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(Journal of Leukocyte Biology. 2000;68:58-64.)
© 2000 by Society for Leukocyte Biology

Monitoring of neutrophil priming in whole blood by antibodies isolated from a synthetic phage antibody library

Leo Koenderman, Deon Kanters, B. Maesen, Jan Raaijmakers, Jan-Willem J. Lammers, John de Kruif*,{dagger} and Ton Logtenberg*,{dagger}

Department of Pulmonary Diseases and
* Immunology, University Medical Center and
{dagger} Utrecht Biotechnology Systems, Utrecht, The Netherlands

Correspondence: Dr. L. Koenderman, Department of Pulmonary Diseases, F.02.333, University Hospital Utrecht, Heidelberglaan 100, NL 3508 GA Utrecht, The Netherlands. E-mail: L.Koenderman{at}hli.azu.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophil activation is a multistep process. In vitro activation of neutrophils with semiphysiological activators is optimal only after preactivation or priming with cytokines, chemotaxins, and/or bacterial products. Until now, no antibodies have been developed that can distinguish between resting and (cytokine) primed neutrophils with a sufficient dynamic range necessary for screening clinical samples. We have isolated two human phage antibodies, designated MoPhab A17 and A27, from a synthetic bacteriophage antibody library. These phage antibodies recognize epitopes that are upregulated on neutrophils present in whole blood treated with low priming concentrations of cytokines, such as GM-CSF and TNF-{alpha}. This induction was time- and concentration-dependent and optimal at concentrations that are sufficient for priming functional responses in neutrophils: GM-CSF (10 pM) and TNF-{alpha} (100 IU/ml). PMNs, isolated from the peripheral blood of chronic obstructive pulmonary disease (COPD) patients with a clinical exacerbation, exhibited a partial in vivo primed phenotype. These antibodies promise to be an ideal tool to monitor disease activity in whole blood of patients with inflammatory diseases.

Key Words: priming • detection • antibodies • neutrophils • monocytes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils play an important role in the host defense against invading microorganisms such as bacteria and fungi [1 ]. For the killing reaction, these cells have an extensive machinery of cytotoxic effector mechanisms, including phagocytosis, production of toxic oxygen metabolites initiated by a membrane-bound reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and degranulation of cytotoxic proteins [for a review, see ref. 2 ]. In addition to being cytotoxic, these cells are involved in maintaining inflammatory reactions by the release of cytokines and bioactive lipids, such as platelet-activating factor and arachidonic acid metabolites [3 , 4 ]. Uncontrolled activation of neutrophils plays an important role in the pathogenesis of diseases, such as adult respiratory stress syndrome and septic shock [5 ]. Syndromes associated with neutrophil dysfunctions can have severe clinical consequences [1 ].

Neutrophil activation is a multistep process. It is widely accepted now that optimal activation of neutrophils by semiphysiological activators requires priming by chemotaxins or cytokines. This priming of functional responses in vitro has been described in detail during the last decade [6 ]. Priming of granulocytes in vivo has been made plausible for eosinophils isolated from the blood of patients with allergic diseases [7 , 8 ]. Despite the recognition of the importance of priming for neutrophil responses, relatively little is known about the intracellular signals responsible for this process.

Priming is also poorly defined in the context of expression of cell-surface markers. Some studies describe upregulation of Mac-1 and CD66b, and down-regulation of L-selectin in response to cytokines and chemotaxins [9 , 10 ]. The interpretation of these studies is hampered by the fact that priming and activation are poorly defined. Low doses of cytokines (picomolar range) do not cause activation of the respiratory burst and degranulation of azurophil and specific granules. Instead, they preactivate or prime these responses in the context of formyl peptides [11 ] and opsonized particles [12 ]. Another important drawback for the study of neutrophil priming in vitro in the context of cell-surface marker expression is the induction of marker expression caused by isolation artifacts [13 ].

Here we describe the isolation of two phage antibodies that recognize cytokine-primed neutrophils. These antibodies enable us to monitor priming in whole blood with very low and priming doses of cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-{alpha}


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Ficoll paque and Percoll were obtained from Pharmacia (Uppsala, Sweden). Human serum albumin (HSA) and pasteurized human serum proteins were from the Central Laboratory of the Blood Transfusion Service (Amsterdam, The Netherlands). Peptone 140, select yeast extract, kanamycin sulfate, and RPMI were purchased from GIBCO (Paisley, UK). Polyethyleneglycol 6000, platelet-activating factor, and N-formyl-methionyl-leucyl-phenylalanine (fMLP) were obtained from Sigma (St. Louis, MO). Tetracyclin HCl and Na-ampicillin were from Boehringer GmbH (Mannheim, FRG). Sulfofluorescein diacetate (SFDA) was bought from Molecular Probes (Junction City, OR). All other chemicals were reagent grade. Incubation medium of the cells consisted of NaCl (132 mM), KCl (6 mM), potassium phosphate (1,2 mM), MgSO4 (1 mM), CaCl2 (1 mM), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES, 20 mM), glucose (5.5 mM), and HSA (0.5% wt/vol), pH 7.4.

Monoclonal antibodies and cytokines
Polyclonal sheep anti-M13 (Pharmacia) and polyclonal polycoerythrin (PE)-labeled donkey anti-sheep (Jackson ImmunoResearch, West Grove, PA) were used to visualize phage antibodies in the flowcytometric assays. GM-CSF was purchased from Genzyme (Cambridge, MA), TNF-{alpha} was bought from Boehringer Mannheim, and interleukin-5 (IL-5) was a kind gift from Dr. M. McKinnon (Cell Biology Unit, GlaxoWellcome, Stevanage, UK). Monoclonal antibodies (mAbs) directed against MAC1 (CD11b, clone 44a) and CR1 (CD35, clone 543) were isolated from the supernatants of hybridomas obtained from American Type Culture Collection (Rockville, MD).

Chronic obstructive pulmonary disease (COPD) subjects
Nine patients with unstable, moderate-to-severe COPD from the clinic of the Heart Lung Center (Utrecht, The Netherlands) were used as subjects. Inclusion criterion for entry was a clinical diagnosis of COPD according to the European Respiratory Society standard consensus. Furthermore, the patients had to have a smoking history of at least 10 pack years, an FEV1 < 70% predicted, an FEV1/FVC < 70%, and a reversibility of < 10% of the predicted value in stable conditions. All patients were treated during a hospital admission for a moderate-to-severe clinical exacerbation with two or all three of the following cardinal symptoms: 1) an increase from baseline of sputum production, 2) sputum purulence or shortness of breath, or 3) based on the judgment of the clinician. Patients were allowed to use glucocorticosteroids and bronchodilators at admission. Patients with uncontrolled, severe disease other than COPD contributing to the deterioration were excluded. During hospitalization, subjects were treated optimally according to the recommendations of the ERS consensus on COPD [14 ], and smoking was forbidden during the time of admission. The study was approved by an institutional review board.

Cell isolations
Blood was obtained from healthy donors from the Red Cross Bloodbank (Utrecht, The Netherlands) or from healthy, nonallergic donors from the laboratory staff. Unprimed leukocytes were isolated from heparinized blood as follows: After venapuncture, the blood was directly cooled to 0°C, and cells were kept on ice. Erythrocytes were lysed via ice-cold lysis with NH4Cl, as previously described [10 ]. Remaining leukocytes were washed with ice-cold phosphate-buffered saline (PBS), supplemented with HSA (0.5%, wt/vol.). Total leukocyte preparations were used for the isolation of phage antibodies and in flowcytometric analysis. Lymphocytes and neutrophils were identified according their specific side-scatter and forward-scatter signals [10 ].

Phage antibody library
The semisynthetic phage antibody display library of human scFv antibody fragments has been described in detail elsewhere [15 ]. Briefly, 49 germline VH genes were fused to semirandomized, synthetic, heavy-chain CDR3 regions, varying in length between 6 and 15 amino acid residues. The resulting products were inserted into phagemid vectors, containing seven different light chains of {kappa} and {lambda} subclasses, resulting in a library of 3.6 x 108 MoPhabs.

Strategy for the isolation of phage antibodies directed against primed neutrophils
Isolation of phages directed against primed granulocytes was performed as follows. In short, the phage library (~1011 phage particles) was precleared with resting/unprimed leukocytes from a nonallergic healthy donor (70x106 cells in 10 ml PBS in the presence of 1% milk) during 90 min at 4°C on a rotating wheel to deplete the library from all phages recognizing epitopes present on unprimed cells. Subsequently, the precleared library was mixed with GM-CSF-primed eosinophils (20x106 cells in 10 ml PBS/1% milk) for 90 min at 4°C on a rotating wheel. We used eosinophil rather than neutrophils, because these latter cells are very sensitive for a specific priming caused by isolation artifacts. By using eosinophils, a better chance was foreseen for obtaining antibodies directed against cytokine-induced priming epitopes. The cell-associated phages were isolated from nonbinding phages via two wash steps and a subsequent centrifugation over isotonic Percoll (d 1.030 g/ml, during 20 min, 1000 g at 4°C). Phages were eluted from the cells by incubation in 76 mM citric acid, pH 2.5, during 5 min at room temperature. This whole procedure was performed three times. Subsequently, the positive phages were expanded, essentially as described before, and screened for epitopes on primed cells, which are absent on resting/unprimed cells.

Procedure for staining neutrophils with phages A17 and A27
Blood was collected immediately after venapuncture, kept on 37°C, and immediately treated with buffer (control) or with different amounts of cytokines during different periods of time, as indicated in the figures. Hereafter, the blood was chilled to 4°C, and red cells were lysed in ice-cold isotonic NH4Cl [10 ]. Then, the cells were washed twice and resuspended in incubation medium. For staining of leukocytes, 25 µl of MoPhab was blocked by adding 75 µl of PBS/4% milk powder for 15 min on ice. Leukocytes (5x105) in 100 µl of PBS/HSA (1%, wt/vol) were added and incubated on ice for 90 min. The cells were washed twice in ice-cold PBS/1% HSA. To detect cell-bound phages, the cells were incubated in 50 µl of a 1:200 dilution of sheep anti-M13 polyclonal antibody (Pharmacia) for 45 min on ice, washed twice, and incubated in 50 µl of a PE-labeled donkey anti-sheep polyclonal antibody solution (20 µg/ml) (Jackson ImmunoResearch) for 20 min on ice. After a final wash step, the cells were analyzed in a FACSvantage Flowcytometer (Becton & Dickinson, Mountain View, CA). Neutrophils were identified according to their specific side-scatter and forward-scatter signals [10 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MoPhabs A17 and A27 recognize a priming epitope on neutrophils treated in vitro with GM-CSF and TNF-{alpha}
After three selection rounds (see Materials and Methods), two bacteriophages (MoPhabs) were isolated that recognized an epitope highly expressed on primed neutrophils. These MoPhabs were designated A17 and A27. Figure 1 shows that neutrophils and monocytes present in total blood leukocyte preparations exhibit a low level of expression of the epitopes recognized by the MoPhab A17. In marked contrast, lymphocytes stained negative with MoPhab A17. The next set of experiments was performed to show the effect of cytokine priming on the fluorescein-activated cell sorter (FACS) histogram, showing the expression of epitopes recognized by MoPhab A17. As can be seen in Figure 1a clear induction of expression is present on neutrophils and monocytes. Again, activated lymphocytes were negative for MoPhab A17 staining. Experiments carried out with MoPhab A27 gave similar results except that a relatively high background binding is present to unprimed cells (see also Figs. 2 3 4 5 6 7 ).



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  		Figure 1. Histograms describing the expression of the epitopes on neutrophils (N), monocytes (M), and lymphocytes (L) recognized by the MoPhab A17. Whole blood was incubated with buffer (control) or TNF-{alpha} (100 IU/ml) during 30 min at 37°C. Hereafter, the red cells were lysed in ice-cold isotonic NH4Cl. Subsequently, the white blood cells were washed, stained with the phage antibody A17, and analyzed by flow cytometry. Neutrophils (N), monocytes (M), and lymphocytes (L) were identified according their forward-scatter and side-scatter characteristics. As control, the TNF-{alpha} signal of an irrelevant MoPhab is shown in the neutrophil panel N (striped line). The histograms shown are representative of 24 experiments.

 


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  		Figure 2. The effect of priming by cytokines on the expression of epitopes on neutrophils recognized by the phage antibodies A27 and A17. Whole blood was treated with different cytokines (100 pM IL-5, 10 pM GM-CSF, and 100 IU/ml TNF-{alpha}), and leukocytes were stained with A27 (A) and A17 (B), as described in the legend of Figure 1 . The median values of 24 different experiments were expressed as means ± SE. *Values differ significantly from the control (37°C) value (P<0.001).

 


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  		Figure 3. Dose-response curves of GM-CSF-induced expression of epitopes expressed on neutrophils recognized by MoPhabs A27 and A17. Whole blood was treated with different amounts of GM-CSF for 30 min at 37°C. Hereafter, red cells were lysed, and the leukocytes were stained with MoPhabs A27 (A) and A17 (B). Data are expressed as means ± SE of eight different experiments.

 


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  		Figure 4. Dose-response curves of TNF-{alpha}-induced expression of epitopes expressed on neutrophils recognized by MoPhabs A27 and A17. Whole blood was treated with different amounts of TNF-{alpha} for 30 min at 37°C. Hereafter, red cells were lysed, and the leukocytes were stained with MoPhabs A27 (A) or phage A17 (B). Data are expressed as means ± SE of eight different experiments.

 


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  		Figure 5. Time courses of TNF-{alpha}- (100 IU/ml) and GM-CSF- (10 pM) induced expression of epitopes recognized by MoPhabs A27 and A17. Whole blood was treated with buffer (A, B), GM-CSF (C, D), or TNF-{alpha} (E, F) for different time periods at 37°C. Hereafter, red cells were lysed, and the leukocytes were stained with MoPhabs A17 (A, C, E) and A27 (B, D, F). Data are expressed as means ± SE of eight different experiments.

 


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  		Figure 6. Comparison of kinetics of cytokine/chemotaxin-induced expression of priming epitopes recognized by the phage antibodies A17 and A27, and complement receptor 1 (CR1, CD35, Mab 543) and Mac-1 (CD11b, Mab 44A). Whole blood was treated with TNF-{alpha} (100 IU/ml), GM-CSF (10 pM), and fMLP (1 µM) at 37°C for the indicated timepoints. Hereafter, the samples were immediately chilled on ice, red cells were lysed, and the leukocytes were stained with MoPhab A17 (solid diamonds), MoPhab A27 (open triangles), CD11b (open circles), and CD35 (solid triangles). Data are expressed as percentage of maximum expression to allow proper comparison of the kinetics. Maximally induced values were 1823 ± 254, 548 ± 16, 53 ± 4, and 270 ± 45 for A17, A27, CD11b, and CD35, respectively. Data are expressed as means ± SE of three independent experiments.

 


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  		Figure 7. In vivo priming of neutrophils in the peripheral blood of patients with a clinical exacerbation of COPD. Whole blood was drawn from normal volunteers or patients with COPD, and part of the blood was immediately chilled on melting ice. Hereafter, the cells were analyzed with MoPhabs A17 (A) and A27 (B), as described in the legend of Figure 1 . Data are expressed as means ± SEM of 20 normal controls compared with 11 patients with a clinical exacerbation of COPD. *<0.001 compared with control value (Student’s t-test for independent samples)

 
Figure 2 shows the summary of the results of a series of experiments evaluating priming at the optimal priming concentrations of GM-CSF (100 pM) and TNF-{alpha} (100 IU/ml; n=20). The control incubation of the blood at 37°C (30 min) did not induce a significant change in expression of both epitopes when compared with the control immediately chilled upon venapuncture. Cytokine-induced expression of the epitope recognized by MoPhab A27 was always higher on resting neutrophils compared with the epitope recognized by MoPhab A17.

Characterization of neutrophil priming by GM-CSF and TNF-{alpha} in the context of expression of the priming epitopes recognized by the MoPhabs A17 and A27
Figures 3 and 4 show dose response curves of the expression of priming epitopes induced by GM-CSF (Fig. 3) and TNF-{alpha} (Fig. 4) . Whole blood was treated with different amounts of the cytokines at 37°C. After 30 min, the blood was chilled, erythrocytes were lysed, and the total leukocyte preparation was treated with the MoPhabs A17 and A27. TNF-{alpha} priming of neutrophils was optimal at 10 IU/ml and 100 IU/ml for MoPhabs A27 and A17, respectively. GM-CSF priming of neutrophils was optimal at 100 pM and 1 nM for phages A17 and A27, respectively.

Figure 5 shows a time course of GM-CSF (100 pM, C, D) and TNF-{alpha} (100 IU/ml, E, F) induced induction of the priming epitopes on neutrophils recognized by A17 and A27. TNF-{alpha}-induced expression of the priming epitopes expressed on neutrophils was optimal after 30 min and remained high up to at least 60 min. Similar kinetics was found for GM-CSF-treated leukocytes.

The next series of experiments was designed to compare the kinetics with other granule-associated markers. As can be seen in Figure 6 , cytokine-induced (GM-CSF and TNF-{alpha}) and chemotaxin-induced (fMLP) expression of the epitopes recognized by A17 and A27 coincide with the expression of MAC-1 (CD11b/CD18) and CR1 (CD35), albeit with a superior dynamic range.

MoPhabs A17 and A27 recognize primed neutrophils in the blood of COPD patients with a clinical exacerbation
The next series of experiments was set out to evaluate whether MoPhabs A17 and A27 were able to detect neutrophil priming in vivo in patients with a neutrophil-driven inflammatory process. As is shown in Figure 7 , neutrophils isolated from patients suffering from COPD with an acute clinical exacerbation exhibited a primed phenotype. A significant difference in binding MoPhabs A17 and A27 to these cells was observed compared with cells from healthy controls. These data demonstrate that the neutrophil compartment of these patients exhibits a phenotype comparable with cells that have interacted with cytokines.

After activation of whole blood with fMLP, a clear, increased binding of A17 and A27 was observed in cells of healthy control subjects and COPD patients (data not shown). These data are consistent with a partial-primed phenotype of the cells obtained from COPD patients. Moreover, these data clearly demonstrate that the MoPhabs A17 and A27 can be used to monitor priming in vivo in patients with a neutrophil-driven inflammatory disease such as COPD.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Activation of phagocytes is a multistep process. Preactivation or priming is a prerequisite for several functions in neutrophils activated by semiphysiological activators such as fMLP [16 ]. Despite the recognition that priming is an important process in phagocyte physiology, surprisingly little is known about the molecular mechanisms that lead to this response. This is partly caused by the difficulties in obtaining unprimed cells. Several environmental factors are important in this respect, including contamination of media and chemicals with endotoxins and effects of shear during density-centrifugation steps with the use of Ficoll and Percoll. Elegant studies by Kuijpers et al. [13 ] show that human neutrophils become activated during isolation of these cells as evidenced by the expression of activation markers. These authors used mAbs directed against granule-associated activation markers, such as the {alpha}m chain of MAC-1 and the glycoproteins recognized by mAbs clustered in CD63 and CD66b. Moreover, they studied the shedding of L-selectin, which is a sensitive marker for neutrophil activation. Unfortunately, these markers turned out to be poor monitors for priming processes in whole blood induced by low amounts of cytokine [10, 17, and unpublished results]. Likely, this is caused by the fact that these processes are, in part, linked to the degranulation of specific granules in neutrophils. CD66b is present in specific granules, and fusion of these granules with the plasma membrane caused upregulation of the protein. So far, no evidence has emerged to support the fact that degranulation of azurophilic or specific granules occurs in peripheral blood of patients with chronic inflammatory pulmonary diseases.

We set out experiments to develop mAbs for priming epitopes from a synthetic phage-antibody library for various reasons. First, until now, no mAbs have been described that recognize primed neutrophils with an appreciable dynamic range in vitro (i.e., large difference in expression between nonprimed and primed cells). Second, the putative priming epitope might not be very immunogenic, which precludes development of useful m{phi}Abs. The synthetic phage library circumvents this latter problem because of the partial randomization of the complementarity-determining region (CDR) III. Finally, the speed of the synthetic-phage technology accelerates the development of new antibodies dramatically. Indeed, we were able to isolate two different phage antibodies that specifically recognize primed cells. The characteristics of these antibodies are superior to the pattern of recognition by established activation markers for neutrophils such as MAC1, CD63, CD66b, and ICAM-1 (unpublished results).

The identity of the epitopes remains to be elucidated. Unfortunately, single-chain Fv fragments of phages A17 and A27 turned out be very poor blotting antibodies and do not immunoprecipitate a protein from membrane preparations. This precludes straightforward-expression cloning of this epitope. Experiments to monitor priming of granulocytes in whole blood performed with these scFv preparations lead to similar conclusions as the experiments performed with MoPhabs (D. Kanters, unpublished results).

The observation that phagoyctes (neutrophils and monocytes), in marked contrast to lymphocytes, express the priming epitopes makes it tempting to speculate on a granular localization of the epitope. As is mentioned above, it is not very likely that the epitope is only present in azurophilic and/or specific granules. A third compartment consists of the small, secretory vesicles localized in the vicinity of the plasma membrane, as described by Borregaard and coworkers [18 ]. This compartment also contains elements of the NADPH oxidase and part of the MAC-1 and CR1 molecules [18 ]. These vesicles are thought to associate with the plasma membrane in response to cytokines and heat shock [for a review, see ref. 19 ]. To explore this hypothesis further, we studied the kinetics of cytokine/chemotaxin-induced expression of the epitopes recognized by A17 and A27 in comparison with the expression profile of CR1 and MAC1/CR3. As can be seen from Figure 6 , the kinetics of expression of all these markers is remarkably similar. Also, heat shock induces the expression of these markers (D. Kanters, unpublished results). These data further support the hypothesis that these markers are present within a similar localization in the neutrophil. The expression range of the phage antibodies is ~100 times better compared with the CD35 and CD11b antibodies. Detailed immunohistochemistry with scFv fragments will elucidate the precise localization of the epitopes within the cell.

Antibodies that recognize neutrophil-priming epitopes may be clinically relevant, because they allow the monitoring of cytokine/chemokine action in the peripheral blood. Priming occurs in vivo in the peripheral blood by proinflammatory cytokines as a first step to recruit granulocytes to the side of inflammation.

Several lines of evidence suggest that priming precedes extravasation and activation of granulocytes in the tissues. Integrins need priming signals for upregulation of their functions [for recent reviews, see refs. 20 and 21]. For eosinophils, indeed it has been made plausible that these cells are primed in the peripheral blood of allergic patients in the context of migration and activation of the respiratory burst before actual activation [7 , 8 ]. The occurrence of primed granulocytes in peripheral blood in patients with different diseases associated with inflammation might predict a possible worsening of the clinical condition. We set out a proof-of-concept study to evaluate the possibility that in vivo priming of neutrophils occurs in vivo in COPD, because of the indications that a neutrophil-mediated inflammatory process contributes to the pathogenesis of this disease [22, and refs. therein]. Our experiments with the cells from COPD patients show a clear, in vivo, partially primed phenotype during a severe clinical exacerbation. This supports the concept that circulating cytokines prime inflammatory cells in the peripheral blood in inflammatory lung diseases. It is tempting to speculate now that priming in vivo is a multistep process and that only the first steps of priming occur in the peripheral blood. Fully primed cells will marginate and extravasate into the tissues and thereby leave the peripheral blood. Flow cytometry with labeled antibodies directed against priming epitopes will contribute to the monitoring of the inflammatory processes in these patients in a rapid and reliable fashion.

Received August 6, 1999; revised February 22, 2000; accepted February 24, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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L. Galibert, G. S. Diemer, Z. Liu, R. S. Johnson, J. L. Smith, T. Walzer, M. R. Comeau, C. T. Rauch, M. F. Wolfson, R. A. Sorensen, et al.
Nectin-like Protein 2 Defines a Subset of T-cell Zone Dendritic Cells and Is a Ligand for Class-I-restricted T-cell-associated Molecule
J. Biol. Chem., June 10, 2005; 280(23): 21955 - 21964.
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E{-}J.D. Oudijk, J{-}W.J. Lammers, and L. Koenderman
Systemic inflammation in chronic obstructive pulmonary disease
Eur. Respir. J., November 2, 2003; 22(46_suppl): 5s - 13s.
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