Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kirveskari, J.
Right arrow Articles by Salmi, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kirveskari, J.
Right arrow Articles by Salmi, M.
(Journal of Leukocyte Biology. 2000;68:243-250.)
© 2000 by Society for Leukocyte Biology

Expression of {alpha}4-integrins on human neutrophils

Juha Kirveskari*, Petri Bono*,{dagger}, Kaisa Granfors*, Marjatta Leirisalo-Repo{ddagger}, Sirpa Jalkanen*,{dagger} and Marko Salmi*,{dagger}

* National Public Health Institute, Department in Turku;
{dagger} MediCity Research Laboratory, University of Turku; and
{ddagger} Helsinki University Central Hospital, Department of Internal Medicine, Helsinki, Finland

Correspondence: Dr. Marko Salmi, MediCity Research Laboratory, University of Turku, Tykistökatu 6A, FIN-20520 Turku, Finland. E-mail: marko.salmi{at}utu.fi


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
{alpha}4 Integrins are important adhesion molecules mediating binding of lymphocytes, monocytes, and eosinophils to multiple cellular and extracellular ligands. Mature neutrophils have been recently suggested to express {alpha}4-integrins as well. We studied whether human neutrophils can synthesize {alpha}4-integrins upon activation in vitro or in vivo. Two anti-{alpha}4 mAbs, but not multiple subclass-matched non-binding controls, reacted with granulocytes in an inducer and time-dependent manner. Nevertheless, staining with Ig subclass-specific second-stage reagents surprisingly revealed that commercial anti-{alpha}4 mAbs contain two distinct Igs, the {alpha}4-specific IgG1 and an IgG2a of an unknown specificity. We showed that in vitro inductions used by us and others only induce the binding of nonspecific IgG2a from the commercial HP2/1 to activated neutrophils. By reverse-transcriptase polymerase chain reaction, {alpha}4 mRNA was not detectable in purified neutrophils. Our results show that {alpha}4 integrin protein and mRNA are absent from normal and stimulated human neutrophils.

Key Words: adhesion molecules • cell trafficking • antibody • FACS


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell-cell and cell-matrix contacts are essential for leukocyte trafficking under normal conditions as well as in the setting of inflammation and hence for maintaining our immune defense. Leukocyte extravasation encompasses a multistep cascade including initial tethering, rolling, activation, stable arrest, and transendothelial migration [1 , 2 ]. Many adhesion molecules belonging to distinct superfamiles are needed for different steps. Integrins are a family of at least 20 heterodimeric molecules consisting of non-covalently associated {alpha} and ß subunits [3 ]. On leukocytes, {alpha}4-containing integrins and members of the ß2 family mediate endothelial cell interactions. On lymphocytes, {alpha}4 integrins have a well-established role in tethering and in firm arrest and transmigration, as well as in subsequent adhesive events within the tissue stroma [4 5 6 7 8 ].

There are two {alpha}4-containing integrins on lymphocytes. {alpha}4ß7 Is the principal mucosal homing receptor, which interacts with the gut-selective endothelial adhesion molecule called mucosal addressin cell adhesion molecule-1 [4 , 9 ]. It can also bind with a lower affinity to vascular cell adhesion molecule-1 (VCAM-1) as well as to fibronectin [9 10 11 ]. {alpha}4 Can alternatively pair with ß1 chain to produce VLA-4 (CD49d/CD29) [12 , 13 ]. When compared with {alpha}4ß7, {alpha}4ß1 binds better to VCAM-1 but weakly if at all to mucosal addressin cell adhesion molecule-1, although CS-1 and other motifs of fibronectin are the preferred ligands [8 , 9 , 14 , 15 ].

{alpha}4 Integrins are expressed on multiple leukocyte subsets [16 ]. On lymphocytes {alpha}4ß1 is found on the majority of peripheral blood lymphocyte (PBL) T and B cells, and its expression level is induced after prolonged in vitro or in vivo stimulation [12 , 17 ]. {alpha}4ß7 Is synthesized at highest levels on the gut seeking subpopulation of PBL [11 , 18 19 20 ]. Activation also up-regulates its expression and functional avidity [21 ]. Monocytes strongly express {alpha}4ß1 but {alpha}4ß7 is absent or only detectable at low levels [12 , 22 ]. On granulocytes much less is known about {alpha}4 integrins. Early hematopoietic precursors express {alpha}4 [23 , 24 ]. Eosinophils and basophils synthesize functional {alpha}4ß1 and {alpha}4ß7 that mediate interactions with vascular endothelium [25 26 27 28 ]. In contrast, neutrophils are thought to lose {alpha}4 during the maturation beyond the band stage [23 , 24 , 29 ] and {alpha}4 is traditionally thought to be absent from circulating neutrophils [12 , 16 , 18 , 23 , 25 26 27 , 29 ]. However, induction of {alpha}4 on human neutrophils has also been reported [30 ]. In those experiments a short exposure of purified granulocytes to C5a [31 ] or to various physiological and nonphysiological stimulators in the presence of the microfilament-disrupting agent dihydrocytochalasin B (DHCB) [32 ] resulted in surface expression of {alpha}4 on the majority of polymorphonuclear neutrophils (PMN). Very recently, even nonperturbed blood neutrophils have been reported to display functional {alpha}4 [33 ]. In animal studies, rat neutrophils have been shown to be uniformly {alpha}4 positive and blocking of {alpha}4 significantly reduced immigration of these cells into inflamed joints [34 , 35 ].

The purpose of our study was to analyze whether inflammation known to induce acute granulocyte-dependent reactive arthritis could induce {alpha}4 expression on human neutrophils in vivo. Immunostaining with anti-{alpha}4 and multiple Ig subclass-matched control mAbs showed consistent reactivity on peripheral blood and joint fluid neutrophils in patients with acute salmonella infection with or without reactive arthritis and on in vitro-stimulated granulocytes. Nevertheless, detailed analyses revealed an unexpected and hitherto unknown cross-reactivity of the most widely used, commercially available anti-{alpha}4 mAb HP2/1. Detailed analyses with subclass-specific reagents and reverse transcriptase-polymerase chain reaction (RT-PCR) with purified granulocyte populations suggest that human neutrophils lack {alpha}4.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
mAbs
Two mouse IgG1 mAbs against human {alpha}4 integrin were used: HP2/1 ([17 ] Immunotech, Marseille, France, catalog no. 0764, and from Chemicon International, Temecula, CA) and B-5G10 [12 ]. HP2/1 was purified IgG1 and B-5G10 was obtained in the form of ascites. Control mAbs included subclass-matched (mouse IgG1) mAbs (3G6 against a chicken T cell antigen [36 ]), NS-1 from a revertant of a myeloma cell line [37 ], 4G4 against CD73 [38 ], IVD12 against HLA-DQ (Becton-Dickinson), and 2D10 against human vascular adhesion protein-1 [39 ], which do not bind granulocytes. All control mAbs were grown in serum-free medium and concentrated by ammonium sulfate precipitation. NS-1 was also obtained as ascites fluid from BALB/c mice. Fluorescein isothiocyanate (FITC)-conjugated sheep anti-mouse IgG [F(ab’)2 fragments, adsorbed with human serum protein, catalog no. F2883] was from Sigma Chemical. IgG subclass-specific reagents [FITC-labeled goat F(ab’)2 anti-mouse IgG1 ({gamma}1 heavy chain specific) and IgG2a ({gamma}2a heavy chain specific) were obtained from Southern Biotechnology (Birmingham, AL)]. These second-stage reagents have been raised against affinity-purified mouse IgG1 or IgG2a and cross-absorbed with mouse IgM, IgA, IgG1/IgG2a, IgG2b, IgG3, pooled human sera, and purified human paraproteins. Phycoerythrin-conjugated anti-CD16 mAb was purchased from Becton-Dickinson. Function blocking mAb 10.1 (mouse IgG1) against human CD64 (Fc{gamma}R1) was from Ancell (Bayport, MN).

Cells and patients
Peripheral blood follow-up samples were collected from seven patients with acute Salmonella-triggered reactive arthritis (ReA). They all had a typical clinical course of ReA. The laboratory diagnosis of Salmonella infection was based on positive stool culture in five of seven patients and solely on elevated serum IgM, IgG, and IgA antibodies against Salmonella [40 ] in two patients. All patients were treated with nonsteroidal anti-inflammatory drugs, and one patient also with ciprofloxacin, oral cortisone, and sulfasalazine. Three to five peripheral blood follow-up samples from eight patients with Salmonella infection without ReA were collected from two small outbreaks caused by S. enteritidis [41 ]. The patients diagnosed with S. enteritidis infection were informed about this study and volunteers spontaneously contacted the research group. All these patients had a typical clinical course of Salmonella infection, including high fever (>38.5°C), gastrointestinal pain, and diarrhea, and they had positive stool culture for S. enteritidis. U937 cells were from ATCC.

Venous blood samples from the patients and healthy volunteers were drawn into heparinized Vacutainer tubes. Mononuclear cells were isolated by Ficoll-gradient centrifugation. For PMN separation 10 mL of blood was mixed with 4 mL 20% Dextran T-500. After a 30-min gravitation on bench, the leukocyte-rich plasma was collected and layered onto 56% Percoll (Pharmacia, Uppsala, Sweden). After a 30-min centrifugation at 1200 g, the pellet was resuspended in Ca-, Mg-free Hanks’ balanced salt solution (HBSS). After two washings, the contaminating erythrocytes were lysed by a hypotonic shock in ammonium chloride (0.73% NH4Cl, pH 7.2, +22°C). After immediate restoration of the isotonicity, the cells were washed once more before further experiments.

Synovial fluid was drawn from swollen knee joints when therapeutically indicated. Synovial fluid samples were then centrifuged, the cells were resuspended in Ca-, Mg-free HBSS and divided on top of Ficoll and Percoll density media as described above.

Stimulation of cells
Isolated granulocytes were cultured in vitro in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin in six-well polystyrene plates (Greiner, 1 x 106 cells/well in 1 mL medium, overnight, +37°C in a humidified cell incubator). The cells were stimulated overnight with 100 IU/mL interferon-{gamma} (IFN-{gamma}), 100 IU/mL GM-CSF, 10 U/mL transforming growth factor ß (TGF-ß; all from Genzyme; recombinant IFN-{gamma} was also obtained as a gift from Dr. Ilkka Julkunen, University of Helsinki, Finland) or for 30 min with 1:20 dilution of C5a (our stock, a concentration known to cause a brisk oxidative burst), 0.1–1 µM N-formyl-methionyl-leucyl-phenylalanine (fMLP; Sigma) and 10 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma). In certain experiments, the granulocytes were activated by allowing them to transmigrate through a confluent endothelial layer (EaHy926 cells [42 ]) grown on a Costar polycarbonate filter (Transwell) with 2-µm pores. Cells (5 x106) were added into the upper compartment and allowed to transmigrate for 2 h at 37°C toward the lower chamber containing 0.1–1 µM fMLP, and the cells from the lower chamber were collected. In another set of experiments, 7.5 µM DHCB was added to the cells to disrupt the microfilaments and to induce exocytosis of storage granules [32 ].

IF staining and FACS analyses
Cells (0.5 x 106) were resuspended in the staining buffer [phosphate-buffered saline (PBS) with 2 mM Ca and Mg, 2% fetal calf serum (FCS), and 0.1% NaN3] and pelleted. One hundred microliters of the primary antibody at 10 µg/mL was added for 15 min. After two washings with the staining buffer, the cells were reacted with FITC-conjugated goat anti-mouse IgG at a dilution of 1:200 +5% normal pooled AB serum for another 15 min. Alternatively, FITC-conjugated goat anti-mouse IgG1 or IgG2a (1:500 dilution) with 5% AB serum were used as the second-stage reagents. Thereafter the cells were washed once in the staining medium and once in the same medium without FCS. Finally the cells were fixed with 500 µL PBS containing 1% formalin. All steps were done on an ice-bath.

In certain experiments, an additional blocking step was included. It consisted of an incubation with 2 mg/mL human {gamma}-globulin (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland) for 15 min before starting the staining.

In further experiments, the cells were first preincubated with anti-CD64 mAb for 30 min to block Fc{gamma}R1, and then stained using the normal protocol with isotype-specific second-stage reagents.

FACS analyses were done on the same day or 1 day after the staining. We used a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA) and CellQuest analysis program. Routinely, 10,000 cells were collected for analyses. Characteristic light scatter parameters were used to distinguish between monocytes and lymphocytes in the samples containing mononuclear cells. The few contaminating mononuclear cells from the granulocyte samples were excluded during the analyses based on the scatter parameters.

Cell separation
To obtain essentially pure neutrophils, contaminating lymphocytes were removed from Percoll gradient isolated PMN by a further centrifugation through a Ficoll gradient. The granulocytes were then subjected to immunoisolation. We used CD16 as a selection criteria because eosinophils, which are known to express {alpha}4, and basophils lack this antigen, whereas neutrophils are brightly CD16-positive [43 , 44 ]. The freshly isolated cells were incubated sequentially with an anti-CD16 mAb and anti-mouse IgG1-specific paramagnetic MACS beads according to the manufacturer’s instructions. The non-bound (= eosinophils and basophils) and bound (= neutrophils) fractions were collected using a BS-type column and VarioMACS as suggested by the manufacturer. The purity of populations was confirmed by an immunostaining with an anti-CD16-PE mAb against another epitope.

RT-PCR
Total RNA was isolated from 5 x 106 lymphocytes, Percoll-purified granulocytes, and CD16+ MACS-selected neutrophils using Ultraspec RNA reagent (Biotecx Lab). Two micrograms of the resulting RNA was reverse-transcribed into cDNA with MMLV reverse transcriptase. The PCR primers used were as follows: {alpha}4 forward CCA CCT TGG TCC TCA TGT CAT, {alpha}4-reverse CAT GCG CAA CAT TCT GAT CCT [45 ], ß-actin sense GAA ATC GTG CGT GAC ATT AAG GAG and ß-actin antisense ATA CTC CTG CTT GCT GAT CCA CAT. PCR were carried out for 94°C 1 min, 50°C 1 min, and extension at 72°C 1 min for 30 cycles in a Perkin-Elmer instrument. The resulting PCR fragments were size-fractionated in 1% agarose and visualized using ethidium bromide staining. To obtain semiquantitative results ß-actin was amplified from the same cDNA lot and run in the same agarose gels.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Human PMN express an anti-{alpha}4 mAb reactive antigen in acute Salmonella infection
{alpha}4 Integrins are expressed on rat neutrophils and mediate homing of these cells into inflamed joints [34 ], and activated human neutrophils have been reported to express {alpha}4 [31 , 32 ]. We hypothesized that in vivo activated neutrophils might express {alpha}4 in conditions associated with arthritis if this integrin is important in targeting these cells into the joints. Because Salmonella-triggered gastroenteritis may lead to ReA we collected peripheral blood and synovial fluid from such patients. mAb HP2/1 and immunofluorescence staining followed by FACS analysis was used to detect surface {alpha}4.

In a subpopulation of the patients {alpha}4 expression was detected on the majority of PMN. The sample was defined as positive when PMN gave 1.5 times higher mean fluorescence intensity (MFI) with mAb HP2/1 than with a subclass of matched non-binding controls. Using these criteria, two out of eight Salmonella outbreak patients and two out of seven ReA patients displayed {alpha}4 integrin on the surface of their blood granulocytes (Fig. 1 ). Furthermore, the induction was time-dependent because the {alpha}4 expression peaked in the first samples drawn 1–2 weeks after the primary infection and declined thereafter (Fig. 1) . Similar {alpha}4 expression was also seen on joint PMN during the acute phase (data not shown). As expected, practically all blood granulocytes also expressed low levels of ß1; notably, it was present already on freshly isolated normal granulocytes in agreement with the constitutive {alpha}5ß1 expression on granulocytes [29 ], but no ß7 was detectable (data not shown). These results show that an antigen reactive with an {alpha}4-specific mAb is transiently induced on blood PMN in patients with acute gastroenteritis or ReA.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 1. An antigen reactive with an {alpha}4 integrin-specific mAb HP2/1 is transiently expressed on human granulocytes during gastroenteritis. A representative sample from one patient with an acute Salmonella-triggered gastroenteritis is shown. PMN and lymphocytes were isolated from the blood during the initial visit and follow-ups. The cells were stained with an anti-{alpha}4 mAb HP2/1 (solid line) and an IgG1 non-binding control (dotted line) and analyzed by flow cytometry. The x-axis is the fluorescence intensity on a log scale; y-axis is the relative number of cells.

 
In vitro stimulation of an {alpha}4 antigen on neutrophils
To study the mechanisms that induce {alpha}4 on neutrophils we activated granulocytes by different stimuli. It has been reported that transendothelial migration and maximal stimulation with the chemotactic agent fMLP and DHCB can rapidly induce {alpha}4 expression on human neutrophils [32 ]. However, we were unable to reproduce these findings because neither trigger alone or in combination yielded in any reactivity with mAb HP2/1 (Fig. 2 ). PMA, TGF-ß, and GM-CSF stimulation did not affect {alpha}4 expression either (data not shown). In contrast, a brief exposure of PMN to the chemotactic agent C5a elicited a threefold increase in {alpha}4 integrin expression on PMN (Fig. 2) . However, the most dramatic induction of {alpha}4 was seen after overnight stimulation of the cells with IFN-{gamma}. After this treatment the whole PMN population was {alpha}4 positive and the MFI was ~25 times higher than with a non-binding control mAb (Fig. 2) . To confirm the specificity of the staining, altogether five isotype-matched non-binding control mAbs were used. The results revealed no reactivity against IFN-{gamma}-stimulated granulocytes with any of these control mAbs (three shown in Fig. 3 ). Thus, human PMN seem to be capable of inducing an antigen reactive with an {alpha}4-integrin-specific mAb after certain chemotactic and proinflammatory stimuli in vitro.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 2. Induction of anti-{alpha}4 integrin mAb HP2/1 epitope on granulocytes during in vitro stimulation. Purified granulocytes were stimulated in vitro by allowing them to transmigrate through endothelial monolayer in absence (Trans) or presence (Trans+fMLP) of chemotactic gradient, with fMLP alone (fMLP), or together with DHCB (fMLP+DHCB), with C5a and with IFN-{gamma} as detailed in Materials and methods. Thereafter, cells were stained with a non-binding IgG1 mAb (neg. co) or with anti-{alpha}4 mAb HP2/1 and subjected to FACS analyses. The x-axis is fluorescence intensity on a log scale; y-axis is relative number of cells. Numbers in top right corner of each histogram are MFI of the given staining.

 


View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. Stimulated granulocytes react with anti-{alpha}4 mAb HP2/1 but not with subclass-matched control mAbs. IFN-{gamma}-stimulated granulocytes were reacted with three non-binding mouse IgG1 mAbs and mAb HP2/1. The x-axis is the fluorescence intensity on a log scale; y-axis is the relative number of cells. MFI values are given in top right corner of each histogram.

 
Because the strongest induction of {alpha}4 antigen was seen after IFN-{gamma} treatment, we chose it for further studies. PMN are known to express various Fc receptors [46 ]. Despite usage of multiple isotype-matched negative control mAbs (Fig. 3) , we were still concerned about the possibility of nonspecific binding of mAb HP2/1 to the stimulated cells. As an additional control the Fc receptors on PMN were blocked by a preincubation with 2 mg/mL human {gamma}-globulin before staining. In these experiments mAb HP2/1 gave reactivity that was stronger than that of any of the five independent subclass-matched controls, but the staining intensity was drastically reduced (Fig. 4A and B ). To address the specificity issue further, we used another anti-{alpha}4 mAb B-5G10, produced as an ascites fluid, to confirm the {alpha}4 expression on the IFN-{gamma}-stimulated cells. Usage of this antibody and a relevant ascitic control also revealed apparently specific {alpha}4 expression on IFN-{gamma}-stimulated PMN, although the specific MFI was lower than when using mAb HP2/1 (Fig. 4A) . However, when we applied B-5G10 instead of HP2/1 after the {gamma}-globulin blocking, no specific {alpha}4 reactivity was any longer detectable (Fig. 4B) .



View larger version (15K):
[in this window]
[in a new window]
  		Figure 4. Anti-{alpha}4 mAbs are contaminated with nonspecific Igs. IFN-{gamma}-stimulated granulocytes were stained with anti-{alpha}4 mAbs HP2/1 and B-5G10 (solid lines) and appropriate non-binding controls (3G6 and ascitic NS-1, respectively; dotted lines). (A) Normal FITC-conjugated sheep anti-mouse IgG second stage. (B) Pre-blocking with an extra {gamma}-globulin treatment and normal FITC-conjugated sheep anti-mouse IgG second stage. (C) IgG subclass-specific FITC-conjugated second-stage reagents. The x-axis is the fluorescence intensity on a log scale; y-axis is the relative number of cells.

 
Anti-{alpha}4 mAb HP2/1 is a mixture of Abs
The diminished intensity and inconsistency of {alpha}4 expression after {gamma}-globulin blocking raised a concern of the specificity of the binding of the anti-{alpha}4 mAbs. Therefore we utilized subclass-specific second-stage reagents for further studies. The IFN-{gamma}-stimulated granulocytes were first reacted with HP2/1 or B-5G10 and then with an FITC anti-mouse IgG1-specific reagent. It was surprising that no specific reactivity was detected (Fig. 4C) , although both anti-{alpha}4 mAbs are of the IgG1 isotype. In contrast, when using FITC anti-mouse IgG2a-specific second-stage reagent a clear positivity was detected with both HP2/1 and B-5G10 (Fig. 4C) . These data unpredictably show that both HP2/1 and B-5G10 contain contaminating IgG2a, which binds specifically to activated PMN. The staining with the specific IgG1 second-stage reagent revealed no {alpha}4 expression on C5a elicited neutrophils either (data not shown). These results unequivocally showed that there is no genuine {alpha}4 expression on PMN after IFN-{gamma} (or C5a) stimulation and that the nonspecific IgG2a component was responsible for the low-level positivity seen after using the normally applied isotype-specific (but not subclass specific) second-stage reagent.

As a final proof about the specificity of the anti-{alpha}4 mAbs used, we stained histocytic U937 cells, which are known to express high levels of {alpha}4 [47 ]. These results yielded the expected results, i.e., FITC-conjugated anti-mouse IgG1 gave positive reactivity with both HP2/1 and B-5G10, whereas U937 cells stained with FITC-conjugated anti-mouse IgG2a as a second stage revealed no reactivity at all (Fig. 5 ).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 5. Specific Ig in mAbs HP2/1 and B-5G10 are of IgG1 subclass. U937 cells were stained with the indicated anti-{alpha}4 mAbs or control mAbs 3G6 and ascitic NS-1. FITC-conjugated second-stage reagent was sheep anti-mouse IgG (total), goat anti-mouse IgG1, or goat anti-mouse IgG2a as indicated. The x-axis is the fluorescence intensity on a log scale; y-axis is the relative number of cells. MFI values are shown in the histograms.

 
We hypothesized that induction of Fc{gamma}R1 on stimulated granulocytes may lead to nonspecific binding of IgG2a. We performed additional staining in which the Fc{gamma}R1 was first blocked and compared HP2/1 preparations from different sources. As shown in Figure 6 , the genuine {alpha}4-specific IgG1 activity from either preparation did not stain anything on stimulated granulocytes. The amount of the contamination in HP2/1 varied between different commercial sources. In activated granulocytes Fc{gamma}R1 was also induced. When the Fc{gamma}R1 was first blocked with an anti-CD64 mAb [48 ], the IgG1 second-stage reagent reacted with the blocking mAb in all samples, as expected. In contrast, no nonspecific Fc-mediated binding of the contaminating IgG2a from HP2/1 preparations was detectable after the blocking of Fc{gamma}R1 (Fig. 6) .



View larger version (31K):
[in this window]
[in a new window]
  		Figure 6. Nonspecific IgG2a binds to Fc{gamma}R1 in stimulated granulocytes. HP2/1 from two sources (#1, Immunotech; #2, Chemicon) was used to stain IFN-{gamma}-stimulated granulocytes. In additional experiments, anti-CD64 mAb (IgG1) was used to block the Fc{gamma}R1 receptor before applying the HP2/1 mAbs. Note that this blockade prevents the nonspecific absorbance of IgG2a component(s) from the HP2/1 preparations to the cells.

 
Thus, the most widely used commercially available purified anti-{alpha}4 integrin mAb HP2/1 contains the specific IgG1 anti-{alpha}4 component and irrelevant IgG2a Ab(s) with an unknown binding specificity. The binding of the nonspecific IgG2a is induced on activated PMN via Fc{gamma}R1.

{alpha}4 mRNA is not found in resting neutrophils
Different leukocyte classes were isolated for analyses of {alpha}4 expression at mRNA level. Because Percoll-purified granulocytes, in addition to contaminating lymphocytes, always contain eosinophils, which are known to express functionally active {alpha}4 [25 26 27 28 ], we sought to further purify neutrophils. To that end the Percoll-purified cells (typically the starting population contained ~10% lymphocytes, 5% eosinophils, and <1% basophils) were subjected to centrifugation over Ficoll-gradient to get rid of contaminating lymphocytes and then reacted with CD16 mAb for immunomagnetic purification with MACS. Using this selection, all lymphocytes were eliminated and the vast majority of CD16-negative eosinophils and all basophils was separated from CD16-positive neutrophils. mRNA was immediately isolated from the CD16-positive neutrophils, Percoll-purified granulocytes, and control lymphocytes. RT-PCR was subsequently used to analyze the {alpha}4 mRNA expression in these three leukocyte populations. A strong signal of the expected size of 1142 bp was seen with {alpha}4 primers from lymphocyte mRNA (Fig. 7 ). A faint, but clearly detectable {alpha}4-specific signal was also obtained using mRNA from Percoll-purified cells. In contrast, the purified CD16-positive neutrophil population gave no signal at all (Fig. 7) . These RT-PCR experiments using purified leukocyte subpopulations thus suggest that the {alpha}4 mRNA detected in PMN population originates from the eosinophils and contaminating lymphocytes, whereas resting neutrophils lack {alpha}4 mRNA synthesis.



View larger version (42K):
[in this window]
[in a new window]
  		Figure 7. Resting neutrophils do not express {alpha}4 mRNA. Ficoll-purified lymphocytes, Percoll-purified PMN, and lymphocyte-depleted, MACS-selected CD16-positive neutrophils were separated. mRNA was isolated from the purified cell populations, reverse transcribed into cDNA, and subjected to PCR using {alpha}4-specific primers and ß-actin-specific primers (to assess the equivalence of the loading). The products were separated in agarose gels and visualized with ethidium bromide staining.

 
{alpha}4 is not found on neutrophils in inflammation in vivo
Taking into account the unanticipated difficulties with the commercially available anti-{alpha}4 reagent, mAb HP2/1, we reanalyzed some of the samples obtained from the original patient material. Using IgG subclass-specific second-stage reagents no expression of {alpha}4 on blood neutrophils was found in these patient cells (data not shown). Thus, human PMN appear to be unable to induce {alpha}4 expression during acute gastrointestinal infection or ReA in vivo. We conclude that other adhesion molecules rather than {alpha}4 must be used to target human PMN into inflamed joints.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We show here that resting human neutrophils neither synthesize {alpha}4 integrin mRNA nor express {alpha}4 protein on the cell surface. These cells also are not able to induce {alpha}4 synthesis during rapid activation by chemotactic agents, by transendothelial migration, or after an overnight stimulation with certain proinflammatory cytokines. Moreover, acute gastrointestinal infection or chronic arthritis in patients fails to induce {alpha}4 on neutrophils. Most importantly, we describe a previously unrecognized complication of using {alpha}4 integrin-specific mAbs in analyzing activated PMN populations in humans.

There are conflicting reports on {alpha}4 expression in human PMN. It is generally thought that neutrophils do not express {alpha}4 [12 , 16 , 18 , 23 , 25 26 27 , 29 ]. This paradigm holds well with the leukocyte-subtype selective trafficking pattern seen in vivo [1 , 2 ]. In particular, the fact that in the congenital ß2-integrin deficiency the granulocytes are unable to infiltrate into sites on inflammation is attributed to the lack of alternative {alpha}4-integrin-dependent adhesion mechanisms on neutrophils, whereas ß2-deficient, {alpha}4-positive lymphocytes are thought to utilize {alpha}4 to bind endothelial VCAM-1 and fibronectin at the inflammation focus [49 ]. However, myelomonocytic precursors have been reported to synthesize {alpha}4 integrin, and the expression level diminishes along with further maturation [23 , 24 , 29 ]. Approximately 50% of CD34-positive bone marrow cells express low levels of {alpha}4. The majority of CFU-GM were among the cells that expressed intermediate levels of {alpha}4. More than ninety percent of cells expressed {alpha}4 still at the band stage but only 20% of cells expressed {alpha}4 thereafter. However, in another study, {alpha}4 expression was restricted to early myeloid precursors [24 ]. Cell lines of myelomonocytic differentiation capacity, like HL60, already synthesize high levels of {alpha}4 in an undifferentiated stage, and {alpha}4 expression level does not change after the cells are induced to differentiate along to a granulocytic-like pathway [50 ]. Eosinophils are a notable exception among PMN because these cells retain {alpha}4 expression in a functionally active form during their maturation [25 26 27 28 ]. There are recent reports that resting human PMN can be very rapidly induced to express {alpha}4 on the cell surface [30 ]. In one series of experiments, transendothelial migration or chemotactic stimulation (fMLP, C5a, PMA) resulted in {alpha}4 expression on the majority of PMN within 10 min [32 ]. Notably, inclusion of a microfilament disrupting agent, DHCB, during the stimulation was a prerequisite for the {alpha}4 induction. Furthermore, the induced {alpha}4 was shown to be able to mediate neutrophil binding to endothelium but not to mediate rolling on VCAM-1 substrate in an in vitro flow chamber assay [51 ]. Even unactivated neutrophils in whole blood have been shown to adhere to recombinant VCAM-1 in an {alpha}4-dependent manner at high shear rates in vitro [33 ]. Another group has reported that a 15-min C5a treatment alone can induce {alpha}4 expression on human neutrophils [31 ].

We found apparent induction of {alpha}4 expression on human PMN by C5a and IFN-{gamma} stimulation when analyzed with a generally accepted method including all relevant controls. However, a more detailed analysis revealed that this was, in fact, a staining artifact caused by an unreported contamination of the commercial preparation of the most widely used anti-{alpha}4 mAb HP2/1 by other Igs. This antibody was originally reported to be against VLA-3 [17 ] but later it was actually found to react with VLA-4 {alpha}-chain. It has been isotyped as a mouse IgG1 [17 ]. We confirmed that the specific anti-human {alpha}4 Ig in HP2/1 preparations is of mouse IgG1 isotype, but that there is a contaminating IgG2a component with an unknown specificity. There are multiple Fc receptors on neutrophils, which are differentially expressed under resting and activated conditions and which bind different IgG subclasses with a different affinity [46 ]. Intriguingly enough, FcR type I is known to be more potently induced by IFN-{gamma} than by many other mediators and it has much higher affinity for IgG2a than for IgG1 [46 ]. Thus, despite using multiple subclass-matched controls (IgG1) we nevertheless detected apparent positivity in the IFN-{gamma}-stimulated cells because this stimulation elicits synthesis of FcRI, which preferentially binds the contaminating non-anti-{alpha}4 Ab of the IgG2a subclass from the HP2/1 preparation. Blocking of Fc{gamma}R1 efficiently prevented the nonspecific staining results. For some unknown reason the binding of the irrelevant IgG2 mAb to IFN-{gamma}-stimulated neutrophils even translates into functional consequences because pretreatment of these cells with mAb HP2/1 resulted in diminished binding to fibronectin-coated surfaces (three assays, inhibition 52 ± 19.5%, mean ± SD).

This unforeseen complication could be circumvented by using IgG1 subclass-specific second-stage reagents. Restaining with the subclass-specific second stage antibody revealed that there is no genuine positivity after IFN-{gamma} of C5a stimulation on human PMN in our hands. Moreover, we could not detect {alpha}4 mRNA through the use of a sensitive RT-PCR technique in resting cells. We interpret our finding so that there is no {alpha}4 message or protein in resting PMN, and hence a short-term stimulation of the cells in vitro or in vivo cannot induce {alpha}4 expression or translocate it from any internal storage granules, at least before the protein synthesis is turned on.

We propose that the different results between our group and others most likely depend on the contamination of the commercial preparations of HP2/1. Because it is the only widely available anti-{alpha}4 reagent, most studies reporting expression and function of {alpha}4-integrin on neutrophils [23 , 32 , 33 , 51 ] have used the very same anti-{alpha}4 mAb. We tested four different HP2/1 batches from Immunotech with similar results. Obviously the origin of the mAb has clear effects on the amount of contaminating Ig because the same HP2/1 reagent from another company showed only marginal nonspecific staining. Application of other anti-{alpha}4 mAbs does not exclude the role of nonspecific binding to FcRI because we in fact demonstrated that B-5G10 used as an ascites also gives false positive results. This is most likely because ascitic fluid is rich in all types of Igs, which then unavoidably co-precipitates during the chromatographic purification of the desired mAb with Protein A or G Sepharose. In fact, our study is the first one studying human granulocytes which utilized anti-{alpha}4 mAbs against different epitopes [52 ] and an extra blocking step with 2 mg/mL human IgG to avoid nonspecific staining results. In the work of Kubes, the authors fixed the stimulated cells for 15 min with 1% formalin and then blocked the cells with polyclonal goat Ig before HP2/1 staining [32 ]. In the other studies with human granulocytes no mention can be found about possible extra blocking steps, specific second stage reagents, or other precautions to avoid nonspecific staining. However, due to the unpredicted cause of the nonspecific staining, not even careful blocking was enough, since the problem could only be overcome with usage of subclass-matched second-stage reagents.

In rat, {alpha}4 integrin has been implicated in endothelial binding and homing of granulocytes into joints, skin, and kidney [34 , 53 ]. Expression of {alpha}4 on resting rat granulocytes and its absence on human, guinea pig, and sheep neutrophils [12 , 16 , 18 , 23 , 25 26 27 , 29 , 54 55 56 ] probably represents genuine species-specific differences. However, also in rats the maximal MFI with the best anti-{alpha}4 mAb is two times that of a control mAb. Thus, alternative explanations of the functional effects of anti-{alpha}4 mAbs, like primary effects on cytokine secretion by highly {alpha}4-positive T lymphocytes and macrophages upon {alpha}4 ligation leading to secondary effects on neutrophil behavior or unknown signal transduction events by the contaminating IgG2a components, may not be completely ruled out. On the other hand, we are aware of the sensitivity limits of our detection systems. In FACS staining >1000 molecules/cell are needed for a positive signal and in RT-PCR the sensitivity is also limited. Therefore, it remains possible that very low levels of {alpha}4 may be expressed on the surface of neutrophils, which might, especially upon suitable activation, still be sufficient for ligand binding. However, our results clearly warrant re-analysis of {alpha}4 integrin expression on cells (especially granulocytes) that may express Fc-receptors upon activation.


   ACKNOWLEDGEMENTS
 
This work was supported by the Finnish Academy, Technology Development Center of Finland, the Finnish Cultural Foundation, and the Paulo Foundation. We thank Dr. M. Hemler for donating mAb B-5G10, Dr. I. Julkunen for providing IFN-{gamma}, Mrs. T. Lähde and Ms. Riikka Lehvonen for excellent assistance, and Mrs. Anne Sovikoski-Georgieva for secretarial help.


   FOOTNOTES
 
Current addresses: Juha Kirveskari, Department of Bacteriology and Immunology, University of Helsinki, Helsinki, Finland; Petri Bono, Center for Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139-4307.

Received December 9, 1999; revised April 14, 2000; accepted April 17, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Butcher, E. C., Picker, L. J. (1996) Lymphocyte homing and homeostasis Science 272,60-66[Abstract]
  2. Springer, T. A. (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm Cell 76,301-314[Medline]
  3. Hynes, R. O. (1992) Integrins: versatility, modulation, and signaling in cell adhesion Cell 69,11-25[Medline]
  4. Holzmann, B., Weissman, I. L. (1989) Peyer’s patch-specific lymphocyte homing receptors consist of a VLA-4-like {alpha} chain associated with either of two integrin ß chains, one of which is novel EMBO J 8,1735-1741[Medline]
  5. Berlin, C., Bargatze, R. F., Campbell, J. J., von Andrian, U. H., Szabo, M. C., Hasslen, S. R., Nelson, R. D., Berg, E. L., Erlandsen, S. L., Butcher, E. C. (1995) {alpha}4 integrins mediate lymphocyte attachment and rolling under physiologic flow Cell 80,413-422[Medline]
  6. Alon, R., Kassner, P. D., Carr, M. W., Finger, E. B., Hemler, M. E., Springer, T. A. (1995) The integrin VLA-4 supports tethering and rolling in flow on VCAM-1 J. Cell Biol. 128,1243-1253[Abstract/Free Full Text]
  7. Hemler, M. E., Elices, M. J., Parker, C., Takada, Y. (1990) Structure of the integrin VLA-4 and its cell-cell and cell-matrix adhesion functions Immunol. Rev. 114,45-65[Medline]
  8. Masumoto, A., Hemler, M. E. (1993) Multiple activation states of VLA-4. Mechanistic differences between adhesion to CS1/fibronectin and to vascular cell adhesion molecule-1 J. Biol. Chem. 268,228-234[Abstract/Free Full Text]
  9. Berlin, C., Berg, E. L., Briskin, M. J., Andrew, D. P., Kilshaw, P. J., Holzmann, B., Weissman, I. L., Hamann, A., Butcher, E. C. (1993) {alpha}4ß7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1 Cell 74,185-195[Medline]
  10. Crowe, D. T., Chiu, H., Fong, S., Weissman, I. L. (1994) Regulation of the avidity of integrin {alpha}4ß7 by the ß7 cytoplasmic domain J. Biol. Chem. 269,14411-14418[Abstract/Free Full Text]
  11. Postigo, A. A., Sánchez-Mateos, P., Lazarovits, A. I., Sánchez-Madrid, F., de Landázuri, M. O. (1993) {alpha}4ß7 integrin mediates B cell binding to fibronectin and vascular cell adhesion molecule-1. Expression and function of {alpha}4 integrins on human B lymphocytes J. Immunol. 151,2471-2483[Abstract]
  12. Hemler, M. E., Huang, C., Takada, Y., Schwarz, L., Strominger, J. L., Clabby, M. L. (1987) Characterization of the cell surface heterodimer VLA-4 and related peptides J. Biol. Chem. 262,11478-11485[Abstract/Free Full Text]
  13. Takada, Y., Elices, M. J., Crouse, C., Hemler, M. E. (1989) The primary structure of the {alpha}4 subunit of VLA-4: homology to other integrins and a possible cell-cell adhesion function EMBO J 8,1361-1368[Medline]
  14. Elices, M. J., Osborn, L., Takada, Y., Crouse, C., Luhowskyj, S., Hemler, M. E., Lobb, R. R. (1990) VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site Cell 60,577-584[Medline]
  15. Strauch, U. G., Lifka, A., Gosslar, U., Kilshaw, P. J., Clements, J., Holzmann, B. (1994) Distinct binding specificities of integrins {alpha}4ß7 (LPAM-1), {alpha}4ß1 (VLA-4), and {alpha}IELß7 Int. Immunol. 6,263-275[Abstract/Free Full Text]
  16. Tanaka, Y., Saito, K. (1998) CD49d Workshop Panel report Kishimoto, T. Goyert, S. Kikutani, H. Mason, D. Miyasaka, M. Moretta, L. Ohno, T. Okumura, K. Shaw, S. Springer, T. A. Sugamura, K. Sugawara, H. von dem Borne, A. E. K. K. Zola, H. eds. Leucocyte Typing VI: White Cell Differentiation Antigens ,396-398
  17. Sánchez-Madrid, F., de Landázuri, M. O., Morago, C., Cebrián, M., Acevedo, A., Bernabeu, C. (1986) VLA-3: a novel polypeptide association within the VLA molecular complex: cell distribution and biochemical characterization Eur. J. Immunol. 16,1343-1349[Medline]
  18. Erle, D. J., Briskin, M. J., Butcher, E. C., García-Pardo, A., Lazarovits, A. I., Tidswell, M. (1994) Expression and function of the MAdCAM-1 receptor, integrin {alpha}4ß7, on human leukocytes J. Immunol. 153,517-528[Abstract]
  19. Lazarovits, A. I., Moscicki, R. A., Kurnick, J. T., Camerini, D., Bhan, A. K., Baird, L. G., Erikson, M., Colvin, R. B. (1984) Lymphocyte activation antigens. I. A monoclonal antibody, anti-Act I, defines a new late lymphocyte activation antigen J. Immunol. 133,1857-1862[Abstract]
  20. Schweighoffer, T., Tanaka, Y., Tidswell, M., Erle, D. J., Horgan, K. J., Luce, G. E. G., Lazarovits, A. I., Buck, D., Shaw, S. (1993) Selective expression of integrin {alpha}4 ß7 on a subset of human CD4+ memory T cells with hallmarks of gut-trophism J. Immunol. 151,717-729[Abstract]
  21. Rott, L. S., Briskin, M. J., Andrew, D. P., Berg, E. L., Butcher, E. C. (1996) A fundamental subdivision of circulating lymphocytes defined by adhesion to mucosal addressin cell adhesion molecule-1. Comparison with vascular cell adhesion molecule-1 and correlation with ß7 integrins and memory differentiation J. Immunol. 156,3727-3736[Abstract]
  22. Tiisala, S., Paavonen, T., Renkonen, R. (1995) {alpha}Eß7 and {alpha}4ß7 integrins associated with intraepithelial and mucosal homing, are expressed on macrophages Eur. J. Immunol. 25,411-417[Medline]
  23. Kerst, J. M., Sanders, J. B., Slaper-Cortenbach, I. C. M., Doorakkers, M. C., Hooibrink, B., van Oers, R. H. J., von dem Borne, A. E. G. K., van der Schoot, C. E. (1993) {alpha}4ß1 and {alpha}5ß1 are differentially expressed during myelopoiesis and mediate the adherence of human CD34+ cells to fibronectin in an activation-dependent way Blood 81,344-351[Abstract/Free Full Text]
  24. Soligo, D., Schiró, R., Luksch, R., Manara, G., Quirici, N., Parravicini, C., Lambertenghi Deliliers, G. (1990) Expression of integrins in human bone marrow Br. J. Haematol. 76,323-332[Medline]
  25. Bochner, B. S., Luscinskas, F. W., Gimbrone, M. A., Jr, Newman, W., Sterbinsky, S. A., Derse-Anthony, C. P., Klunk, D., Schleimer, R. P. (1991) Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules J. Exp. Med. 173,1553-1557[Abstract/Free Full Text]
  26. Weller, P. F., Rand, T. H., Goelz, S. E., Chi-Rosso, G., Lobb, R. R. (1991) Human eosinophil adherence to vascular endothelium mediated by binding to vascular cell adhesion molecule 1 and endothelial leukocyte adhesion molecule 1 Proc. Natl. Acad. Sci. USA 88,7430-7433[Abstract/Free Full Text]
  27. Schleimer, R. P., Sterbinsky, S. A., Kaiser, J., Bickel, C. A., Klunk, D. A., Tomioka, K., Newman, W., Luscinskas, F. W., Gimbrone, M. A., Jr, McIntyre, B. W., Bochner, B. S. (1992) IL-4 induces adherence of human eosinophils and basophils but not neutrophils to endothelium. Association with expression of VCAM-1 J. Immunol. 148,1086-1092[Abstract]
  28. Sriramarao, P., von Andrian, U. H., Butcher, E. C., Bourdon, M. A., Broide, D. H. (1994) L-selectin and very late antigen-4 integrin promote eosinophil rolling at physiological shear rates in vivo J. Immunol. 153,4238-4246[Abstract]
  29. Lund-Johansen, F., Terstappen, L. W. M. M. (1993) Differential surface expression of cell adhesion molecules during granulocyte maturation J. Leukoc. Biol. 54,47-55[Abstract]
  30. Johnston, B., Kubes, P. (1999) The alpha4-integrin: an alternative pathway for neutrophil recruitment? Immunol. Today 20,545-550[Medline]
  31. Gao, J.-X., Issekutz, A. C. (1997) The ß1 integrin, very late activation antigen-4 on human neutrophils can contribute to neutrophil migration through connective tissue fibroblast barriers Immunology 90,448-454[Medline]
  32. Kubes, P., Niu, X.-F., Smith, C. W., Kehrli, M. E., Jr, Reinhardt, P. H., Woodman, R. C. (1995) A novel ß1-dependent adhesion pathway on neutrophils: a mechanism invoked by dihydrocytochalasin B or endothelial transmigration FASEB J 9,1103-1111[Abstract]
  33. Reinhardt, P. H., Kubes, P. (1998) Differential leukocyte recruitment from whole blood via endothelial adhesion molecules under shear conditions Blood 92,4691-4699[Abstract/Free Full Text]
  34. Issekutz, T. B., Miyasaka, M., Issekutz, A. C. (1996) Rat blood neutrophils express very late antigen 4 and it mediates migration to arthritic joint and dermal inflammation J. Exp. Med. 183,2175-2184[Abstract/Free Full Text]
  35. Davenpeck, K. L., Sterbinsky, S. A., Bochner, B. S. (1998) Rat neutrophils express {alpha}4 and ß1 integrins and bind to vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) Blood 91,2341-2346[Abstract/Free Full Text]
  36. Salmi, M., Jalkanen, S. (1992) A 90-kilodalton endothelial cell molecule mediating lymphocyte binding in humans Science 257,1407-1409[Abstract/Free Full Text]
  37. Cowan, N. J., Secher, D. S., Milstein, C. (1974) Intracellular immunoglobulin chain synthesis in non-secreting variants of a mouse myeloma: detection of inactive light-chain messenger RNA J. Mol. Biol. 90,691-701[Medline]
  38. Airas, L., Salmi, M., Jalkanen, S. (1993) Lymphocyte-vascular adhesion protein-2 is a novel 70-kDa molecule involved in lymphocyte adhesion to vascular endothelium J. Immunol. 151,4228-4238[Abstract]
  39. Kurkijärvi, R., Adams, D. H., Leino, R., Möttönen, T., Jalkanen, S., Salmi, M. (1998) Circulating form of human vascular adhesion protein-1 (VAP-1): increased serum levels in inflammatory liver diseases J. Immunol. 161,1549-1557[Abstract/Free Full Text]
  40. Isomäki, O., Vuento, R., Granfors, K. (1989) Serological diagnosis of salmonella infections by enzyme immunoassay Lancet 1,1411-1414[Medline]
  41. Kirveskari, J., Jalkanen, S., Mäki-Ikola, O., Granfors, K. (1998) Increased synovial endothelium binding and transendothelial migration of mononuclear cells during Salmonella infection Arthritis Rheum 41,1054-1063[Medline]
  42. Edgell, C.-J. S., McDonald, C. C., Graham, J. B. (1983) Permanent cell line expressing human factor VIII-related antigen established by hybridization Proc. Natl. Acad. Sci. USA 80,3734-3737[Abstract/Free Full Text]
  43. Terstappen, L. W. M. M., Hollander, Z., Meiners, H., Loken, M. R. (1990) Quantitative comparison of myeloid antigens on five lineages of mature peripheral blood cells J. Leukoc. Biol. 48,138-148[Abstract]
  44. Mul, F. P. J., Knol, E. F., Roos, D. (1992) An improved method for the purification of basophilic granulocytes from human blood J. Immunol. Meth. 149,207-214[Medline]
  45. Zhang, Z. H., Vekemans, S., Aly, M. S., Jaspers, M., Marynen, P., Cassiman, J. J. (1991) The gene for the alpha 4 subunit of the VLA-4 integrin maps to chromosome 2Q31-32 Blood 78,2396-2399[Abstract/Free Full Text]
  46. Deo, Y. M., Graziano, R. F., Repp, R., van de Winkel, J. G. J. (1997) Clinical significance of IgG Fc receptors and FcgR-directed immunotherapies Immunol. Today 18,127-135[Medline]
  47. Bauvois, B., Rouillard, D., Sanceau, J., Wietzerbin, J. (1992) IFN-{gamma} and transforming growth factor-ß 1 differently regulate fibronectin and laminin receptors of human differentiating monocytic cells J. Immunol. 148,3912-3919[Abstract]
  48. Dougherty, G. J., Selvendran, Y., Murdoch, S., Palmer, D. G., Hogg, N. (1987) The human mononuclear phagocyte high-affinity Fc receptor, FcRI, defined by a monoclonal antibody, 10.1 Eur. J. Immunol. 17,1453-1459[Medline]
  49. Anderson, D. C., Springer, T. A. (1987) Leukocyte adhesion deficiency: an inherited defect in the Mac-1, LFA-1, and p150,95 glycoproteins Annu. Rev. Med. 38,175-194[Medline]
  50. Bohnsack, J. F., Chang, J. (1994) Activation of ß1 integrin fibronectin receptors on HL60 cells after granulocytic differentiation Blood 83,543-552[Abstract/Free Full Text]
  51. Reinhardt, P. H., Elliott, J. F., Kubes, P. (1997) Neutrophils can adhere via {alpha}4ß1-integrin under flow conditions Blood 89,3837-3846[Abstract/Free Full Text]
  52. Schiffer, S. G., Hemler, M. E., Lobb, R. R., Tizard, R., Osborn, L. (1995) Molecular mapping of functional antibody binding sites of {alpha}4 integrin J. Biol. Chem. 270,14270-14273[Abstract/Free Full Text]
  53. Mulligan, M. S., Johnson, K. J., Todd, R. F., III, Issekutz, T. B., Miyasaka, M., Tamatani, T., Smith, C. W., Anderson, D. C., Ward, P. A. (1993) Requirements for leukocyte adhesion molecules in nephrotoxic nephritis J. Clin. Invest. 91,577-587
  54. Abraham, W. M., Sielczak, M. W., Ahmed, A., Cortes, A., Lauredo, I. T., Kim, J., Pepinsky, B., Benjamin, C. D., Leone, D. R., Lobb, R. R., Weller, P. F. (1994) {alpha}4-integrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep J. Clin. Invest. 93,776-787
  55. Neeley, S. P., Hamann, K. J., White, S. R., Baranowski, S. L., Burch, R. A., Leff, A. R. (1993) Selective regulation of expression of surface adhesion molecules Mac-1, L-selectin, and VLA-4 on human eosinophils and neutrophils Am. J. Respir. Cell. Mol. Biol. 8,633-639
  56. Weg, V. B., Williams, T. J., Lobb, R. R., Nourshargh, S. (1993) A monoclonal antibody recognizing very late activation antigen-4 inhibits eosinophil accumulation in vivo J. Exp. Med. 177,561-566[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
S. R. Barthel, M. W. Johansson, D. M. McNamee, and D. F. Mosher
Roles of integrin activation in eosinophil function and the eosinophilic inflammation of asthma
J. Leukoc. Biol., January 1, 2008; 83(1): 1 - 12.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
G. W. Sullivan, D. D. Lee, W. G. Ross, J. A. DiVietro, C. M. Lappas, M. B. Lawrence, and J. Linden
Activation of A2A adenosine receptors inhibits expression of {alpha}4/{beta}1 integrin (very late antigen-4) on stimulated human neutrophils
J. Leukoc. Biol., January 1, 2004; 75(1): 127 - 134.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
E. Kudlacz, C. Whitney, C. Andresen, A. Duplantier, G. Beckius, L. Chupak, A. Klein, K. Kraus, and A. Milici
Pulmonary Eosinophilia in a Murine Model of Allergic Inflammation Is Attenuated by Small Molecule alpha 4beta 1 Antagonists
J. Pharmacol. Exp. Ther., May 1, 2002; 301(2): 747 - 752.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
T. M. Carlos
Leukocyte recruitment at sites of tumor: dissonant orchestration
J. Leukoc. Biol., August 1, 2001; 70(2): 171 - 184.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Pereira, M. Zhou, A. Mocsai, and C. Lowell
Resting Murine Neutrophils Express Functional {{alpha}}4 Integrins that Signal Through Src Family Kinases
J. Immunol., March 15, 2001; 166(6): 4115 - 4123.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow