Abstract
The aim of this study was to evaluate the immunoregulatory effects of recombinant human lactoferrin (rhLF) in two in vitro models: (1) the secondary humoral immune response to sheep erythrocytes (SRBC); and (2) the mixed lymphocyte reaction (MLR). We compared the non-sialylated glycoform of rhLF as expressed by glycoengineered Pichia pastoris with one that was further chemically sialylated. In an earlier study, we showed that sialylated rhLF could reverse methotrexate-induced suppression of the secondary immune response of mouse splenocytes to SRBC, and that the phenomenon is dependent on the interaction of lactoferrin (LF) with sialoadhesin (CD169). We found that the immunorestorative activity of sialylated rhLF is also dependent on its interaction with the CD22 antigen, a member of the immunoglobulin superfamily that is expressed by B lymphocytes. We also demonstrated that only sialylated rhLF was able to inhibit the MLR reaction. MLR was inhibited by bovine lactoferrin (bLF), a glycoform that has a more complex glycan structure. Desialylated bLF and lactoferricin, a bLF-derived peptide devoid of carbohydrates, did not express such inhibitory activity. We showed that the interaction of LF with sialic acid receptors is essential for at least some of the immunoregulatory activity of this glycoprotein.
Highlights
Lactoferrin (LF) is an iron-binding protein contained in the exocrine fluids of mammals
In the immunosuppressed immune response mediated in vitro by MTX, which acts as a proapoptotic agent [43, 44], B cells may require triggering of a stimulatory signal via CD22 to develop the optimal immune response
CD22 receptors on most B cells are occupied by endogenous sialylated ligands mediating the inhibitory signals, a proportion of B cells exist with an activated phenotype with unmasked CD22 [45], and are available for recombinant human lactoferrin (rhLF) binding
Summary
Lactoferrin (LF) is an iron-binding protein contained in the exocrine fluids of mammals. LF, as a cationic molecule, interacts with lipopolysaccharide (LPS) in the serum It effectively competes with LPS-binding protein (LBP), preventing the binding of the LPS–LBP complex to the CD14 receptor [29]. We identified the receptor responsible for the upregulatory action of hLF as the CD169 antigen ( known as sialoadhesin or siglec-1) [28], which has an N-terminal domain that contains the sialic acid-binding site, mainly found on macrophages [32] acting as professional accessory cells [33] and antigen-presenting cells [34]. To further explore LF–cell receptor interactions, we evaluated the role of the CD22 molecule, another siglec family lectin that is present on B cells [35], in the in vitro immunorestorative activity of rhLF in methotrexate-induced suppression of the humoral immune response. We attempt to assess the importance of sialic acid in human and bovine milk-derived LFs in the suppression of the human and mouse two-way mixed lymphocyte reaction
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