1994 84: 1640-1649   

 P Eggleton, B Ghebrehiwet, JP Coburn, KN Sastry, KS Zaner and AI Tauber effects of free ligand on activated neutrophilsCharacterization of the human neutrophil C1q receptor and functional

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Characterization of the Human Neutrophil C l q Receptor and Functional Effects of Free Ligand on Activated Neutrophils

By Paul Eggleton, Berhane Ghebrehiwet, John P. Coburn, Kedarnath N. Sastry, Ken S. Zaner, and Alfred I. Tauber

The partial characterization and expression of the Clq recep- tor (Clq-R) in relation to other complement receptors pres- ent on the surface of neutrophils has been examined, as well as the effects of free C l q on cell function. A polyclonal anti- Clq-R antibody recognizes a 68-kD neutrophil surface pro- tein. Clq-R expression was not upregulated upon warming, priming, or exposure to FMLP, but decreased after exposure to phorbol myristate acetate (PMA), because of shedding of the receptor into the extracellular medium, as detected by enzymelinked immunosorbent assay. CR3 and CR1 ex- pression was upregulated from intracellular pools after cell stimulation by PMA. No evidence of intracellular pools of Clq-R was found, as assessed by immunoblotting of subcel- lular fractions. But Clq-R appeared to be expressed early in cell differentiation, was detected on undifferentiated HL-60 cells, and like CR3 expression, increased upon 5 days differ- entiation towards a neutrophil lineage. However, Clq-R ex- pression decreased upon additional culture, whereas CR3 expression continued to increase. A large variation in the percentage of peripheral cells expressing C l q receptors in donors was observed, ranging from 13% to 10050, con-

I Q IS A 460-kD molecular weight glycoprotein’ that circulates in the plasma as a calcium (Ca’+)-dependent

complex with C lr2 and C l s2 (Cl).’ Upon activation of C 1, Cl r , and C Is are disassembled by C l inactivator, leaving complex-bound C Iq with its collagenous region exposed and potentially available for binding to C lq-R. The physiologic levels of free monomeric C l q available for binding to its receptor is uncertain. C l q not only binds avidly to antigen- antibody complexes, but presumably has nonspecific associ- ations with denatured proteins, soluble, altered Igs and other polyanionic substances.’ A wide range of values for C l q in human serum have been reported. Total serum C l q levels measured by radial immunodiffusion (RID) of about 200 pgl mL have been reported: with a range of 34 to 246 pgl d.’ However, under high ionic conditions that aid in the dissociation of C l q from immune aggregates or soluble al- tered Igs, a lower mean concentration of 136 yglmL has been reported.6 Upon reevaluation of the RID technique by

C

From The William B. Castle Hematology Research Laboratory, Boston City Hospital; the Departments of Pathology and Medicine, Boston University School of Medicine, Boston, MA: and the Depart- ments of Medicine and Pathology, State University of New York, Stony Brook.

Submitted August 18, 1993; accepted May 12, 1994. Supported in purr by Public Health Service Grunfs No. GM-37590,

HLB-33565, A133130, and a grant from Applied Immune Sciences Inc.

Address reprint requests to Alfred I . Tauber, MD, Boston Univer- sity School of Medicine, S-Building, Room 301, 80 E Concord St, Boston, MA 021 18.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1994 by The American Society of Hematology. 0006-4971/94/84M-0024$3.00/0

1640

trasting with CR3 receptors that exhibited less variability. Interactions between free monomeric Clq and neutrophils were also studied. Incubation of stimulated neutrophils with 10 to 100 pglmL Clq resulted in a further increase in CR3 expression and adherence to albumin-coated surfaces. Staphylococci opsonized with low quantities of Clq (0.1 to 1 pg/mL) mediated a moderate and sustained respiratory burst in neutrophils, whereas a burst of similar magnitude was generated only with free Clq at concentrations 10- to 100-fold higher. Stimulation was only partially inhibited if cells were first treated with anti-Clq-R antibody, sug gesting other C l q binding proteins may be present on the cell surface. In summary, neutrophil Clq receptor is approxi- mately 68-kD, exhibits varying expression on different sub- jects, and is not upregulated from intracellular stores on exposure to soluble stimuli. Stimulated, but not resting, neu- trophils selectively respond to raised levels of free Clq, re- sulting in altered cell function and enhanced CR3 receptor expression. These studies thus suggest complex roles for Clq in neutrophil function. 0 1994 by The American Society of Hematology.

Ziccardi and Cooper,’ a value of 70 p g l d was obtained. No value for free C l q has been made, but Ziccardi and Cooper’ hypothesized that relatively small amounts of free monomeric C l q are present in serum. Sjoholm et a1* have suggested between 9% and 30% of C l q in a free state in normal serum. Irrespective of methodology, free C l q has been found to be elevated in various pathophysiologic condi- tions!.* I”

Specific receptors for human C lq (Cl q-R) are expressed on the surface of B lymphocytes,” monocytes/macro- phages,” endothelial cells,” fib rob last^,'^ platelets,”~” and neutrophils.18 Most of these early findings were based on radioligand binding studies of a putative cell surface receptor for Clq. Clq has been found to be capable of releasing superoxide and other free radicals from the Previous studies have shown that homogeneous monomeric free C l q binds only to a small number of neutrophils under physio- logic conditions,’* and monomeric ‘2’I-Clq binding specifi- cally to the Clq-R is reduced by 74% under physiologic conditions.’* However, when C l q is bound to latex beads or immobilized on plastic, it apparently binds to the neutrophil under normal ionic strength conditions, as will C l q bound to pathologically relevant compounds such as immune com- plexes, viruses, and lipopolysaccharides.2” It is believed such binding may lead to some structural alteration in the C I q molecule that facilitates binding of the ligand to its receptor. In contrast, ErdeiZ4 has shown that free unbound C l q binds strongly to pre-B-cell lymphomas, cell lines of myeloid origin, and to ~ 5 0 % of mature B-cell lymphomas, showing that physical alteration in the structure of the ligand may not always be necessary for binding.

The present study was designed to provide further molecu- lar characterization of the Clq-R and determine its distribu- tion on human neutrophils. In addition, we have reevaluated the interaction of monomeric C 1 q on resting cells, as typical of previous studies, but also examined, for the first time, the

Blood, Vol 84, No 5 (September l), 1994: pp 1640-1649

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HUMAN NEUTROPHIL C l q RECEPTOR 1641

effect of free Clq on the function of stimulated neutrophils under physiologic conditions.

MATERIALS AND METHODS

Materials. Dulbecco’s phosphate buffered saline (PBS) with and without calcium and magnesium (lox); FMLP, phorbol myristate acetate (PMA), superoxide dismutase, ferricytochrome-c, human se- rum albumin, diisopropyl fluorophosphate, Phosphatadylcholine-@- acetyl-y-0-alkyl (platelet-activating factor; PAF), Dextran (503-kD molecular weight) were purchased from Sigma Chemical CO (St Louis, MO). Ficoll-Hypaque was obtained from Pharmacia Fine Chemicals (Piscataway, NJ). Monoclonal antibodies (MoAbs) to CR1 (CD35) was purchased from Becton Dickinson (Mountain View, CA), CD15, IgG-l, and IgG-2a were obtained from Sigma and antibody purified from an OKMI cell line was a gift of Dr M. Wilkinson (Hunterian Institute, London, UK). Fluorescein isothiocy- anate (FITC)-labeled rabbit and mouse antihuman IgG and IgM were purchased from Jackson Immunoresearch Laboratories (West Grove, PA).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) analysis and sucrose-gradient ultracentrifugation of Clg prepurations. Hemolytically active Clq was purchased from Sigma. The preparations were examined by SDS-PAGE under re- duced conditions in the presence of 4 m o w urea, to ensure homoge- neity of the preparation. As expected, three distinct bands of 30 kD or less were observed. In addition, Clq gels were immunoblotted and probed with antifibronectin to ensure no contaminating fibro- nectin was present in the commercially purchased Clq samples. Linear sucrose gradients of 5% to 31% (wt/vol) sucrose were pre- pared in PBS in a mixing chamber with a constant volume. Commer- cially purified samples of Clq, IgG, IgM, and anti-Clq in 200 pL voiumes were applied to 11.5-mL gradients and centrifuged at 41,000 rpm at 4°C for 18 hours using a TH641 Sorvall rotor in a Sorvall OTD55B ultracentrifuge. Two hundred microliter fractions were collected by puncturing the bottom of the tube with an ISCO piercing mechanism (Lincoln, NE); samples were obtained by pump- ing of the gradient from the base of the gradient with 50% (wt/vol sucrose). Protein content was monitored using an on-line absorbance monitoring apparatus (280 nm) attached to a fraction collector. Pro- teins content within the gradient were confirmed using the Pierce BCA protein assay (Pierce, Rockford, IL). A single narrow homoge- neous peak was seen for Clq, IgG, and IgM at different positions within each gradient. Clq complexed deliberately with anti-Clq IgG has the greatest sedimentation rate as expected (Fig 1).

Isolation andpreparation of neutrophils and HL-60 cells. Whole blood was taken from consenting healthy volunteers into sodium citrate, and neutrophils were isolated by erythrocyte sedimentation in dextran citrate, followed by hypotonic lysis and Ficoll-Hypaque density gradient centrifugation as previously described.25 The iso- lated neutrophils were then resuspended in PBS without calcium and magnesium at 4°C until required, then they were suspended in the appropriate buffer. Neutrophil membranes were prepared from isolated, unstimulated neutrophils disrupted by nitrogen bomb cavi- tation and the subcellular fractions separated into bands by density centrifugation on Percoll gradients. Each band was assayed for marker enzymes to assume their respective homogeneity by pre- viously published methods2? the LY band (primary granules) by mye- loperoxidase, the @ band (secondary granules) by vitamin B,z-bind- ing proteins, and the y-fraction (plasma membrane) by alkaline phosphatase. Other workers have shown the presence of intracellular vesicles that also contain alkaline phosphatase; therefore, care is required to avoid cross-contamination of each fraction, or further separation by free-flow electrophoresis can eliminate the pr~blem.~’ Each fraction was then stored in liquid nitrogen. The y band, which

represents the plasma membrane, was analyzed by SDS-PAGE on 5% to 15% acrylamide gels from both resting and 10” m o a FMLP- stimulated cells. HL-60 cells obtained from the American Type Cul- ture Collection (ATCC) were maintained at l X IO6 cells/mL in RPMI medium supplemented with L-glutamine containing 20% fetal bovine serum and 200 U/mL penicillin and streptomycin. After maintaining the culture for 1 week in a humidified atmosphere of 95% air/5% COz, cells were stimulated to differentiate towards a neutrophil lineage by addition of RPMI containing 1.3% dimethyl sulfoxide (DMSO). Cells were sampled at 0, 5, and 7 days after DMSO induction.

Western blot of Clq-R. The two polyclonal antibodies raised against purified Clq-R originating from Raji cells used in these studies for immunoblotting and fluorescence-activated cell sorting (FACS) analysis were prepared as previously described.’* Either 5 , 10, 20, or 40 pg of each neutrophil subcellular samples were sus- pended in sample buffer (62 mmoVL TRIS-Cl, pH 6.8, 2% SDS, 10% glycerol, and 0.01% bromophenol blue with 5% 2-mercaptoeth- anolz9) and incubated for 4 minutes at 100°C and then applied to individual lanes of a 5% to 15% polyacrylamide gel; electrophoresis was performed in TRIS-buffered saline (TBS) at a constant current of 35 mA on an 8- X 7-cm gel. Molecular-weight standards pur- chased from Sigma, were run simultaneously. The separated proteins were then electrotransferred onto polyvinyl difluoride (PVDF) trans- fer membrane (Immoblin-P; Millipore Corp. Bedford, MA) using a Semiphor transfer unit. After saturation of the nonspecific sites with 5% nonfat dry milk-TBS overnight, the proteins were probed with a 1:30 dilution of rabbit antihuman Clq-R antibody in TBS-5% milk overnight or 1:250 dilution of mouse antihuman CD15 MoAb or 1:250 dilution of CR3 in TBS-5% milk for 1 hour at 37°C. The strips were then washed in 20 mmolL ”RIS-HCl, pH 7.5,0.14 mow L NaCl containing 0.4% Tween-20 (TBS-Tween) and then incubated for I hour in an antirabbit or antimouse peroxidase conjugate IgG antibody diluted 1:1000 in TBS-Tween with 5% milk. After washing five times in TBS-Tween, the immunoblots were exposed to ECL immunoassay substrate reagent (Amersham International plc, UK) for 1 minute to detect any signal, and then the membranes were exposed to x-ray film (XAR-film, Eastman-Kodak, Rochester, NY).

Analysis of Clq-R, CRI, and CR3 expression on neutrophils by Jlow cytometry. Human neutrophils (1 X lo6) were resuspended in PBS with calcium and magnesium (pH 7.4) and were incubated at 37°C for 15 minutes with either PMA (100 pg/mL), FMLP (lo” mmol/L) or PBS. The cells were then immediately washed in 4°C PAB (phosphate buffered saline without Caz+ and Mg2+, containing 0.1% bovine serum albumin (BSA), 0.02% sodium azide, and 1% human heat inactivated serum), before incubation with antibodies raised against Clq-R, CRl, or CR3 prepared in the same buffer. In addition to the experiments outlined above, neutrophils in some experiments were incubated with the above stimuli at 4°C as well as at 37°C before addition of the above antibodies. In other experi- ments, free Clq (100 to 1 pg/mL) was also incubated with the neutrophils under resting and Stimulated conditions before addition of the antibodies. All cells were resuspended in 100-pL volumes of the different antibodies tested prepared at the following concentra- tions: in the case of CRl, 0.3 &l00 pL; CR3, 5 pg/100 pL; Clq- R, 5 pg/100 pL; CD15,0.4 and mouse isotype (0.4 pg/lOO pL) controls IgGl and IgGZa, 0.4 pg/lOO pL. Cells were incubated on ice for 30 minutes, washed twice with cold PAB, and pelleted by centrifugation. The pellets were resuspended in 100 pL of either FITC goat-antirabbit IgG or FITC goat-antimouse IgG (1 pg/mL) for 30 minutes at 4°C. Cells were then washed twice with cold PAB and fixed with 1 % paraformaldehyde. Fluorescent staining was assessed using a FACScan flow cytometer (Becton Dickinson) and 5,000 events were acquired. Data analysis was performed on the population of fluorescent cells, and subtracting out the autofluores-

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1642 EGGLETON ET AL

clq - Clq + anti-Clq

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

Fraction no

cence of an unstained control to determine the mean fluorescent intensity (MFI) of positive cells for each antibody of interest.

Enzyme-linked immunosorbent assay (ELSA). Neutrophils at a concentration of 107/mL PBS were incubated at 37°C for 15 minutes in the presence and absence of either FMLP (lo” moVL) or PMA (100 ng/mL). Aliquots of the cell suspension media were then plated in microtitre wells in sodium carbonate buffer (pH 9.6) at 4°C over- night. Plates were washed in PBSK’ween 20 and nonspecific binding sites blocked with 5% milWl00 mmoVL glycine. Polyclonal rabbit antibody against the human Clq receptor (1:200 dilution) was added to each well for 2 hours at 37°C followed by 1:lOOO horseradish peroxidase-conjugated antirabbit IgG. After washing, 150 pL of 3,3‘,5,5’ tetramethyl benzidine peroxidase substrate was added and the color allowed to develop for 20 minutes. The reaction was termi- nated by adding 100 pL 2N H2S04 and the plate read at 450 nm. BSA was used as an irrelevant protein and rabbit nonimmune IgG was used as a control for polyclonal antibodies.

Superoxide production. The basal and Clq-mediated respiratory burst of neutrophils was measured by the 0; dismutase-inhibitable reduction of fenicytochrome c as previously described?’ Briefly, neutrophils were exposed to aliquots of heat-killed Staphylococcus aureus (ATCC 29213) that had been treated with various concentra- tions of Clq (100 to 0.01 pg/mL-final concentration) or PBSl BSA (100 pg/mL-final concentration) or opsonized with undiluted serum for 30 minutes at 4°C. and were used in a final ratio of 25 bacteria:cell. The coated bacteria were washed twice in PBS before use. Noncomplexed Clq (10 to 0.1 pg/mL) was also used as a respiratory burst stimulus. PMA (100 ng/mL)-stimulated neutrophils were used as positive controls and consistently generated between 300 to 500 nmoles 0; over a 30-minute period. The change in absorbance at 550 nm was monitored over a 30-minute period and the amount of 0; generated after 30 minutes was recorded in a UVI vis double-beam spectrophotometer (Perkin-Elmer model 559) and the rate of 0; generation expressed as nmol 0;/30 min/106 cells calculated from an extinction coefficient for reduced cytochrome c of 21.2 x 1o3(m01~)-’ cm”.

Measurement ofneutrophil adhesion. Neutrophil suspensions (5

Fig 1. Analyaia on 31% to 5% (wtlvoll suwoae gradient of 200 pL of various proteins in PBS. Samplea (200 pL) of the fractions were teated by BCA protein aaray.

X lo5 cells in 50 @well) were incubated on either BSA (5 pgl mL), human fibronectin (30 pg/mL), or human laminin- (10 pg/mL) coated 96-well plates, and cell adhesion assayed by the absorbance method of Kueng et al.” Briefly, neutrophils were added to each well with and without various concentrations of Clq, mol/L Fh4LP, or PBS. After 30-minute incubation at 37T , nonadherent cells were removed by vigorous washing in PBS and the adherent cells fixed by adding 100 pL of 10% formaldehyde solution for 10 minutes at room temperature. The plates were then washed six times in deionized water and stained by adding 50 pL of 0.5% crystal violet in 0.85% (wt/vol) NaCl in 25% (vol/vol) ethanol for 10 mi- nutes; excess stain was washed off and the adherent cells solubilized in 0.5% SDS in 0.5 m o m acetic acid, before reading optical ab- sorbance at 550 nm. Neutrophil adherence to the various protein- coated surfaces was expressed as a percentage of cells adhering to nontreated plastic control wells, which was regarded as 100% adherence.

Statistical analysis. Statistical analysis of the data expressed as mean 5 SEM was done by paired and unpaired t-test. The signifi- cance threshold was set at P < .OS for all comparisons.

RESULTS

Identification and expression of the neutrophil CIq-R. Western blot analysis of 5, 10, 20, and 40 pg of neutrophil plasma membrane protein ( y fraction) using a 1:30 dilution of polyclonal anti-Clq-R antibody, identified a single im- munoreactive band with an apparent molecular weight of 68 W, under reducing conditions (Fig 2).

To determine if alteration in Clq-R expression on the neutrophil cell surface follows a similar pattern to CR1 and CR3 receptors as assessed by flow cytometry, neutrophils were isolated, maintained at 4°C and pretreated with selected stimulatory and priming agents as indicated in Tables 1 and 2. After labeling and staining with the various antibodies, the expression of each complement receptor was analyzed

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HUMAN NEUTROPHIL C lq RECEPTOR 1643

Table 2. Effect of PAF Priming on Clq-R, CRl, and CR3 ReCeDtOr ExDression

MFI FMLP-Induced 0, Production

Treatment Clq-R CR1 CR3 tminllO' cells)

None 141 t 22 21 c 2 130 2 20 27c 4nmol lo - * mol/L PAF 137 t 22 34 -c 4 217 c 16 42 2 5 nmol lO""ol/L PAF 131 t 14 26 c 6 163 2 10 29 c 10 nmol

Neutrophils (10' cells) were incubated at 37°C for 15 minutes with and without PAF. Separate aliquots were assayed for complement receptor expression, or exposed for an additional 15 minutes with 10" mol/L FMLP and superoxide assayed. The results represent the mean of three separate experiments t SD.

S l b r n ~

Clq-R Mr x 10'

Fig 2. Western blot analysis of neutrophil plasma membrane ( y fraction) probed with polyclonal rabbit lgG antiserum raised against purified Raji cell Clq-R. Lanes l, 2, 3, and 4 show binding of a 1:30 dilution of the anti Clq-R t o 5, 10, 20. and 40 p g of protein, respec- tively; lane 5 shows proteins used as molecular-weight standards: myosin heavy chain, 200 kD; Escherichia coli-galactosidase, 116 kD; phosphorylase a, 97.4 kD; BSA, 66 kD.

on a Becton Dickinson FACScan. Cells kept at 4°C for the duration of the experiment displayed a mean fluorescent in- tensity for Clq-R, CRI, and CR3 of 137 U, 20 U, and 133 U, respectively. Both CR 1 and CR3 receptor expression appeared to increase upon warming of the isolated cells to 37"C, consistent with the finding of Van Epps et a13' who showed that both buffy-coat and Ficoll-Hypaque-isolated

Table 1. Expression of Clq-R, CR1, and CR3 Receptors in Stimulated and Unstimulated Neutrophils

MFI'

Treatment Clq-R CR1 CR3

0°C 137 -c 22 (3) 20 2 2 (3) 133 2 13 (3) 37°C 114 2 15 (8) 28 2 3 (8)* 184 2 18 (8)* 37°C + PMA 53 2 13 (7)t 35 t 4 (6)§ 310 c 42 (711

Numbers represent the MFI for each stained population. The MFI for unstained cells or FITC-labeled cells were 3.7 2 0.4 and 3.3 2 0.8, respectively. Isotype-matched control MoAbs for FITC lgGl and IgG2a always gave MFI values of less than 10. Each value represents the mean t SEM for the number of subjects given in parenthesis.

t Treatment of neutrophils with PMA (10 ng/mL) at 37°C signifi- cantly reduced the MFI of Clq-R expression, P < .01.

*Warming of the cells did not significantly enhance any comple- ment receptor expression.

§ Treatment of neutrophils with PMA (10 ng/mL) at 37°C signifi- cantly enhanced the MFI of CR1 and CR3 receptor expression above 0°C control cell levels ( P < .05 and .01, respectively).

cells increased these surface receptors to similar degrees. However, the difference in our study was not statistically significant. Furthermore, warming of the cells had no effect on the 68-kD Clq-R expression.

When cells were stimulated with PMA ( I O ng/mL), the expression of CR3 was increased by twofold to threefold ( P < .05) above cells suspended at 4°C. Only a moderate in- crease in CRI was observed upon stimulation with PMA ( P < .05). whereas PMA treatment significantly decreased Clq- R expression on neutrophils (Table I , P < .01). stimulation of the cells with FMLP (IO" mol/L), as expected, led to a twofold enhancement in both CR 1 and CR3 expression (data not shown), but failed to affect Clq-R expression in any way. Neutrophils were also treated with the superoxide prim- ing agent, PAF (IO-' mol/L and IO"" mol/L), and comple- ment receptor expression analyzed by flow cytometry. Once again, Clq-R expression similarly was not altered by the treatment with PAF. In contrast, IO-' mol& PAF led to an increase in both CRI and CR3 levels with MFI mean values of 21 and 130 to 34 and 217, respectively (Table 2). FMLP- stimulated 0; production rates in neutrophils increased from 27 nmoles to 42 nmoles/mL/min/107 cells after pretreatment with IO-' mol& PAF. This enhancing effect on CRI and CR3 expression and 0; generation deminished substantially when subpriming levels of PAF (IO"" mol/L) were used. Levels of fluorescence observed by analysis of control cells not treated with primary antibodies, or treated with isotype- matched IgG mouse antibodies together with FITC IgG sec- ondary antibody, were consistently less than 10 U (data not shown).

The mean number of purified neutrophils confirmed by dot-plot analysis and visual examination staining positive for Clq-R was also examined in eight different donors and compared with the number of neutrophils expressing CR3 receptors. Approximately 63% -+ 9.6% (mean 2 SEM) of neutrophils stained positive for Clq-R. Whereas a greater number of cells expressed CR3 (86% 2 3.6%). there was no significant difference.

Alteration in the expression of Clq-R expression in pe- ripheral neutrophils and undifferentiated and dicferentiated HL-60 cells. To determine whether the decrease in Clq-R expression from the surface of human neutrophils was caused by shedding of the receptor upon stimulation with PMA, an ELISA assay was used to detect the presence of

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1644 EGGLETON ET AL

*

Treatment

Fig 3. ELISA analysis of soluble Clq-R expression in the cell sus- pension buffer of isolated neutrophils under nonstimulated and stim- ulated conditions. The data are expressed as absorbance values at 450 nm representing the relative amount of receptor released/106 cells, given as mean ? SD from four subjects.

receptor in the cell suspension buffer. Neutrophils were maintained in PBS, or stimulated with mol/L FMLP or 100 ng/mL PMA for 15 minutes at 37"C, and then the cell supernatants were collected and fixed onto microtitre plates. The anti-Clq-R antibody was used to detect the presence of released Clq-R. Cells from four subjects were screened in this way and the results are presented in Fig 3. Cell suspen- sion medium from both nonstimulated and FMLP-stimulated cells showed low mean 2 SD absorbance values for Clq-R (0.13 i 0. I ; 0.13 i 0.18, respectively). In contrast, medium from PMA-stimulated cells showed a twofold increase in C lq-R absorbance values (0.33 -t 0. l), which was significant ( P < .OS).

A series of experiments were performed to study the ex- pression of Clq-R with CR3 during HL-60 differentiation. Undifferentiated and differentiated HL-60 cells (towards a neutrophil lineage) were compared. As shown in Fig. 4, Clq- receptors were present on the surface of 2-week cultured undifferentiated, nonsuperoxide-producing HL-60 cells (MFI = 34); after S days of differentiation with DMSO, the HL-60 cells were able to generate superoxide, and their Clq- R expression increased twofold (MFI = 70). A similar profile was seen with CR3 expression. However after 7 days of culture in the presence of DMSO, the Clq-R expression was observed to diminish to basal levels (MFJ = 37), whereas CR3 expression continued to increase.

Subcellular analysis to identify localization of Clq-R in the neutrophil. Neutrophil subcellular fractions were stud- ied by immunoblotting with the polyclonal rabbit anti-Clq- R to see if the receptor could be detected in cytoplasm gran- ules (Fig 5) ; however, the 68-kD protein was limited to the plasma membrane. In addition, immunoblots of FMLP- stimulated cell membranes were examined, but showed no evidence of an increase in density or subcellular location of the single protein band detected by anti-Clq-R. In contrast,

the CR3 was associated with the plasma membrane fraction, but in resting cells was most evident as a large subcellular pool within the specific granules (P-band) and cytoplasm fraction (S, band). As expected, the control CD15 antibody identified multiple proteins in the plasma membrane and secondary granule fractions.

Modulation of CR3 expression by Clq treatment qf neu- trophils and effect on cell adhesion. C 1 q has been reported to enhance phagocytosis of IgG-opsonized red blood cells via modulation of Fc receptor function in monocytes." Here, the ability of free soluble C l q to increase C Iq-R, CRI , and CR3 expressions on resting and PMA-stimulated neutrophils was examined. Under resting conditions, the presence of free C I q (1 to I00 pg/mL) did not affect the expression of any of the complement receptors as determined by mean fluo- rescence intensity measurements on the flow cytometer. As expected, exposure of the cells to PMA alone (10 ng/mL) resulted in a mean increase in CR3 MFT from 191 -+ 29 (mean i SEM; N = S) to 38.5 2 75, representing a twofold increase in CR3 expression. The simultaneous addition of I O and I00 pg/mL free C l q led to further increases in PMA- stimulated expression of CR3 by 24% and SO% ( P < .OS), respectively. The use of 1 pg/mL C l q or 100 pg/mL human serum albumin did not affect the stimulated level of CR3 expression as determined by flow cytometry. Neither Clq- R or CRI receptor expression in the presence of PMA was enhanced in the presence of Clq at the concentrations tested.

Because CR3 (CD 1 1 b/CD18) is associated with increases in the adherence of cells, the ability of neutrophils to adhere to BSA, fibronectin, and laminin-coated microtitre wells in the presence and absence of free C l q was assessed (Table 3). In the presence of 10 pg/mL C1 q, 72% of neutrophils

%

U)

0

.- c

.-

c

8o 1

l: 20 0

0 Clq-R

CR3

0 5 7

Days in culture

Fig 4. Expression of Clq-R and CR3 during differentiation of HL- 60 cells over 7 days. HL-60 cells were cultured for 14 days at an initial concentration of 1 x 106cells/mL in an undifferentiated state (0 days). Then cells were grown in the same media, but containing 1.3% DMSO to induce differentiation towards a neutrophilic lineage. Cells were harvested at 5 and 7 days and assayed for Clq-R and CR3 expression by flow cytometry.

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HUMAN NEUTROPHIL C lq RECEPTOR 1645

A B

200 - 116 - 97.4 - 66-

45 - 29 -

C D A B C D A B C D

P

Mr x 10' ClQR CR3 CD15

Fig 5. Western blots of anti Clq-R, CR3, and CD15 of neutrophil subcellular fractions. Neutrophils were disrupted by nitrogen cavitation, fractionated over discontinuous Percoll gradients, separated by SDS-PAGE in a 5% t o 15% polyacrylamide gel under reducing conditions, transferred to PVDF transfer membranes, and immunoblotted with 1:30 dilution of polyclonal anti Clq-R, 1:250 monoclonal CR3,1:250 mouse monoclonal CD15, and a 1:250 dilution of a mouse-specific irrelevant lgG as detailed in Materials and Methods. Protein (40 and 20 pg l containing samples of (A) plasma membrane fraction; (B) primary granules; (C) secondary granules; and (Dl cytoplasm fractions were immunoblotted.

adhered to the BSA-coated wells, which was similar to the degree of adherence observed when the neutrophils were incubated in the presence of the chemotactic peptide, FMLP, which is known to upregulate the CR3 antigen complex. There was no increase in adherence of neutrophils in the presence of 1 pg/mL Clq above control levels on BSA- treated wells. Neutrophils allowed to adhere on fibronectin or laminin-coated wells in the presence of 10 pg/mL Clq did not show any significant difference in the level of adherence above PBS-treated control levels.

High doses of free Clq and lower doses of bound Clq induce neutrophil 0-, production. Clq at concentrations

Table 3. Effect of Free Clq on Activated Neutrophil Adherence*

Substratum Agonist X Neutrophil Adherencet

BSA (5 pg/mL) PBS lo-' mol/L FMLP 10 pg/mL C lq 1 pg/mL C lq

10 pglmL Clq

10 pg/mL Clq

Laminin (10 pglmL) PBS

Fibronectin (30 pg/mL) PBS

~~ ~

27.4 2 5 $85.0 t 5 $72.1 2 15

10.0 ? 6 38.9 -c 6

932.0 2 11 48.0 z 9

935.0 t 6

* Plates were treated with BSA, laminin, or fibronectin in PBS with- out Ca' and Mg' for 45 minutes at 37°C as detailed in Materials and Methods.

t Adhesion is expressed as the percentage of adhering neutrophils, determined by crystal violet staining. Adhesion was measured after the cells were incubated for 30 minutes at 37°C in 5% CO, humidified incubator. Each result represents the mean ? SEM determinations from four subjects.

*The difference in the number of adherent cells in the presence of 10.' mol/L FMLP ( P < .01) and 10 pg/mL Clq ( P < .05) is statistically significant compared with PBS-treated control cells.

9 There was no significant difference in the number of neutrophils adhering to laminin and fibronectin in the presence of free Clq com- pared with PBS control.

of 2.2 pg or less per 5 X 10" latex beads have been reported to stimulate chemiluminescence and the hexose monophos- phate shunt, but not 0; production of neutrophils in suspen- ion.'^.^" The relative ability of free and bound Clq to stimu- late neutrophils has now been reexamined by stimulating cells with either Clq-opsonized S aureus. opsonized with between 0.01 and 100 pg/mL Clq or free Clq (ranging between 1 and 100 pg/mL). The presence of free Clq alone acts as an activator of 0; production by neutrophils (Fig 6A). Resting cells generated a basal amount of Or ( 5 t 1.8 nmoles) during incubation at 37°C for 30 minutes; neutro- phils exposed to relatively high doses of free Clq, ie, I O pg/mL and 100 pg/mL Clq over the same period led to the generation of significantly more 0; production by the neu- trophils: 31 ? 1 I and 63 ? 29 nmoles of 0; IO' cells, respectively ( P < .01). One microgram per milliliter of free Clq did not stimulate an increase in 0; above basal levels generated by nontreated control cells in any subjects tested. Next, the ability of Clq to enhance the respiratory burst of neutrophils in the presence of bacteria was determined. As shown in Fig 6B, cells exposed to unopsonized bacteria were stimulated sufficiently to generate a mean OF production of 26 t 4 nmoles (mean ? SEM) over 30 minutes/lO' neutro- phils, whereas neutrophils exposed to bacteria opsonized with fresh human serum generated 75 ? 7 nmoles over the same period. Exposure of neutrophils with bacteria opso- nized with as little as 0.1 pg/mL Clq resulted in the genera- tion of 57 ? l 1.6 nmoles 0; , which represented a significant increase in the level of 0; ( P < .05) achieved by cells exposed to unopsonized bacteria alone. When neutrophils were treated with anti-Clq-R antibody for 30 minutes at 4°C before the addition of 10 pg/mL free Clq, the generation of 0; evoked by Clq was partially inhibited. Cells treated with a control IgG antibody (antihuman CD15) continued to generate increased levels of 0; above PBS-treated control cells. Some enhancement of 0; was also seen when a high concentration of BSA ( I O pg/mL) was used. but this proved

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1646 EGGLETON ET AL

0 1 1 0 100

[Clq] pg/ml alone

1 2 3

Treatment

Fig 6. (AI Stimulation of neutrophil 0; production by free soluble Clq. Neutrophils were warmed to 37°C and exposed to various con- centrations of free soluble C lq and the generation of superoxide recorded after 30 minutes incubation as detailed in Materials and Methods. The results are presented as the mean 2 SEM from experi- ments of three to eight different donors. The rate of 0; produced by cells incubated with 10 and IO0 pg/mL free C lq was significantly greater than nontreated cells (P< .01). (B) Effect of Clq on Staphylo- cocci-stimulated neutrophils.

statistically nonsignificant, (Fig 7); these experiments were performed in the presence of 1,000 U/mL polymyxin B to ensure no basal levels of 0; production over the 30-minute assay period were caused by the presence of endotoxin.

To determine whether the binding site occupied by anti- ClqR was located near the site occupied by Clq on the receptor, nitrocellulose blots of isolated neutrophil mem- brane were pretreated with free Clq and then immunoblotted with anti-Clq-R. A 68-kD band was not observed under these conditions, suggesting bound ligand was occupying the antibody binding site. As a control, MoAb against Clq confirmed the presence of C lq bound to the neutrophil mem- brane protein at 68-kD.

DISCUSSION

Human neutrophils possess a number of complement re- ceptors: CRI (C3WC4b receptor, CD35, 160 to 250 kD) and

CR3 (Mac-l, C3bi receptor, CDllb/CDl8,280 kD) are high molecular-weight proteins that are stored in granules and can be mobilized upon cell activation and subsequently inter- na l i~ed .” .~~ In contrast, the CSa receptor (SS kD) is not upregulated from internal stores upon stimulation, but its presence on the cell surface is reduced after s t im~la t ion .~~ Similar expression characteristics were observed with the 68-kD Clq-R protein. The receptor for Clq has some unique features as compared with CRI, CR3, and the CSa receptor. Complement peptides bind specifically to these latter recep- tors; however, the Clq-R is distributed on a diverse number of cell types, and several forms of the Clq-R have been f o ~ n d . ~ ~ . ’ ~ C l q - R is believed to bind a number of different proteins, such as mannan binding protein, lung surfactant protein (SP-A) and bovine conglutinin, which all show a structural similarity to Clq, each having both collagenous and globular domain^.^' These distinct domains may be re- sponsible for different functional messages upon binding to the Clq-R.’l Clq itself can be found in human serum in three different forms: (1) bound to the C1 complex,” (2) bound to immune complexes,39 or (3) in a free soluble form?’ It is able to bind to the surface of a number of cell types via the Cl q receptor or form complexes or aggregates with other serum proteins in the microvascular environment.

The present study was designed to identify the neutrophil membrane Clq-R, analyze its coexpression in association with other complement receptors under resting and stimu- lated conditions, and examine the effect of free Clq upon neutrophil function under similar conditions. Western blot

40 1 W PBS

to€ ANTI-C1QR ANTI-CD15

ANTIBODY TREATMENT

Fig 7. Effect of anti-Clq-R antibody on Clq-mediated neutrophil respiratory burst. Neutrophils were pretreated with anti-Clq-R anti- body (5 pg/106 PMNs) or control antibody (antihuman CD15 IgG) at the same concentration and then incubated with PBS, Clq, or BSA for 30 minutes at 37°C. 0; was measured as detailed in Materials and Methods. Values are the mean k SEM of between three and six separate experiments. The results were compared by using the Student‘s t-test: the respiratory bursts generated by the Clq-treated cells were significantly reduced in the presence of anti-ClqR anti- body (P < .05), whereas a control antibody (antihuman CD15) did not have an inhibitory effect. BSA-treated cells generated signifi- cantly less (P < .05) superoxide than Clq-treated cells.

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HUMAN NEUTROPHIL Clq RECEPTOR 1647

analysis of plasma membrane extracts of human neutrophils by a polyclonal anti-Clq-R showed the presence of a single protein of 68 kD under reducing conditions. Recent evidence by Guan et al;l have suggested that Clq, via its collagenous domain, can bind to several sialoglycoproteins present on neutrophils, which may play a role in modulation of Clq- R-mediated responses. The number of isolated resting neu- trophils staining positive for the 68-kD Clq-R varied be- tween donors, with a range of between 13% to loo%, depending on the individual, with a mean ? SEM value of 63% ? 9.6% whereas CR3 expression appeared more uni- form, mean 86% ? 3.6% (range, 55% to 99%).

Clq-R expression decreased after treatment of the cells with PMA (Table 1). Other studies using similar concentra- tions of PMA (5 to 10 ng/mL) as a stimulant, have observed upregulation of both CR1 and CR3;' whereas the use of greater concentrations of PMNphorbol dibutyrate (> 10 ngl mL) has resulted in a reduction in CR1, but not CR3 expres- ion.‘''.^^ An explanation for the differing responses of vari- ous complement receptors in response to phorbol esters is not yet fully understood. Immunoblots of subcellular compo- nents of neutrophils failed to detect any granule or cytoplasm stores of Clq-R, making it unlikely that the receptor is inter- nalized or localized in these subcellular compartments. How- ever, Percoll gradient extracts of nitrogen cavitated cells has limited ability to fractionate tertiary and secretory granules that may be a source of the 68-kD membrane protein." Alternatively, reduction in Clq-R surface expression could be caused by shedding of the receptors from the cell surface. This is one mechanism by which CR1 and FcRIII down modulation occurs.45 As shown in Fig 3, the decrease in C lq-R could be explained by release of the receptor into the cell media upon stimulation. This observation, together with the donor variation in Clq-R expression suggests there may be a limited pool of Clq-R that is not replenished upon release from the membrane. Further insight as to the pheno- typic expression of Clq-R was gained from studying the human promyelocytic HL-60 cell line. When HL-60 cells were cultured in an undifferentiated form, both Clq-R and CR3 expression were detectable in low levels (Fig 4), sug- gesting that the Clq-R receptor like the CR3 receptor is expressed at an early stage of development. Induction of HL-60 cell differentiation of cells for 5 days with DMSO led to a twofold increase in both Clq-R and CR3 expression. Evidence that the cells were differentiating normally, was confirmed by PMA-inducible 0; generation. Interestingly, after 7 days of differentiation, the Clq-R expression began to diminish, whereas the CR3 expression continued to increase, suggesting expression of Clq-R occurs early in differentia- tion, and is not further upregulated from an intracellular

The effect of free soluble Clq on neutrophil function un- der resting and stimulated conditions was performed in nor- mal ionic strength buffer. Incubation of neutrophils in sus- pension with various concentrations of Clq, with and without PMA for 15 minutes, led to a significant increase in PMA-stimulated upregulation of CR3 expression above levels achieved by PMA alone, suggesting a role for Clq- R in modulation of other complement receptor expression,

pool.

but only when the cells are in an activated state, as similar concentrations of free Clq added to resting cells did not alter CR3 expression significantly, which is consistent with the recent studies of Goodman and Tenner,*' who similarly observed that free Clq did not alter CR3 expression on unactivated neutrophils. Increased expression of CR3 recep- tors is a marker of increased cellular adhesion, therefore the adherence of neutrophils to protein-coated surfaces in the presence of free Clq was assessed. Work in our laboratory,& and others4' has shown that adherence of neutrophil to un- coated polystyrene itself triggers a sustained respiratory burst, whereas coating of the wells with BSA can reduce the activation partially, whereas adhesion to laminin or fibro- nectin does not lead to respiratory burst stim~lation.4~"~ However, it should be noted that laminin can stimulate a respiratory burst in suspended cells.50 Based on these obser- vations, microtitre wells were coated with either BSA to mimic moderate stimulation, or fibronectin or laminin to allow adherence of neutrophils without inducing a respira- tory burst. As expected, neutrophils incubated in the pres- ence of FMLP led to a significant increase in adherence above control levels. Clq (10 pg/mL) was also a potent stimulator of neutrophil adherence on BSA-coated wells. In contrast, free Clq at the same concentration did not enhance cell adherence above baseline levels on laminin or fibronec- tin-coated surfaces. This supported the notion that free Clq can enhance neutrophil adherence in activated cells, but not in resting ones.

Clq-bound to latex is unable to enhance the generation of 0; in neutrophils in suspen~ion ,~~ whereas neutrophils adhering to Clq-coated plastic wells do generate O;." In our study, the ability of free Clq to stimulate a respiratory burst in both resting and bacteria-stimulated neutrophils was studied. Staphylococci, fully opsonized with fresh serum, generated the greatest respiratory burst, and unopsonized bacteria induced a moderate amount of 0; production during the 30-minute incubation period, but the response was poor compared with serum or Clq-treated bacteria. A low concen- tration of free Clq (0.1 p g / d ) used to opsonize Staphylo- cocci was sufficient to significantly enhance the respiratory burst, suggesting that the quantities of free Clq thought to exist in the circulation may be sufficient, in combination with other opsonizing proteins, to activate the neutrophil respiratory burst synergistically. When used alone, 100 to 1,000-fold higher concentrations of free C lq are required to trigger a respiratory burst of similar magnitude. Such in- creases in free Clq may only arise in inflammatory disease states such as rheumatoid arthritis and primary biliary cirrho- sis, where higher levels of Clq have been reported. As ob- served in other even the ability of surface-bound Clq to evoke a 0; response by neutrophils from different donors has differed significantly. This may be explained by the number of cells from an individual staining positive for the Clq-R, or alternatively the density of the receptor on the cell surface. Addition of the anti-Clq-R antibody led to a 50% reduction in the 0; production induced by 10 pg/mL free Clq, suggesting the burst was mediated partially through a Clq-Clq-R interaction. Qualitative evidence that the anti- body was directly binding to the site occupied by the Clq

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1648 EGGLETON ET AL

protein was provided by immunoblots of the neutrophil membrane proteins. When Western blots were exposed to Clq before immunoblotting with the anti-Clq-R antibody, the ability of the antibody to detect the 68-kD protein was inhibited, whereas control immunoblots confirmed Clq bind- ing to a 68-kD protein. Interestingly, another Clq-binding protein of 126-kD has been isolated from monocytes, neutro- phils, and U937 cells4' that appears to be involved in Clq- mediated enhancement of phagocytosis in monocytes. The presence of other Clq-binding proteins on the cell surface not detected by the antibody used in this study may also play a role in Clq-mediated respiratory burst. In support of this possibility, a marked heterogeneity in the number of neutrophils expressing the 68-kD Clq-R was observed, which is in agreement with previous binding studies per- formed under nonphysiologic ionic strength conditions,18 which noted the number of binding sites for Clq per neutro- phil varied considerably amongst donors, and which supports the already substantial evidence that neutrophils are hetero- geneous with respect to cell membrane proteins and func- tional responsivene~s.~"~~

ACKNOWLEDGMENT

We wish to thank Tanya Mealy for her excellent technical assis- tance in the isolation of subcellular fractions of neutrophils by nitro- gen cavitation.

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