary investigations demonstrated that standard protein purification procedures can be used to separate LEF from its CD1 antagonist normally
LEUKOKINESIS-ENHANCING
FACTOR
IN
HUMAN
SERUM
389
-30 1
FRACTIONS (% SATURATION
OF APMONIUM
SULFATE)
2. LEF activity in ammonium sulfate fractions of human serum. Serum was fractionated seqwntially by increasing ammonium sulfate concentrations. The percentage change from the control value was determined as described in the legend to Fig. 1. FIG.
present in human serum. The CDI-free LEF preparations were found to be suitable for further studies of LEF functional activity on PMN as described below. Work is now in progress to refine the application of these techniques so that they may be used as part of a practical purification procedure for LEF. Stimulation of Monocyte Migration
by Serum and by Partially PuriJied LEF
To determine whether LEF has activity on the migration of cells other than PMN, normal human monocytes were tested in the presence of normal human serum and in the presence of partially purified LEF obtained by DEAE chromatography of normal serum. Each chamber contained 5 x lo5 monocytes. Techniques for assessment of monocyte activity were similar to those performed with PMN, except that B-pm pore-size filters were used and chamber incubation was continued for 2 to 3 hr. When the effect of normal serum was tested in the absence of chemotactic attractant in the lower compartment, the locomotion indexes obtained at 2 hr were 10.4 ? 0.9 pm (mean * SD) without normal serum in the upper chamber, and 13.2 -c 1.8 pm with 10% normal serum in the upper chamber [26% increase, Student’s t = 3.425, degrees of freedom (df)= 10, P < 0.011. On the other hand, when the chemotactic response to zymosan-activated normal serum in the lower compartment was measured, the locomotion indexes obtained were 11.8 + 0.6 pm without normal serum in the upper compartment, and 15.7 + 1.0 pm with 10% normal serum in the upper compartment (33% increase, t = 10.032, df = 16, P < 0.001). Thus, 10% normal serum in the cell compartment increases both the non-
390
MADERAZO
El- AL.
ENiiANCFMENT
_L
INBIBITION
10
20
30
90
100
110
120
FRACTIONS
FIG. 3. DEAE-cellulose chromatography of LEF. The nonsoluble fraction of normal serum at 60% ammonium sulfate saturation was applied to a DEAE column. LEF activity was found in fractions with 0.5 h4 phosphate buffer (pH 6.8) (arrow). The initial peak (fractions 12-20) was rich in IgG and contained inhibitor activity.
chemotactic and the chemotactic responses of normal human monocytes. In separate experiments using a 3-hr incubation time, an increased response was also observed when 0.1 ml of an LEF fraction (Azso = 0.77) prepared by DEAE chromatography was used with monocytes in the cell compartment, with activated normal serum in the lower compartment. In this case the LI was 22.7 + 0.8 pm without LEF and 31.5 t 1.7 pm with LEF (39% increase, f = 14.768, df= 15;P < 0.001).
LEUKOKINESIS-ENHANCING
FACTOR
IN
HUMAN
SERUM
392
MADERAZO
ET AL.
Effect of LEF on Phagocytosis and NBT Reduction by PMN Pooled partially purified LEF fractions (A 28,,= 2.25) obtained by DEAE chromatography of whole serum enhanced phagocytosis of opsonized zymosan by PMN as determined by NBT reduction, when compared with PMN treated with Medium 199 alone (AA,,, = 0.52 + 0.02 without LEF versus 0.55 -+: 0.01 with LEF, t = 3.171, df = 4, P < 0.05). Phagocytic activity was also augmented by treatment of the cells with normal serum (AA,,, = 0.58 t 0.01 with serum, P < 0.01). These results indicate that LEF is not only a specific stimulator of leukocyte migration, but also a weak stimulator of phagocytosis. Dysfunction of Leukocyte Migration Due to a Deficiency of Serum LEF Activity Three patients, one child (PI) and two adults (P2 and P3), were evaluated because of recurrent Staphylococcus aureus infections (PI and P2) or recalcitrant periodontitis (P3). The association between leukotactic dysfunction and periodontal disease was shown previously (11). Initial studies of granulocyte migration showed no intrinsic abnormality of the cells (Table 4, Experiment A), but a serum-associated defect resembling cell-directed inhibitor activity was noted (Table 4, Experiment B). Further studies of their sera, however, demonstrated some differences from the previously described dysfunctions caused by increased inhibitor activity (14). For instance, unlike patients with high serum cell-directed inhibitor activity, where washed cells continue to be abnormal until reincubated with normal serum (14), washed cells of these patients showed normal nonstimulated
TABLE 4 PRESENCE OF A SERUM-ASSOCIATED DEFECI OF LEUKOCYTE MICRAIION IYX PATIENTS WITH RECURRENT OR CHRONIC INFECTIONS" Materials Upper compartment cells + serum
Experiment A
B
added to:
Locomotion Lower compartment
1 2
Normal Patient’s
+ none + none
Medium Medium
199 199
3 4
Normal Patient’s
+ none + none
Bacterial Bacterial
factor factor
I 2
Normal Normal
+ normaId + patient’s
Medium Medium
199’ 199
3 4
Patient‘s Patient’s
+ normal + patient’sd
Medium Medium
199 199
Patient 1
index (pm)*
Patient 2
30.1 t 8.0 28.1 -r 4.9 (>0.5) Not done Not done
29.6 _f 27.4 + (>O.l) 43.1 * 42.1 k (BO.7)
1.3 2.1
52.9 2 2.1 36.2 2 5.4 (
49.0 k 1.9 35.1 2 4.6 (
4.3 1.4
Patient 3 21.4 ? 22.5 A (>0.5) 23.1 ? 23.5 r (>0.5)
1.5 1.8 2.0’ 2.0c
40.4 + 3.9 25.8 + 4.5 (
@Normal controls were tested simultaneously with each experiment to correct for day-to-day variability of the leukotactic assay. The control and patient’s values were compared and the significance of the difference was determined using Student’s f test. Numbers in parentheses are P values. b Mean + SD.
LEUKOKINESIS-ENHANCING
FACTOR
IN
HUMAN
393
SERUM
and chemotactic responses (Table 4, Experiment A). Secondly, LEF-deficient sera from all three patients lost their abnormality and became indistinguishable from normal serum after storage at -90, -10, and 4°C for 1 to 2 weeks. This reversal towards normal after storage was confirmed in all three patients by simultaneous testing of aged and fresh sera. The most convincing evidence that these sera were deficient in LEF rather than possessing high inhibitor activity was obtained by heat treatment of sera at 56°C for 1 hr. As shown in Table 5, heat treatment reduced the serum leukokinetic activity of all serum samples (both normal and abnormal). Activity of heated normal sera and LEF-deficient set-a were reduced to approximately similar levels, whereas that of inhibitor-rich sera were reduced to significantly lower levels. The explanation of this observation is that because LEF is heat labile, heat treatment of the serum permits one to measure the activity of unopposed heat-stable cell-directed inhibitor. DISCUSSION
The data presented above demonstrate the presence in normal serum of a stimulator of leukocyte migration. This factor, the leukokinesis-enhancing factor (LEF) is heat labile and nondialyzable. It can be fractionated from whole serum using a variety of techniques including ammonium sulfate fractionation, DEAEcellulose chromatography, and isoelectric focusing in granulated G-75 gel. The sedimentation coefficient of LEF is estimated to be about 2.9. The spectrum of activity of LEF appears to be wide and includes stimulatory effects on PMN and monocyte locomotion and on PMN phagocytosis. Every known activity of the cell-directed inhibitor appears to have a corresponding antagonistic LEF activity. The stimulatory effect of LEF on monocytes suggests its importance as a regulator for both acute and chronic inflammatory reactions. In TABLE HEAT
TREATMENT
OF SERA
CELL-DIRECTED
Diagnosis
Serum Donor
5
T O DIFFERENTIATE
LEF
INHIBITION
DEFICIENCY
FROM
INCREASED
ACTIVITY
Locomotion
index (prn)O
Unheated
Heatedb
Normal:
Normal 1 Normal 2
52.4 2 0.8 50.7 + 1.6
33.4 k 1.4 29.4 f 1.0
4 LEF:
Patient 1 Patient 2 Patient 3
49.9 2 1.4’ 42.8 2 1.7’ 53.0 2 0.9’
30.0 zt 0.6’ 30.0 2 1.4’ 27.7 k 0.8’
T CDI:
Patient 4d Patient 5 Patient 6
31.6 ? 1.7 24.2 ” 0.6 25.9 k 0.8
23.6 k 0.8 15.6 k 0.9 19.8 _’ 0.8
a Three fields on each of two filters were counted. Values are mean 2 SD. b Sera were incubated at 56°C for 1 hr. c Stored frozen sera were used. These results indicate complete (patients 1 and 3) or partial (patient 2) reversal of LEF-deficient serum to normal after storage in a freezer for 1 week or longer. See Table 4, Experiment B-2 for the LI values obtained under the same conditions on fresh serum from these patients. d This patient was previously reported [Ref. (14)].
394
MADERAZO
ET
AL.
viva studies with experimental animals should further clarify its role in inflammation. The recognition of the existence of LEF naturally implied the possible existence of abnormalities of leukocyte migration owing to a deficiency of this factor in serum. Such an abnormality has now been identified, as described in the preceding sections, in humans with recurrent or chronic infections. In vitro laboratory observations which were found useful for the differentiation of LEF deficiency from increased inhibitor activity include: (i) heat treatment of sera at 56°C for 1 hr, where there was reduction of stimulatory activity such that the difference between abnormal and normal sera was lost, unlike serum with high cell-directed inhibitor activity where heat treatment produced reduction of PMN locomotion greater than that produced in similarly heat-treated normal serum: (ii) nonstimulated locomotion of PMN treated with LEF-deficient serum and washed was similar to the response of PMN pretreated with normal serum, whereas, nonstimulated response of cells treated with inhibitor and washed (PMN preincubated in serum with high cell-directed inhibitor activity and subsequently washed) was lower than the corresponding control (PMN preincubated in normal serum and washed); and (iii) reversal to normal of LEF-deficient sera upon storage at -90, -10, and 4”C, whereas this was not observed in sera containing high celldirected inhibitor activity. It should be noted that normal serum also showed increased enhancing activity upon storage, indicating that LEF may have an inactive (or less active) precursor from which it is derived in serum, and that apparent deficiencies of LEF can result either from a lack of conversion of this precursor or from a rapid metabolism or destruction of LEF once formed from this precursor. Presumably, there is a net gain of LEF in serum upon storage and this net gain is higher in the three patients with apparent LEF deficiency studied so far. A substance that functionally resembles LEF was described previously by Jacobs and Goetzl in the serum of a patient with recurrent sterile pyoderma gangrenosum (29). Their studies revealed the presence of a migration-enhancing factor found in the serum of the patient, which increased both PMN and monocyte migration. The effect on monocytes was minor compared with the effect on PMN. Based upon results of studies using Sephadex G-200. they estimated the molecular weight of their stimulator to be 160,000. This is different from the LEF we have found in normal serum which has a sedimentation coefficient of approximately 2.9 S and an estimated molecular weight of approximately 37,000. Nevertheless, it is possible that this higher molecular weight LEF of Jacobs and Goetzl does occur normally. The latter LEF preparation and CD1 have molecular weights that are similar and can therefore be expected to be present in the same serum fractions prepared by Sephadex G-200. Because the two preparations have antagonistic activities, inhibitory or stimulatory activity will then be observed in these fractions depending upon the relative concentrations of each of these opposing factors. In normal serum this zone, which is easily detected because of its immunoglobulin G content, is inhibitory. In the patient described by Jacob and Goetzl it is likely that the abnormal elevation of the heavier LEF was sufficient to overcome the activity of normal CDI, and thus, LEF activity became more apparent. This possible contamination of CDI-rich serum fractions with heat-labile higher molecular weight LEF can explain the augmentation of CD1 activity by heat treatment as observed previously by two groups of investigators (17,30).
LEUKOKINESIS-ENHANCING
FACTOR
IN
HUMAN
SERUM
395
Recently, Wilkinson and Allan (31) have shown the chemokinetic effect of bovine serum albumin. This is of interest because LEF activity is frequently found in albumin-rich fractions. However, as shown by the results of the ultracentrifugation studies (Fig. l), maximum LEF activity was not found in the fraction containing the highest concentration of albumin.Additionally, some bovine serum albumin preparations tested in our laboratory do not contain appreciable chemokinetic activity. These findings suggest that LEF is distinct from albumin. The effects of removing albumin from our LEF-rich fractions on LEF activity is presently being studied. The results of these studies should prove or disprove our suspicion that LEF and albumin are not identical. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
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