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A simple assay to measure phagocytosis of live bacteria.

Phagocytosis, the engulfment and degradation of particles such as bacteria, is an essential function of the immune system. The impairment of phagocytosis leads to severe infections, as can be seen in patients with primary and secondary immunodeficiencies (1-6). Phagocytosis assays currently used in medical diagnostics analyze only the engulfment of dead bacteria, the surfaces of which are loaded with fluorescent stains (7,8). Other phagocytosis tests can be applied to measure only bacterial degradation, but these methods are based on counting viable bacteria after phagocyte lysis and thus are costly in terms of time and effort (9). Therefore, there is a need for a simple and reliable assay to measure both bacterial engulfment and bacterial degradation under conditions similar to those in vivo.

Enhanced green fluorescent protein (EGFP) is a mutant form of the wild-type green fluorescent protein from the jelly fish Aequorea victoria that has been optimized for brighter fluorescence and higher expression in cells (10). The EGFP excitation maximum is 488 nm, and its emission maximum 507 nm (10). We hypothesized that engulfment of living bacteria with intracellular expressed EGFP would cause an increase in phagocyte green fluorescence representing the engulfment of these cells. Over time this fluorescence intensity will decline owing to bacterial destruction and release of EGFP to an environment characterized by low pH and activated proteases. The percentage reduction of the green fluorescence will reflect the degradation ability of the phagocytes. Based on this idea we attempted to create a simple and reliable flow cytometric assay to measure both engulfment and degradation of living bacteria by monocytes and granulocytes in human whole blood.

To generate EGFP-expressing bacteria we transformed Eschericha coli with the plasmid vector pEGFP. In this vector, the lac promoter expresses the EGFP fusion protein, and the vector contains an ampicillin resistance gene for propagation and selection of transformed bacteria. (For detailed information on the generation of EGFP-expressing E. coli and control-E. coli, see the Data Supplement that accompanies the online version of this Brief Communication at http://www.clinchem.org/content/vol54/issue5.)

We established the test for use on whole blood samples to avoid time-consuming cell separation and also to perform the test under near in vivo conditions. Based on results of preliminary studies (see Supplemental Fig. 1a in the online Data Supplement), we used heparinized venous blood, diluting 100 [micro]L of a freshly drawn sample with 400 [micro]L of assay medium (for data about the influence of whole blood dilution on test values, see Supplemental Fig. 1b in the online Data Supplement). The assay medium was composed of RPMI 1640 medium supplemented with heat-inactivated fetal calf serum, L-glutamine, heparin, and penicillin/ streptomycin. We used penicillin/streptomycin in the assay medium to inhibit further bacterial growth during the assay (see Supplemental Fig. 1c in the online Data Supplement). Next, we added 10 [micro]L of freshly recultivated EGFP- or control-E. coli suspension, corresponding to a bacterial concentration of 6 X [10.sup.6] E. coli/mL in the assay (for data about the influence of bacterial number on test values, see Supplemental Fig. 1d in the online Data Supplement). Samples were then carefully resuspended and incubated at 37[degrees]C and spun at 600 rpm for 15 min ([t.sub.15 min]) or for an additional 3 hours and 45 min at 37[degrees]C without rotation ([t.sub.4 h]). Based on kinetic study results, which showed that the maximum green fluorescence intensity of monocytes and granulocytes incubated with EGFP-E. coli was highest after 15 min and thereafter diminished continuously, we assigned 15 min ([t.sub.15 min]) to be the measuring time-point for bacterial engulfment and 4 hours ([t.sub.4 h]) to be the measuring time-point for bacterial degradation (see Supplemental Fig. 2 in the online Data Supplement).

After 15 min and 4 hours, respectively, engulfment and degradation reactions were stopped by cooling the samples for 10 min on ice, followed by washing with ice cold PBS (1X, without calcium and magnesium). The remaining cell pellet was then incubated with the antibody mix anti-CD14 Phycocyanin 5 (PC5)/anti-CD33 PC5 for 20 min at 4[degrees]C in darkness, followed by erythrocyte lysis and leukocyte fixation via incubation with 1 mL fluorescence-activated cell-sorting (FACS) lysing solution for 15 min. After removal of the lysing solution by centrifugation for 5 min at 200g and decantation of the supernatant, another washing step with PBS supplemented with heat-inactivated fetal calf serum and sodium azide was performed. Cells were then analyzed using a FACS Calibur (Becton Dickinson), measuring at least 50 000 phagocytes by setting a live cell gate according to the characteristic scatter properties of monocytes and granulocytes. A detailed protocol of the assay and further information about human blood sample origin, instrumental setup of the flow cytometer, and statistical analysis can be found in the online Data Supplement.

The commercially available Phagotest[R] reagent set (Orpegen Pharma), which analyzes the uptake of FITC-labeled heat-inactivated E. coli, was frequently used in parallel with the EGFP assay described herein. To allow better comparison between both tests, the Phagotest reagent set was used in a manner partially modified from the manufacturer's instructions (as described in the online Data Supplement). In samples from both patients and healthy individuals, FITC intensity of monocytes and granulocytes in the Phagotest reagent set increased beyond that of our assay after 15 min (data not shown).

Data analysis was performed as follows. First, phagocytes were separated from lymphocytes using respective forward- and side-scatter properties. Then, monocytes were distinguished from granulocytes by means of CD14 and CD33 expression and respective side-scatter properties (Fig. 1A). Next, the mean green fluorescence intensities (mfi) of monocytes and granulocytes incubated with EGFP- and control-E. coli were analyzed (Fig. 1, B and C). Bacterial engulfment and degradation were then calculated using the following formulas:

bacterial engulfment (mfi) = specific mfi ([t.sub.15 min])

= [mfi.sub.EGFP-E. coli] ([t.sub.15 min]) - [mfi.sub.control-E. coli] ([t.sub.15 min])

bacterial degradation (%) = specific mfi ([t.sub.4 h])/specific

mfi ([t.sub.15 min]) X 100, where specific mfi ([t.sub.4 h])

= [mfi.sub.EGFP-E. coli] ([t.sub.4 h]) - [mfi.sub.control-E. coli] ([t.sub.4 h]).

It should be noted that the changes seen in phagocytic green fluorescence intensities are most probably caused by bacterial engulfment and degradation, respectively. However, these data can also be interpreted as initial bacterial association and subsequent dissociation from the surface of the phagocytes.

Next we investigated intraassay and interassay variances, the influence of blood storage before test initiation on test values, and the stability of the samples before flow cytometric measurement. CVs were significantly lower than 20% for all evaluation parameters, indicating that our assay was reproducible. For example, the mean (SE)/CV for engulfment of monocytes was 56.2 (6.9)/12.3 for sample 1, and 76.7 (4.2)/5.5 for sample 2; engulfment by granulocytes was 55.4 (2.9)/ 5.3 for sample 1, and 64.3 (1.8)/2.8 for sample 2; degradation by monocytes was 52.6 (7.4)/14.1 for sample 1, and 29.2 (2.0)/6.8 for sample 2; and degradation by granulocytes was 66.0 (2.5)/3.9 for sample 1, and 80.6 (0.7)/0.9 for sample 2 (n = 5/sample). Interassay values can be found in Supplemental Fig. 3 in the online Data Supplement. Furthermore, we found that the storage condition of blood samples before test initiation should be constant, and flow cytometric measurement should be performed immediately after completion of the test procedure, because phagocytosis values decrease with time (see Supplemental Fig. 3 in the online Data Supplement).

[FIGURE 1 OMITTED]

Subsequently, we analyzed bacterial engulfment and degradation by human blood monocytes and granulocytes from 12 healthy individuals. In contrast to granulocytes, the monocytic engulfment decreased with age (r = -0.614, P < 0.05, Spearman rank correlation; see Supplemental Fig. 4 in the online Data Supplement). Interestingly, in both monocytes and granulocytes bacterial degradation was negatively correlated with age (monocytes: r = -0.661, P < 0.05, granulocytes: r = -0.697, P < 0.05, both Spearman rank correlation; Fig. 1D).

Wethen investigated whether the test detected differences between healthy individuals and patients with secondary immunodeficiencies. We measured the phagocytosis of monocytes and granulocytes from immunosuppressed kidney transplantation patients and patients with postoperative sepsis in immunoparalysis [a state in which the immune system is not able to successfully fight off infections (11-13)] and compared these results to those for samples from respective age-matched healthy individuals. As shown in Table 1, granulocytes from septic patients in immunoparalysis demonstrated significantly decreased bacterial engulfment. Furthermore, monocytes from kidney transplantation patients showed significantly decreased bacterial degradation. Interestingly, no significant differences were found between healthy individuals and the analyzed patients as detected with the Phagotest reagent set (data not shown).

We have shown that this flow cytometric assay can measure the engulfment and degradation of live bacteria by human blood monocytes and granulocytes. By means of this assay we detected significant differences between healthy controls and patients with secondary immunodeficiencies. These differences may contribute to the increased incidence of infection complications seen in these patients (14,15).

It would be interesting to test whether our assay would reveal subtle engulfment values, but severely diminished values for the degradation capacity, in both monocytes and granulocytes in patients with chronic granulomatous disease caused by deficiency in NADPH oxidase activity (16). Another interesting application would be the analysis of patients undergoing therapy with anti-tumor necrosis factor [alpha] (17).

Grant/Funding Support: We would like to thank the Deutsche Forschungsgemeinschaft for financial support of our work.

Financial Disclosures: None declared.

Acknowledgment: We would like to thank Elizabeth Wallace for accurately proofreading the manuscript.

References

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(3.) Serlenga E, Garofalo AR, De Pergola G, Ventura MT, Tortorella C, Antonaci S. Polymorphonuclear cell-mediated phagocytosis and superoxide anion release in insulin-dependent diabetes mellitus. Cytobios 1993;74:189 -95.

(4.) Hornef MW, Wick MJ, Rhen M, Normark S. Bacterial strategies for overcoming host innate and adaptive immune responses. Nat Immunol 2002; 3:1033- 40.

(5.) Lei B, DeLeo FR, Hoe NP, Graham MR, Mackie SM, Cole RL, et al. Evasion of human innate and acquired immunity by a bacterial homolog of CD11b that inhibits opsonophagocytosis. Nat Med 2001;7:1298-305.

(6.) Ojielo CI, Cooke K, Mancuso P, Standiford TJ, Olkiewicz KM, Clouthier S, et al. Defective phagocytosis and clearance of Pseudomonas aeruginosa in the lung following bone marrow transplantation. J Immunol 2003;171:4416-24.

(7.) Foukas LC, Katsoulas HL, Paraskevopoulou N, Metheniti A, Lambropoulou M, Marmaras VJ. Phagocytosis of Escherichia coli by insect hemocytes requires both activation of the Ras/ mitogen-activated protein kinase signal transduction pathway for attachment and beta3 integrin for internalization. J Biol Chem 1998;273: 14813-8.

(8.) Grunwald U, Fan X, Jack RS, Workalemahu G, Kallies A, Stelter F, Schutt C. Monocytes can phagocytose Gram-negative bacteria by a CD14-dependent mechanism. J Immunol 1996;157: 4119-25.

(9.) Cuffini AM, Tullio V, Giacchino F, Bonino A, Mandras N, Bianchi N, et al. Improved phagocyte response by co-amoxiclav in renal transplant recipients. Transplantation 2001;71:575-7.

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(11.) Docke WD, Randow F, Syrbe U, Krausch D, Asadullah K, Reinke P, et al. Monocyte deactivation in septic patients: restoration by IFN-gamma treatment. Nat Med 1997;3:678-81.

(12.) Wolk K, Hoflich C, Zuckermann-Becker H, Docke WD, Volk HD, Sabat R. Reduced monocyte CD86 expression in postinflammatory immunodeficiency. Crit Care Med 2007;35:458-67.

(13.) Docke WD, Hoflich C, Davis KA, Rottgers K, Meisel C, Kiefer P, et al. Monitoring temporary immunodepression by flow cytometric measurement of monocytic HLA-DR expression: a multicenter standardized study. Clin Chem 2005;51: 2341-7.

(14.) Tolkoff-Rubin NE, Rubin RH. The interaction of immunosuppression with infection in the organ transplant recipient. Transplant Proc 1994;26: 16-9.

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(16.) Pallister CJ, Hancock JT. Phagocytic NADPH oxidase and its role in chronic granulomatous disease. Br J Biomed Sci 1995;52:149-56.

(17.) Philipp S, Wolk K, Kreutzer S, Wallace E, Ludwig N, Roewert J, et al. The evaluation of psoriasis therapy with biologics leads to a revision of the current view of the pathogenesis of this disorder. Expert Opin Ther Targets 2006;10:817-31.

DOI: 10.1373/clinchem.2007.101337

Heike Bicker, [1] Conny Hoflich, [1] Kerstin Wolk, [2] Katrin Vogt, [1] Hans-Dieter Volk, [1] and Robert Sabat [2] *

[1] Institute of Medical Immunology and [2] Interdisciplinary Group of Molecular Immunopathology, Dermatology/Medical Immunology, University Hospital Charite, Berlin, Germany; * address correspondence to this author at: Interdisciplinary Group of Molecular Immunopathology, Dermatology/Medical Immunology, University Hospital Charite, Campus Charite Mitte, Chariteplatz 1, D-10117 Berlin, Germany. Fax +49 30 450 518964; e-mail robert.sabat@charite.de.
Table 1. Bacterial engulfment and degradation by peripheral
blood monocytes and granulocytes from kidney transplantation
patients and septic patients in immunoparalysis compared
to respective age-matched healthy controls. (a)

 Kidney transplantation patients
 vs age-matched healthy controls

 Healthy
 Patients controls
Parameter (n = 7) (n = 7) P

Engulfment, mfi
 Monocytes 55.2 (6.06) 53.4 (9.52) NS
 Granulocytes 27.7 (3.21) 32.3 (3.09) NS
Degradation, %
 Monocytes 39.9 (5.96) 68.4 (11.40) <0.05
 Granulocytes 57.1 (7.13) 77.2 (3.62) NS

 Sepsis patients in immunoparalysis
 vs age-matched healthy controls

 Healthy
 Patients controls
Parameter (n = 7) (n = 7) P

Engulfment, mfi
 Monocytes 27.9 (5.72) 43.5 (4.73) NS
 Granulocytes 17.5 (3.91) 36.1 (4.25) <0.05
Degradation, %
 Monocytes 34.4 (6.81) 53.8 (6.40) NS
 Granulocytes 53.5 (4.92) 68.8 (6.02) NS

(a) Engulfment and degradation of EGFP-E. coliby monocytes
and granulocytes analyzed with the phagocytosis assay as
described in the text. Data are given as mean (SE)
(P indicates significance level between patient and
respective healthy control group; NS, not significant,
Mann Whitney U-test)
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Title Annotation:Brief Communications
Author:Bicker, Heike; Hoflich, Conny; Wolk, Kerstin; Vogt, Katrin; Volk, Hans-Dieter; Sabat, Robert
Publication:Clinical Chemistry
Date:May 1, 2008
Words:2395
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