Mechanisms for induction of acquired host immunity by neutrophil peptide defensins

Abstract

Human neutrophil peptide (HNP) defensins were studied to determine their potential effects on adaptive mucosal immunity. Intranasal delivery of HNPs plus ovalbumin (OVA) enhanced OVA-specific serum IgG antibody (Ab) responses. However, OVA-specific IgA Abs were not induced in mucosal secretions or in serum. CD4(+) T cells of intranasally immunized mice displayed higher OVA-specific proliferative responses and elevated production of interferon gamma, interleukin (IL) 5, IL-6, and IL-10 when compared with control groups receiving OVA alone. In vitro, HNPs also enhanced both proliferative responses and T helper (Th) cytokine secretion profiles of CD3epsilon-stimulated spleen- and Peyer's patch-derived naive CD4(+) T cells. HNPs modulated the expression of costimulatory molecules by lipopolysaccharide- or CD3epsilon-stimulated splenic and Peyer's patch B or T cell populations, respectively. These studies show that defensins enhance systemic IgG, but not IgA, Ab responses through help provided by CD4(+) Th1- and Th2-type cytokines and foster B and T cell interactions to link innate immunity with the adaptive immune system.

Figure 1

Kinetics of serum OVA-specific IgA, IgM, and IgG and IgG subclass Ab responses. After anesthesia, groups of C57BL/6 mice were intranasally immunized three times on days 0, 7, and 14 with 50 μg of OVA and 0.0 or 1.0 μg of HNPs in 10 μl of PBS. The data presented are the mean Ab titers ± SEM of these experiments. Experimental groups consisted of five mice, and studies were repeated three times. The data distribution of OVA-specific IgA, IgM, and IgG serum Abs collected on day 14 (A) and day 21 (B) was determined by ELISA. The profile of Ag-specific IgG subclasses of serum samples taken on day 14 (C) and day 21 (D) also was determined by ELISA. The data presented are the mean Ab titer ± SEM. Asterisks indicate statistically significant differences (∗, P < 0.05; ∗∗, P < 0.01) relative to Ab titers of mice immunized with OVA alone.

Figure 2

Numbers of Ag-specific AFCs in peripheral and mucosal tissues after intranasal immunization with HNPs and OVA. After anesthesia, groups of C57BL/6 mice were intranasally immunized on days 0, 7, and 14 with 50 μg of OVA and 0.0 or 1.0 μg of HNPs in 10 μl of PBS. Experimental groups consisted of five mice, and studies were repeated three times. OVA-specific IgA, IgM, and IgG AFCs present in cervical lymph nodes, lower respiratory tract and associated lymphoid tissues, Peyer’s patches, mesenteric lymph nodes, and spleens were determined by ELISPOT analysis. The data presented are the mean AFCs ± SEM 1 week after the last immunization of these experiments. Asterisks indicate statistically significant differences (∗, P < 0.05; ∗∗, P < 0.01) relative to Ab titers of mice immunized with OVA alone.

Figure 3

OVA-specific proliferation and induction of Th1- and Th2-type cytokine secretion by spleen- and mucosa-derived CD4⁺ T cells. After anesthesia, groups of C57BL/6 mice were intranasally immunized on days 0, 7, and 14 with 50 μg of OVA and 0.0 (empty boxes) or 1.0 μg (filled boxes) of HNPs in 10 μl of PBS. One week after the last immunization, lower respiratory tract, Peyer’s patches, cervical, mesenteric, and spleen lymphoid tissue-derived CD4⁺ T cells were purified and cultured at a density of 5 × 10⁶ cells/ml with 500 μg/ml of OVA for 3 days with T-cell-depleted and -irradiated splenic feeder cells (1 × 10⁶ cells/ml) in complete medium. Experimental groups consisted of five mice, and studies were repeated three times. Proliferation was measured by ³H-thymidine incorporation. The stimulation index corresponds to the cpm of cell cultures containing OVA divided by the cpm of cultures with no additions. The data presented are the mean stimulation index ± SEM. ∗ indicate statistically significant differences (∗, P < 0.05; ∗∗, P < 0.01) relative to the stimulation index of mice immunized with OVA alone. Cytokine protein production of these cultured supernatants was determined by ELISA. The data presented are the mean cytokine levels (pg/ml) ± SEM in each group. Asterisks indicate statistically significant differences (∗, P < 0.05; ∗∗, P < 0.01) relative to cytokine levels of mice immunized with OVA alone.

Figure 4

Anti-CD3ɛ and HNP-mediated proliferative responses and cytokine secretion by naive CD4⁺ T lymphocytes. Spleen- (⧫) or Peyer’s patch-derived (◊) cells were isolated from naive mice and stimulated in vitro in rat anti-mouse CD3ɛ-coated 96-well plates with 0, 10, 100, or 1,000 ng/ml of HNPs. Cytokine protein production of cultured supernatants containing a suboptimal dose of anti-mouse CD3 mAb and 0 (□) or 10 ng/ml (■) of HNPs was determined by ELISA. Experimental studies were repeated three times, and the data presented are the mean proliferation ± SEM measured by ³H-thymidine incorporation and illustrated as cpm or the mean cytokine levels ± SEM in each group. Asterisks indicate the statistically significant differences (P < 0.05) relative to the cpm of or cytokine levels of CD4⁺ T cells incubated without HNPs.