FcgammaRI (CD64) contributes substantially to severity of arthritis, hypersensitivity responses, and protection from bacterial infection.

The high-affinity receptor for IgG, FcgammaRI, shares its capacity to bind IgG2a immune complexes (IgG2a-IC) with the low-affinity receptor FcgammaRIII and complement factors, hampering the definition of its biological role. Moreover, in vivo, FcgammaRI is occupied by monomeric IgG2a, reducing its accessibility to newly formed IgG2a-IC. By using a variety of FcgammaR(-/-) mice, we demonstrate that in the absence of FcgammaRI, the IgG2a-IC-induced cellular processes of phagocytosis, cytokine release, cellular cytotoxicity, and antigen presentation are impaired. FcgammaRI(-/-) mice showed impaired hypersensitivity responses, strongly reduced cartilage destruction in an arthritis model, and impaired protection from a bacterial infection. We conclude that FcgammaRI contributes substantially to a variety of IgG2a-IC-dependent immune functions and immunopathological responses.

Fc receptor-mediated immunity against Bordetella pertussis.

The relevance of specific Abs for the induction of cellular effector functions against Bordetella pertussis was studied. IgG-opsonized B. pertussis was efficiently phagocytosed by human polymorphonuclear leukocytes (PMN). This process was mediated by the PMN IgG receptors, FcgammaRIIa (CD32) and FcgammaRIIIb (CD16), working synergistically. Furthermore, these FcgammaR triggered efficient PMN respiratory burst activity and mediated transfer of B. pertussis to lysosomal compartments, ultimately resulting in reduced bacterial viability. Bacteria opsonized with IgA triggered similar PMN activation via FcalphaR (CD89). Simultaneous engagement of FcalphaRI and FcgammaR by B. pertussis resulted in increased phagocytosis rates, compared with responses induced by either isotype alone. These data provide new insights into host immune mechanisms against B. pertussis and document a crucial role for Ig-FcR interactions in immunity to this human pathogen.

Immunoglobulin A-mediated protection against Bordetella pertussis infection.

Infection with Bordetella pertussis, the causative agent of pertussis (whooping cough) in humans, is followed by the production of antibodies of several isotypes, including immunoglobulin A (IgA). Little is known, however, about the role of IgA in immunity against pertussis. Therefore, we studied targeting of B. pertussis to the myeloid receptor for IgA, FcalphaRI (CD89), using either IgA purified from immune sera of pertussis patients or bispecific antibodies directed against B. pertussis and FcalphaRI (CD89 BsAb). Both IgA and CD89 BsAb facilitated FcalphaRI-mediated binding, phagocytosis, and bacterial killing by human polymorphonuclear leukocytes (PMNL) and PMNL originating from human FcalphaRI-transgenic mice. Importantly, FcalphaRI targeting resulted in enhanced bacterial clearance in lungs of transgenic mice. These data support the capacity of IgA to induce anti-B. pertussis effector functions via the myeloid IgA receptor, FcalphaRI. Increasing the amount of IgA antibodies induced by pertussis vaccines may result in higher vaccine efficacy.

Targeting to Fcgamma receptors, but not CR3 (CD11b/CD18), increases clearance of Bordetella pertussis.

In the absence of opsonizing antibodies, Bordetella pertussis, the causative agent of pertussis, readily binds to phagocytes via complement receptor 3 (CR3). After opsonization with antibodies, binding is mediated by IgG receptors (FcgammaR). The effect of targeting B. pertussis to either FcgammaR or CR3 was studied. The fate of unopsonized B. pertussis, IgG-opsonized B. pertussis, and B. pertussis opsonized with bispecific antibodies (BsAbs) directed to CR3 or FcgammaRII/-III was compared. IgG antibodies mediated binding and phagocytosis of B. pertussis via FcgammaR by polymorphonuclear leukocytes (PMNL) in vitro. Opsonization of B. pertussis with BsAbs directed against either CR3 or FcgammaRII/-III facilitated PMNL phagocytosis; however, in vivo studies with BsAb revealed that FcgammaR-mediated uptake facilitates B. pertussis clearance, in contrast to uptake via CR3. Targeting of B. pertussis to FcgammaRII/-III in mice deficient in FcgammaRII or FcgammaRIII indicated that the protective effect is attributable to FcgammaRIII. Competition between uptake via CR3 or FcgammaR may determine the outcome of natural infection.

Evidence for an intracellular niche for Bordetella pertussis in broncho-alveolar lavage cells of mice.

Bordetella pertussis can attach, invade and survive intracellularly in human macrophages in vitro. To study the significance of this bacterial feature in vivo, we analyzed the presence of viable bacteria in broncho-alveolar lavage (BAL) cells of mice infected with B. pertussis. We found B. pertussis to be present in a viable state in BAL fluid cells until at least 19 days after infection, suggesting B. pertussis to be able to survive in those cells. This intracellular niche may play an important role in the pathogenesis of pertussis. Pertussis toxin and the RGD sequence of the virulence factor filamentous hemagglutinin (FHA) both play a role in the attachment of B. pertussis to human and mouse macrophages in vitro and we hypothesized these virulence factors to be required for invasion and subsequent intracellular survival of B. pertussis in macrophages in vivo. A B. pertussis double mutant, in which the FHA RGD motif was changed to RAD and the ptx genes were deleted, was also found in a viable state in BAL fluid cells, albeit at lower levels than the wild-type strain. In our model, uptake of B. pertussis by alveolar phagocytes in vivo is thus, at least in part, determined by the bacterial virulence factors FHA and pertussis toxin.

CD44 is involved in tumor angiogenesis; an activation antigen on human endothelial cells.

CD44 is described to be an activation molecule in a number of different cell types. We investigated the role of CD44 on human endothelial cells (EC) and in tumor angiogenesis. Using flow cytometry we showed that EC from the vasculature of human solid tumors display an enhanced expression of CD44 as compared to EC from normal tissue. This finding was confirmed by immunohistochemical studies on frozen tissue sections. Because tumors are dependent on angiogenesis, the role of angiogenic stimuli in the enhanced CD44 expression was investigated. We found that basic fibroblast growth factor (bFGF) and vascular endothelial growth factor were able to efficiently upregulate CD44 expression on cultured human EC. The upregulation reached maximal levels after treatment for 3 days with 10 ng/mL bFGF. The physiological impact of this upregulation was shown by the enhanced binding of EC to hyaluronate after pretreatment with bFGF. In a next set of studies that were designed to unravel the regulation of CD44 expression on EC we concluded that CD44 is an activation antigen on human EC since (1) human umbilical vein derived endothelial cells, which in vivo do not express CD44, begin to express CD44 when plated and cultured, (2) CD44 expression is enhanced after subculture of confluent cultures, (3) CD44 is predominantly expressed on the BrdU incorporating subset of cultured EC. The specific expression of CD44 on activated and tumor EC prompted us to study the usefulness of CD44 as an endothelial target for therapy with immunotoxins. In vitro experiments showed that EC are efficiently killed after targeting immunotoxin to CD44.

Endothelial CD34 is suppressed in human malignancies: role of angiogenic factors.

We report the suppressed vascular CD34 expression in renal cell carcinoma. This was found by quantitatively analyzing CD34 expression on normal and tumor derived EC by flow cytometry. In vitro studies revealed that culture of umbilical cord or dermis derived microvascular EC with angiogenic factors such as basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor induced downregulation of CD34. This angiogenesis-induced downregulated expression of CD34 adhesion molecule may contribute to the tumor mediated escape mechanism from immune surveillance. It is concluded that there are quantitative differences in expression of endothelial CD34 in different compartments of the vasculature, that angiogenic factors affect this expression and that subpopulations of EC exist with differences in EAM expression.