Both persistence of FNf and a low percentage of CD49e-positive monocytes in the blood of HIV-infected patients were associated with increased long-term mortality. In both cases, these associations were independent of viral load or CD4 cell count. All but two of the deaths in this study occurred for 1.3 or more years after we stopped testing the patients’ blood, suggesting that once they commence, the mechanisms responsible for generating FNf and depleting monocytes’ CD49e [1
] continue for many years, despite therapy.
We emphasize that it is the fragments of fibronectin, not the native molecule, that are associated with adverse outcomes. Our previous measurements and those of others have demonstrated that concentrations of native 450-kDa plasma fibronectin are not abnormal in HIV-infected patients [16
Recent research has linked inflammation, exemplified by high levels of acute phase proteins, D-dimer and IL-6 with increased all-cause mortality in HIV infections [17
]. Our studies help to explain how inflammation, normally considered a beneficial response to infection, could accelerate disease progression. A central feature of inflammation is leukocyte activation. Lysosomal proteases, released by activated neutrophils and monocyte/macrophages, readily degrade plasma and matrix fibronectin, producing FNf and other fragments that can stimulate leukocytes and modulate host–pathogen interactions. For example, FNf-stimulated monocytes produce tumor necrosis factor-alpha (TNFα) [18
]. TNFα and other proinflammatory cytokines stimulate the synthesis of the lysosomal proteases that breakdown fibronectin and release FNf. FNf, acting together with TNFα, stimulate HIV-infected and uninfected leukocytes to cross endothelial tight junctions and cluster together in the perivascular matrix, creating an environment wherein transmission of virus from infected to uninfected leukocytes readily occurs [5
]. In effect, a positive feedback loop involving TNFα and other proinflammatory cytokines, leukocyte proteases, fibronectin breakdown products [19
], including the III1-C fibronectin fragments that can stabilize budding virus and promote cell-to-cell virus transmission [12
], may help to produce the depots of infected cells, relatively inaccessible to antiretroviral drugs, that perpetuate this disease.
In addition to the effects of FNf on the propagation and dissemination of virus-infected cells, these fragments may also accelerate disease progression by interfering with adaptive immune responses to opportunistic infections. Exposure to FNf for as little as 15 min in vitro
causes monocytes to produce sufficient IL-10 and TGFβ to suppress proliferation of activated T cells [6
]. Thus, FNf, produced as a result of the proteolysis of plasma or tissue fibronectin by leukocyte [3
] or viral proteases [20
], may cause host monocytes to become ‘suppressor’ cells that further impair the already compromised immune response system of HIV-infected hosts.
Although more than 60% of the HIV-infected patients had FNf in their plasma at least once, we never found FNf in serial samples collected concurrently from 17 normal controls [3
]. As FNf are generated by the unopposed actions of diverse proteases, this comparison suggests that the generation of protease by HIV can outstrip the host’s ability to produce proteinase inhibitors. The concentrations of active, functional α-1 antitrypsin in patients with advanced HIV infections are reportedly low to normal [22
]. As α-1 antitrypsin is an acute phase reactant, its concentration should rise with inflammation [22
]. Low-to-normal α-1 antitrypsin levels in plasma from HIV-infected people, beset by a persistent inflammatory response engendered by this chronic virus infection, may indicate either that this proteinase inhibitor is produced in subnormal amounts or that it is being consumed and/or inactivated at an accelerated rate. Prior experience favors the latter explanation. In 15 plasma samples that contained between 38 and 100 μg/ml FNf, we previously found that α-1 antitrypsin was often taken up in high molecular weight complexes with elastase, proteinase-3 or cathepsin G [3
]. In these blood samples, the fraction of monocytes that expressed CD49e was reciprocally related to the quantity of α-1 antitrypsin that was incorporated in these complexes [3
]. As α-1 antitrypsin in complexed form is not available to bind and inactivate proteinase 3, as it is expressed on the monocyte cell surface, it follows that CD49e on monocytes in blood containing FNf is at risk of being hydrolyzed.
Thus, deficiency of CD49e on patients’ circulating monocytes likely indicates that their microenvironment is deficient in functional proteinase inhibitors, most particularly α-1 antitrypsin, the inhibitor that opposes the protease responsible for FNf-induced loss of monocyte CD49e [3
]. Increased mortality was as significantly associated with persistently reduced monocyte CD49e as with the persistence of FNf in these patients’ blood samples. Unfortunately, we were unable to systematically measure the relationship between monocyte CD49e display and α-1 antitrypsin function in the plasma samples of all patients; the potential importance of such an analysis became apparent only when we examined the 5-year survival results, by which time the antiprotease activity of the stored plasma could no longer be reliably measured.
α-1 antitrypsin, by itself, has antiretroviral activity. Physiological concentrations of this antiproteinase block HIV-1 infection of permissive target cells and inhibit viral replication in previously infected peripheral blood mononuclear cells [24
]. Delivery of α-1 antitrypsin genes into cell lines and into primary human lymphocytes blocks HIV gp160 and p55 processing [25
]. A carboxyterminal fragment of α-1 antitrypsin reportedly suppresses HIV transcription [26
]. Thus, loss of functional α-1 antitrypsin in HIV-1-infected people may deprive the host of an element of the innate immune system that helps to control HIV replication. Increased viral replication, in turn, likely increases the inflammatory responses producing the proteases that generate FNf and inactivate α-1 antitrypsin.
Although HIV-specific aspartyl protease inhibitors have clearly had a major impact on the treatment of HIV-1 infections [27
], the results of this study suggest that agents that suppress tissue injury mediated by other classes of proteolytic enzymes [28
] or that amplify naturally occurring proteinase inhibitors may also contribute significantly to the control of this retroviral infection [24
]. Indeed, if future studies show an association among FNf, depressed monocyte CD49e expression and depletion of functional α-1 antitrypsin, exploration of the effects of treatment with α-1 antitrypsin should be considered. In the interim, monitoring circulating FNf, or more easily, from a technical standpoint, the fraction of blood monocytes that express CD49e, may help to identify patients in whom the inflammatory response to this chronic infection may adversely affect prognosis.