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Antibody conjugates bispecific for intercellular adhesion molecule 1 and allergen prevent migration of allergens through respiratory epithelial cell layers.

Madritsch C, Eckl-Dorna J, Blatt K, Ellinger I, Kundi M, Niederberger V, Valent P, Valenta R, Flicker S - J. Allergy Clin. Immunol. (2015)

View Article: PubMed Central - PubMed

Affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

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It is becoming increasingly evident that intact epithelial barrier function is important for preventing allergens to reach tissues involved in allergic inflammation... In this study we produced an antibody conjugate bispecific for intercellular adhesion molecule 1 (ICAM1) and a major respiratory allergen (ie, the major grass pollen allergen Phl p 2) termed P2/ICAM1 to investigate whether such a conjugate can inhibit the migration of allergens through respiratory cell layers and reduce the activation of the inflammatory cells underneath... When P2/ICAM1 was added to the cells, it was detected with Alexa Fluor 488 goat anti-mouse IgG specific for αICAM1 mouse IgG (Fig 1, A-C, green)... When P2/ICAM1 was omitted, detection antibodies did not bind (Fig 1, D)... No cell staining was found when only secondary antibodies were applied (data not shown)... In a subsequent series of experiments, we demonstrated that P2/ICAM1 can prevent the apical-to-basolateral penetration of Phl p 2 but not of the birch pollen allergen Bet v 1, a similarly sized but not related allergen, through a layer of the cultured respiratory epithelial 16HBE14o- cells by using a well-established Transwell culture system (Costar Corning Incorporated, Corning, NY; see Figs E5 and E6 in this article’s Online Repository at www.jacionline.org), as described in the Methods section in this article’s Online Repository... Furthermore, we showed that P2/ICAM1 also reacted specifically with natural grass pollen–derived Phl p 2 (data not shown)... In the Transwell experiments we found that the apical addition of P2/ICAM1 prevented the penetration of Phl p 2 over the full period of analysis (ie, for 72 hours) into the basolateral compartment (see Fig E5, A, gray bars, +) when compared with conditions without addition of P2/ICAM1 (Fig E5 A, gray bars, −)... When Phl p 2 and P2/ICAM1 conjugates were omitted, no Phl p 2 signal was detected (see Fig E5, A, no Phl p 2, 72 hours)... Additionally, no relevant penetration of P2/ICAM1–Phl p 2 complexes into the basolateral wells was found (Fig E5, B, gray bars, +)... The extent of basophil activation induced with basolateral samples showed a significant and consistent reduction for samples taken from cultures in which P2/ICAM1 had been added to the apical compartments (Fig 2, gray bars, +) compared with those in which P2/ICAM1 conjugates had been omitted (Fig 2, gray bars, −)... This effect was observed at each of the analyzed time points... Cell-culture supernatants were applied, and P2/ICAM1–bound Phl p 2 was detected with rabbit anti–Phl p 2 antibodies, as described above... The effect of P2/ICAM1 on Phl p 2 apical-to-basolateral transepithelial migration was assessed by comparing P2/ICAM1-1–treated wells with untreated control wells.

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Inhibition of transepithelial migration of Phl p 2 by P2/ICAM1 conjugates leads to decreased basophil activation with basolateral samples. Samples obtained from apical and basolateral compartments of Transwell cultures were incubated with blood samples from 3 patients allergic to Phl p 2 (A-C), and basophil activation was measured by determining upregulation of the surface marker CD203c on basophils by using flow cytometry. CD203c upregulation expressed as the stimulation index is displayed on the y-axis. Results are means of triplicates, and error bars indicate SDs. *P < .05, **P < .01, and ***P < .001, ANOVA and linear contrasts.
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Figure 2: Inhibition of transepithelial migration of Phl p 2 by P2/ICAM1 conjugates leads to decreased basophil activation with basolateral samples. Samples obtained from apical and basolateral compartments of Transwell cultures were incubated with blood samples from 3 patients allergic to Phl p 2 (A-C), and basophil activation was measured by determining upregulation of the surface marker CD203c on basophils by using flow cytometry. CD203c upregulation expressed as the stimulation index is displayed on the y-axis. Results are means of triplicates, and error bars indicate SDs. *P < .05, **P < .01, and ***P < .001, ANOVA and linear contrasts.

Mentions: Finally, we tested whether a reduction of transepithelial allergen migration by P2/ICAM1 has an effect on basophil activation to assess the migration of Phl p 2, capable of activating mast cells or basophils. Results from basophil activation tests (see the Methods section and Fig E7 in this article’s Online Repository at www.jacionline.org) performed with samples from 3 independent Transwell experiments with basophils from 3 representative allergic patients are shown in Fig 2. The extent of basophil activation induced with basolateral samples showed a significant and consistent reduction for samples taken from cultures in which P2/ICAM1 had been added to the apical compartments (Fig 2, gray bars, +) compared with those in which P2/ICAM1 conjugates had been omitted (Fig 2, gray bars, −). This effect was observed at each of the analyzed time points. Basophil activation induced by samples taken from the apical compartments (Fig 2, black bars) was comparable and almost identical with that obtained with basolateral samples from cell-free preparations (Fig 2, no cells). Basophil activation was not observed when Phl p 2 was absent from the cultures (Fig 2, no Phl p 2). Concentrations of soluble ICAM1 in 16HBE14o- cell cultures were around 1 ng/mL in apical wells and not detectable in basolateral wells in the experiments and had no influence on allergen-induced basophil activation (data not shown).


Antibody conjugates bispecific for intercellular adhesion molecule 1 and allergen prevent migration of allergens through respiratory epithelial cell layers.

Madritsch C, Eckl-Dorna J, Blatt K, Ellinger I, Kundi M, Niederberger V, Valent P, Valenta R, Flicker S - J. Allergy Clin. Immunol. (2015)

Inhibition of transepithelial migration of Phl p 2 by P2/ICAM1 conjugates leads to decreased basophil activation with basolateral samples. Samples obtained from apical and basolateral compartments of Transwell cultures were incubated with blood samples from 3 patients allergic to Phl p 2 (A-C), and basophil activation was measured by determining upregulation of the surface marker CD203c on basophils by using flow cytometry. CD203c upregulation expressed as the stimulation index is displayed on the y-axis. Results are means of triplicates, and error bars indicate SDs. *P < .05, **P < .01, and ***P < .001, ANOVA and linear contrasts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4530582&req=5

Figure 2: Inhibition of transepithelial migration of Phl p 2 by P2/ICAM1 conjugates leads to decreased basophil activation with basolateral samples. Samples obtained from apical and basolateral compartments of Transwell cultures were incubated with blood samples from 3 patients allergic to Phl p 2 (A-C), and basophil activation was measured by determining upregulation of the surface marker CD203c on basophils by using flow cytometry. CD203c upregulation expressed as the stimulation index is displayed on the y-axis. Results are means of triplicates, and error bars indicate SDs. *P < .05, **P < .01, and ***P < .001, ANOVA and linear contrasts.
Mentions: Finally, we tested whether a reduction of transepithelial allergen migration by P2/ICAM1 has an effect on basophil activation to assess the migration of Phl p 2, capable of activating mast cells or basophils. Results from basophil activation tests (see the Methods section and Fig E7 in this article’s Online Repository at www.jacionline.org) performed with samples from 3 independent Transwell experiments with basophils from 3 representative allergic patients are shown in Fig 2. The extent of basophil activation induced with basolateral samples showed a significant and consistent reduction for samples taken from cultures in which P2/ICAM1 had been added to the apical compartments (Fig 2, gray bars, +) compared with those in which P2/ICAM1 conjugates had been omitted (Fig 2, gray bars, −). This effect was observed at each of the analyzed time points. Basophil activation induced by samples taken from the apical compartments (Fig 2, black bars) was comparable and almost identical with that obtained with basolateral samples from cell-free preparations (Fig 2, no cells). Basophil activation was not observed when Phl p 2 was absent from the cultures (Fig 2, no Phl p 2). Concentrations of soluble ICAM1 in 16HBE14o- cell cultures were around 1 ng/mL in apical wells and not detectable in basolateral wells in the experiments and had no influence on allergen-induced basophil activation (data not shown).

View Article: PubMed Central - PubMed

Affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

It is becoming increasingly evident that intact epithelial barrier function is important for preventing allergens to reach tissues involved in allergic inflammation... In this study we produced an antibody conjugate bispecific for intercellular adhesion molecule 1 (ICAM1) and a major respiratory allergen (ie, the major grass pollen allergen Phl p 2) termed P2/ICAM1 to investigate whether such a conjugate can inhibit the migration of allergens through respiratory cell layers and reduce the activation of the inflammatory cells underneath... When P2/ICAM1 was added to the cells, it was detected with Alexa Fluor 488 goat anti-mouse IgG specific for αICAM1 mouse IgG (Fig 1, A-C, green)... When P2/ICAM1 was omitted, detection antibodies did not bind (Fig 1, D)... No cell staining was found when only secondary antibodies were applied (data not shown)... In a subsequent series of experiments, we demonstrated that P2/ICAM1 can prevent the apical-to-basolateral penetration of Phl p 2 but not of the birch pollen allergen Bet v 1, a similarly sized but not related allergen, through a layer of the cultured respiratory epithelial 16HBE14o- cells by using a well-established Transwell culture system (Costar Corning Incorporated, Corning, NY; see Figs E5 and E6 in this article’s Online Repository at www.jacionline.org), as described in the Methods section in this article’s Online Repository... Furthermore, we showed that P2/ICAM1 also reacted specifically with natural grass pollen–derived Phl p 2 (data not shown)... In the Transwell experiments we found that the apical addition of P2/ICAM1 prevented the penetration of Phl p 2 over the full period of analysis (ie, for 72 hours) into the basolateral compartment (see Fig E5, A, gray bars, +) when compared with conditions without addition of P2/ICAM1 (Fig E5 A, gray bars, −)... When Phl p 2 and P2/ICAM1 conjugates were omitted, no Phl p 2 signal was detected (see Fig E5, A, no Phl p 2, 72 hours)... Additionally, no relevant penetration of P2/ICAM1–Phl p 2 complexes into the basolateral wells was found (Fig E5, B, gray bars, +)... The extent of basophil activation induced with basolateral samples showed a significant and consistent reduction for samples taken from cultures in which P2/ICAM1 had been added to the apical compartments (Fig 2, gray bars, +) compared with those in which P2/ICAM1 conjugates had been omitted (Fig 2, gray bars, −)... This effect was observed at each of the analyzed time points... Cell-culture supernatants were applied, and P2/ICAM1–bound Phl p 2 was detected with rabbit anti–Phl p 2 antibodies, as described above... The effect of P2/ICAM1 on Phl p 2 apical-to-basolateral transepithelial migration was assessed by comparing P2/ICAM1-1–treated wells with untreated control wells.

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