Intratracheal exposure to Fab fragments of an allergen-specific monoclonal antibody regulates asthmatic responses in mice

Abstract

Fab fragments (Fabs) maintain the ability to bind to specific antigens but lack effector functions due to the absence of the Fc portion. In the present study, we tested whether Fabs of an allergen-specific monoclonal antibody (mAb) were able to regulate asthmatic responses in mice. Asthmatic responses were induced in BALB/c mice by passive sensitization with anti-ovalbumin (OVA) polyclonal antibodies (pAbs) (day 0) and by active sensitization with OVA (days 0 and 14), followed by intratracheal (i.t.) challenge with OVA on day 1 and days 28, 29, 30 and 35. Fabs prepared by the digestion of an anti-OVA IgG1 (O1-10) mAb with papain were i.t. administered only once 30 min before antigenic challenge on day 1 or day 35. The results showed that i.t. administration of O1-10 Fabs with OVA markedly suppressed the early and/or late phases of asthmatic responses caused by passive and active sensitization. Similar results were obtained when Fabs of anti-OVA IgG2b mAb (O2B-3) were i.t. administered. In contrast, neither i.t. injection of intact 01-10/O2B-3 nor systemic injection of O1-10 Fabs suppressed the asthmatic responses. In vitro studies revealed that the capture of OVA by O1-10 Fabs prevented the subsequent binding of intact anti-OVA pAbs to the captured OVA. These results suggest that asthmatic responses may be down-regulated by the i.t. exposure to Fabs of an allergen-specific mAb via a mechanism involving the capture of allergen by Fabs in the respiratory tract before the interaction of intact antibody and allergen essential for the induction of asthmatic responses.

Keywords: Fab; asthma; intratracheal exposure; monoclonal antibody.

Figure 1

Intratracheal (i.t.) exposure to O1-10 Fabs 30 min before antigenic challenge suppresses asthmatic responses. Mice sensitized (S) with anti-ovalbumin (OVA) polyclonal antibodies (pAbs) on day 0 were i.t. challenged (C) with 40 μg OVA on day 1 (a) followed by measurement of specific airway resistance (sRaw) (b). One hundred and twenty micrograms of O1-10 Fabs (S-C-O1-10 Fabs) and control Fabs (S-C-control Fabs) were i.t. administered 30 min before the antigenic challenge. Non-sensitized (NS) mice given OVA were also i.t. treated with O1-10 Fabs (NS-C-O1-10 Fabs). Mice were also sensitized (S) with OVA on days 0 and 14 and i.t. challenged (C) with 200 μg of OVA on days 28, 29 and 30, and with 40 μg on day 35 (c). After the fourth antigenic challenge, sRaw (d), inflammatory cells including macrophages, lymphocytes, neutrophils and eosinophils in bronchoalveolar lavage fluid (BALF) (e), mouse mast cell protease-1 (mMCP-1) in sera and C3a in BALF (f) were measured. One hundred and twenty micrograms of O1-10 Fabs (S-C-O1-10 Fabs) and control Fabs (S-C-control Fabs) were i.t. administered only once 30 min before the fourth challenge with OVA on day 35. Non-sensitized (NS) mice given OVA were also i.t. administered O1-10 Fabs (NS-C-O1-10 Fabs). Data are means ± SEM of five to seven mice. *P < 0·05 versus control Fabs. Data are representative of three independent experiments.

Figure 2

Intratracheal (i.t.) exposure to O1-10 Fabs reduces the infiltration of neutrophils and production of interleukin-1β (IL-1β) in the lung tissue of mice with asthmatic responses. Mice sensitized (S) with ovalbumin (OVA) on days 0 and 14 were i.t. challenged (C) with 200 μg OVA on days 28, 29 and 30, and with 40 μg on day 35 as shown in Fig. 1(a). One hundred and twenty micrograms of O1-10 Fabs (S-C-O1-10 Fabs) and control Fabs (S-C-control Fabs) were i.t. administered once 30 min before the fourth challenge with OVA on day 35. Non-sensitized (NS) mice given OVA were also i.t. treated with O1-10 Fabs (NS-C-O1-10 Fabs). Twenty-four hours after the fourth antigenic challenge, histological changes in the lung (a–c, ×40) were examined by haematoxylin and eosin staining. Neutrophils and eosinophils are shown by red and black arrows, respectively. Gr-1⁺ cells (e–g, ×40) and IL-1β (i–k, ×20) in the lung were also immunohistochemically stained. Scale bars in all photographs represent 100 μm. Neutrophil infiltration and IL-1β production in the lung tissue were also scored as shown in Materials and methods (d, h, l). Data are means ± SEM of five mice. *P < 0·05 versus S-C-control Fabs. Data are representative of two independent experiments.

Figure 3

Effect of the intratracheal (i.t.) exposure to O1-10 Fabs on the production of various chemokines and cytokines in the lung of mice with asthmatic responses. Mice were sensitized (S) with ovalbumin (OVA) on days 0 and 14 and i.t. challenged (C) with 200 μg OVA on days 28, 29 and 30, and with 40 μg on day 35 as shown in Fig. 1(a). One hundred and twenty micrograms of O1-10 Fabs (S-C-O1-10 Fabs) and control Fabs (S-C-control Fabs) were i.t. administered only once 30 min before the last (fourth) challenge with OVA on day 35. Non-sensitized (NS) mice given OVA were also i.t. administered O1-10 Fabs (NS-C-O1-10 Fabs). Keratinocyte-derived chemokine (KC) (a), macrophage inflammatory protein 2 (MIP-2), (b), interleukin-1β (IL-1β) (c), IL-6 (d), IL-4 (e), IL-5 (f), IL-13 (g), IL-17A (h), and IL-23 (i) from the lung homogenate obtained 24 hr after the last antigenic challenge were measured as described in the Materials and methods. Data are means ± SEM of five to seven mice. *P < 0·05 versus control Fabs.

Figure 4

Intratracheal (i.t.) exposure to O1-10 Fabs suppresses anti-ovalbumin (OVA) IgE monoclonal antibody (mAb) OE-1-mediated asthmatic responses. Mice sensitized (S) with 100 μg anti-OVA IgE mAb OE-1 on days 0, 1, 2, 7, 8, 9 and 15 were i.t. challenged (C) with 200 μg OVA on days 1, 2, 3, 8, 9 and 10 and with 40 μg OVA on day 16 (a). After the last (seventh) antigenic challenge, specific airway resistance (sRaw) (b), inflammatory cells including macrophages, lymphocytes, neutrophils, and eosinophils in bronchoalveolar lavage fluid (BALF) (c), mouse mast cell protease-1 (mMCP-1) in sera, and C3a in BALF (d) were measured. One hundred and twenty micrograms of O1-10 Fabs (S-C-O1-10 Fabs) or control Fabs (S-C-control Fabs) were i.t. administered 30 min before the seventh challenge with OVA. Non-sensitized (NS) mice given OVA were also i.t. treated with O1-10 Fabs (NS-C-O1-10 Fabs). Data are means ± SEM of five mice. *P < 0·05 versus S-C-control Fabs. Either 50 μg/ml OE-1 alone or a mixture of 50 μg/ml OE-1 and 0·5, 5, 50 or 500 μg/ml O1-10 Fabs was added to OVA coated on 96-well plates followed by further addition of anti-Fc of IgE (e). Anti-OE-1 Fabs were used as a positive control. Keyhole limpet haemocyanin was used as a control antigen. Data are representative of two independent experiments.

Figure 5

Intratracheal (i.t.) exposure to O2B-3 Fabs suppresses asthmatic responses. Mice sensitized (S) with ovalbumin (OVA) on days 0 and 14 were i.t. challenged (C) with 200 μg of OVA on days 28, 29 and 30 and with 40 μg of OVA on day 35 (a). After the fourth antigenic challenge, specific airway resistance (sRaw) (b), inflammatory cells including macrophages, lymphocytes, neutrophils and eosinophils in bronchoalveolar lavage fluid (BALF) (c), mouse mast cell protease-1 (mMCP-1) in sera, and C3a in BALF (d) were measured. One hundred and twenty micrograms of O2B-3 Fabs (S-C-O2B-3 Fabs) or control Fabs (S-C-control Fabs) were i.t. administered once 30 min before the last antigenic challenge with OVA on day 35. Non-sensitized (NS) mice given OVA were also i.t. treated with O1-10 Fabs (NS-C-O1-10 Fabs). Data are means ± SEM of five mice. *P < 0·05 versus control Fabs. Fifty micrograms of intact O1-10 alone or a mixture of 50 μg/ml intact O1-10 and 0·5, 5, 50 or 500 μg/ml O2B-3 Fabs was added to OVA coated on 96-well plates followed by further addition of anti-Fc of IgG to the plates (e). Anti-O1-10 Fabs were used as a positive control. Keyhole limpet haemocyanin was used as a control antigen. Data are representative of two independent experiments.

Figure 6

The capture of ovalbumin (OVA) by O1-10 Fabs in advance prevents the subsequent binding of small but not large amounts of intact anti-OVA polyclonal antibodies (pAbs) or intact OE-1 to the captured OVA. The indicated amounts of O1-10 Fabs were added to OVA coated on 96-well plates. This was followed by further addition of 0·05, 0·5 and 5 μg/ml intact anti-OVA pAbs (a) or 1, 3 or 30 μg/ml intact OE-1 (b) to the plates. Then anti-Fc of IgG or IgE was added to investigate the ability of these intact antibodies to bind OVA. Anti-OVA pAb Fabs or anti-OE-1 Fabs were used as a positive control. *P < 0·05 versus control Fabs. Data are representative of three independent experiments.