CD13 and CD10 Expression of Granulocytes as

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6 ________________________________ CD13 and CD10 Expression of Granulocytes as Markers for the Functioning of the Immune System Quantification of the Expression of Membrane Moleeules Using 1: 1 Labeled Monoe/onal Antibodies and Flow Cytometry

Patrick Schroeter, Arnim Sablotzki, and Dagmar Riemann . \\1

Summary

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HLA - DR expression on monocy tc s as a marker für Ihe functioning of the immune syslem is known 10 be scverely depressed in immunodeficiency. Up 10 now. olhe r markers for Ihe rllfl ction 01' the immune system are sea rce. In the peripheral bl ood 01' patienls with ope n hea rt surgerl' the expressi on 01' the membrane peptidases. ncprilysinlCD IO. and aminopeptidase NICD 13. was determined o n granulocytes in comparison to the mo noe)'ti c HLA-DR expression. Wc uscd the QuantiBRITET\I flolV cytornetry system. whie h )'i clds an absolute antigen expression value (antibodics bo und per cell) and mal' bc useful in stan­ dardizing surface antigen expression a nalysis. This system makes use 01' a highll' purified phycoe rl'thrin-Iabeled antibo
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in terms of calibration reagents. standardizcd sampie preparation. and data analysis to ensure intcrlaboratory comparability and reproducibility. The design and use of ealibration beads labeled with predefined amounts of dye allows instrument-independent expression of fllloreseence intensity in units of mole­ cules of equivalent soluble flllorochrome (1). This method was refincd by the combined lIse of such standards with monoelonal antibodies (MAb) conjllgatcd II I with phycoerythrin (PE), allOwing translation of !luorescence intensity into numbers of antibodics bound pcr cell (ABC) (2). Commercially available I: I labeled MAb have been used in patient diagnostics for several years nolV. In HIV infection. for example, antigen density changes in CD38 expression of CD8-positive T-cells may be an important indicator of disease progression (3). In an earlicr study, we quantitatively determincd the simliitaneous expression of HLA-DR and aminopeptidase N/CD 13 on peripheral blood monocytcs of patients suffcring major trauma (4). Trauma paticnts are known to have an early onset depression of the overall cellular immune response. which is associated with a high rate of infection and mortality. Several investigators have shown that the expression of monoeyte HLA-DR as a marker for their antigen-present­ ing eapacity is severely impaired and eorrelates with the outeome of paticnts (5.6). The mcchanisms involved in thc regulation of HLA -DR include shedding of HLA-DR from the cell surfaee and rcgulation of HLA-DR gene transcription (7). We described that thc recovery of thc attenuated monocytie HLA-DR expression after trauma was aecompanied by a strong increase in the membrane enzyme aminopcptidase N/CD 13 on monocytes (4). Fourteen days after trauma, the monocytie expression of CDI3 was still mueh highcr than in normal voluntcers used as a control group. Membrane peptidases are multifunc­ tional moleeules. Not onty do these enzymes hydrolyse small peptide media­ tors, resulting in aetivation or inaetivation; thcy also function as receptors and as moleeules participating in cell motility and in adhesion to cxtraccllular matrix (8). Therefore. the exprcssion of thcse antigens on leukocytes eould be lIseful for charaeterizing the function of the immune system. We investigatcd the expression of two membrane peptidases on granulocytes in patients lIndcr­ going open he art surgery. Using quantitative immunofluoreseenee and flow eytometry. we showed a similar time-course both of the expression of HLA-DR on monocytes and of the membrane peptidases Aminopeptidase N/CD 13 and ncprilysin/CD I 0 on granuloeytes.

2. Materials 2.1. Specimen Requirements ED1A antieoagulated whole blood is the rcquircd spccimen. After collection, keep blood at room temperature. Slaining and cell lixation must be cornpletcd within 4 h of blood drall' in the case of HLA-DR quantilieation of monocytes

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Flow Cytometric Q(Jantification of S(Jrface Molecules

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(see Note 1). For othcr leukocyte surface molecules, thc stability of antigen cxpression has to bc investigated.

2.2. Patients Fifty four patients (mean age of 64 ± 7.5 yr, 72% wcre mcn) lIndergoing open heart slirgery owing to coronary heart disease or heart valvc rcplaeement were prospcctively enrollcd in this study, which was pClformed with thc approval of the ethics commiUee of the Univcrsity HaIle- Wittenbcrg. Informed consent was obtaincd from the paticnts. Thc paticnts spent an average of 3.4 ± 1.2 d in intensive earc. Patient's mcan whitc blood cell counts ranged from 8.3 to 13.7 cclls/mm.1 with a peak on day I. Preoperatively, as weil as on days 1,3, 7, and 10, 2.7 mL EDTA-trcated \'cnous blood was obtaincd. Fourtccn healthy, agc -matched individuals sen'cd as contmls.

2.3. Reagents

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I. BI) J-oACS HI Lysing Solution (BO Biosciences , San lose, CA). 2. Phosphate-buffered saline (PBS): prcpare 10X stock with 1.37 M NaCI. 27 mM ~ KCI, 100 mM Na,HPO,1' 18 mM KH,P0 4 (adjllst to p1-l7.4 with HCI if nccessary). I ' ., ,, 1, and filtrate hcfor~ storage at 4°('. Prepare workin5!'-' solution by dilution of one part •..1 ," :, \ Id, ~ . ' I' I with ninc P,lriS of filtratcd water. 11 I, .1 -, 3. QuantiBRITe~1 PE heads (RD Bioscicnccs). a set of four precalibrated bead lev - 'H,II ", eis in thc form of Iyophilized pellet to calibrate thc nuorescencc (FL)2 axis in I,I:~~!" ':' :'; ' terms of nlllnbers of PE molcellles. 11;11 , 4. HLA-OR I/I PE (clone L243)/anti COl4 PerCP-Cy5,5 MAb (BO Bioscienecs, ~ cat. no, 340827), 5. COI3 111 PE (clone L138: prepared as a eustomer service. BO Bioscienccs, cat. no. 347830), 6. COIO 111 PE (clone HIIOn: preparcd as a Clistolllcr service. BO Bioscicnecs),

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3. Methods 3.1. Principle of the Test PE-Iabeled beads are commereially availablc in a kit, which contains one tube with a mixture of beads with four different prcdefineu levels of PE. The PE-Iabcled antibouies uscd for staining of surface antigens on blood cclls are at a I: I fluorochrome-to-antibody ratio, This allows determination of the ABC when beaus are run unuer the same photomultiplier anu compensation settings as blood cells (see Notcs 2 and 3). All sampies werc analyzcu on a FACS Calibur (BD Biosciences) using the software CcllQuest™. The monocytcs anu granulocytes were scparateu on thc basis of their forward scattcr and side scatter patterns, anu thc staining with CDI4 was useu to check thc identification of the monocytes. At least 3000 monocylcs were analyzcd per sampie, Auuilionally, 10,000 events in the granulocyte gatc

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Schroeter; Sablotzki, and Riemann

were eounted, The PE f1uoreseenee within the gates was measured as speeifie geometrie mean fluoreseenee intensity 01' the whole population 01' eells (see Note 4). QuantiQuest is a quantitative ealibration feature within the CellQuest aequisition software that ealculates the linear funetion relating fluoreseenee to PE molecules. So with help of the Mierosoft ExeeI™-spreadsheet, geometrie mean fluorescenee was converted into the term ABC.

3.2. Monoclonal Antibody Staining I. Pipel 50 ~L 01' whole hlood into each 01' (thrcc) 5-rnL tuhcs (see Note 5). Add 20 ~L 01' the HLA -DR/CDI4 MAb mixturc to tube I (see Note 6). Add 10 ~tL of CDI3 MAb to tube 2 and 10 ~lL 01' CDIO MAb to tube 3. 2. Incubate for 30 min in thc dark at 4°C (see Note 7). 3. Add 2 mL 01' fACS Lysc (see Note 8), mix, and incubatc for 30 min in thc dark at 4°C. 4. Ccntrifuge for 5 rnin at :WOK and dccant supcrnatant (sce Note 9). 5. Analyzc cells in 0.5 rnL PBS. Store at 4°C in thc dark until measurcmcnt.

3.3. Acquisition and Analysis All sampies were analyzed on a fACS Calibur rM using the software CellQuest (BD Bioseienees). After launehing the aequisition software, all instru­ ment parameters have to be adjustcd for the cclls you want to analyze. One has to make sure that the instrument is eompensated properly (see Note 10). Thc PE-stained beads are run first , at least 10,000 events should be acquired. Plaee an aetive gate on the bead singlet population using forward seatter (FSC) and side seatter (SSC). The instrument setting should remain unchanged with respeet to fluoreseence onee the beads have been run. The analysis 01' the sam­ pies should bc performed using the same settings.

3.3.1. Beads I. Rcconstitute one tube of QuantiBRITE beads with 0.5 rnL 01' PBS and vortex. Run the QuantiBRITE bead tube thresholding on fSC or SSC and colleet 10.000 cvents. Displaya FSC vs SSC dot plot and adjust thc gate around the si nglet bead population. 2. Displaya FL-2 histogram and adjust markers around thc four bead populations. View histogram slatistics (makc sure geometrie means are displayed). 3. C1ick thc "Copy means" button to copy the geometrie means of the four bead peaks from the histogram statisties windoll'. 4. Seleet histogram statistics viell' and choose quantitative calibration from the "Aequirc" menu. Enter the lot-specific PE/bcad values from the flyer in the kit. 5. Cliek "Calibrate" for CellQuest to perform regression analysis and to display the slope, interccpt. anel eorrelation coeffleicnt. Savc thc doeUlnent 6. 00 not acljust photomu ltipliers or compcnsation after acquiring these cvents.

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