In humans, infection of the cornea with HSV results in a recurrent
immune-mediated inflammatory response, HSK, which is one of the most
common infectious cause of blindness in many countries. There seems to
be general agreement that HSK largely represents an immunopathologic
disease, and clinicians usually treat the lesions with
anti-inflammatory drugs along with or even without anti-herpesvirus
drugs. The mechanistic nature of HSK remains uncertain, but from
studies in experimental animals, evidence for a variety of mechanisms
has been forthcoming. These include toxic immune complexes,
delayed-type hypersensitivity, and lymphocyte
cytotoxicity.
3 27 Productive infection with HSV is crucial
for HSK manifestation. NO production by different cellular types in
response to viral infection or cytokines may be one of the mechanisms
of innate immunity involved in limiting viral replication after ocular
infection with HSV.
In this work, we investigated the effect of AMG, an iNOS inhibitor, on
the ocular infection of Balb/c mice that had been infected with HSV-1
or HSV-2. We observed an increase in viral titers in ocular washings,
eye tissue, and brains of treated animals. Viral DNA also was detected
earlier in trigeminal ganglia of treated mice. This correlates with the
observed augmentation in mortality and neuropathologic signs in the
treated groups. All these data agree with previous reports from other
investigators showing antiviral activity in vivo of NO against
different viruses,
8 9 15 17 20 thus indicating a role of
NO in the natural resistance against ocular infection with HSV. This
was supported by RT-PCR experiments showing iNOS mRNA induction in
corneas of infected mice. In treated animals, we also observed an
earlier appearance together with an aggravation of the symptoms
associated with HSK, such as blepharitis, edema, neovascularization,
and ulceration.
HSK in mice involves a chronic immune-mediated inflammatory response
that leads to total destruction of the corneal architecture and to
corneal perforation. The corneal inflammation characterized
predominantly by polymorphonuclear neutrophil (PMN) infiltration and T
cells belonging to the CD4
+ population is
regulated by the Th1 cytokine interleukin (IL)-2, tumor necrosis factor
(TNF)-α, and interferon (IFN)-γ.
28 The PMNs cause
progressive destruction of the corneal tissue, which appears to be
responsible for the blinding complications of HSV corneal infections in
humans. The Th2 cytokines IL-4 and IL-10 are not detected in cells that
infiltrate the HSV-infected corneas. IL-10 injection in the infected
cornea was shown to inhibit corneal inflammation.
29 A
replication-induced proinflammatory milieu in the cornea may be crucial
for the subsequent progression of HSK, possibly because of enhancement
of the expression of corneal agonists that drive HSK
manifestation.
30 Productive infection with HSV resulted in
rapid upregulation and sustained expression of chemokines such as
N51/KC, macrophage inflammatory protein (MIP)-1β, MIP-2, monocyte
chemotactic protein (MCP)-1, or such cytokines as IL-1, IL-6, IL-8,
IL-12, and TNF-α.
31 32 33 In particular, IL-8 has been
pointed out as an important chemokine during HSK.
34
In this work, we observed that AMG-treated animals showed increased PMN
infiltration in corneas at PI days 3 and 5 compared with
control animals. Recently, it has been reported that peroxynitrite,
formed by the reaction between NO and superoxide, regulates cytokine
function during inflammation. Peroxynitrite attenuates neutrophil and
monocyte chemotaxis induced by MIP-1α and IL-8 in a dose-dependent
manner, possibly by inhibition of chemokine binding to neutrophils and
monocytes.
35 36 Thus, we hypothesize that the observed
increase in the influx of PMNs to corneas of treated mice could be due
to inhibition of NO production by AMG treatment during HSV infection,
thus suppressing the NO-inhibitory effect on PMN chemotaxis. In the
same way, preliminary data from our laboratory obtained by the RT-PCR
technique indicate an earlier TNF-α response in corneas of
AMG-treated animals, although no differences were observed in IFN-γ
induction between treated and control animals (data not shown). These
data could be consistent with previous reports indicating an important
chemokine (MIP-2, IL-8)-inducing activity of TNF-α
37 38 39 and enhanced Th1 responses as a consequence of NO
impairment.
40 41 We are currently investigating this
issue.
Last, although we observed that inhibition of iNOS during the first
days of infection increased HSV ocular infection, according to Fuji et
al.,
42 intraperitoneal treatment of HSV-1 rats infected
intranasally with
N-monomethyl-
l arginine (
l-NMMA) from PI days 3 through 7
decrease neurologic symptoms and increased survival of treated animals.
They demonstrated that NO production is related to histopathologic
changes in the brain during infection. Thus, although NO production can
be beneficial as an antiviral effector against HSV and other viruses,
it also may be detrimental by contributing to disease during immune
responses, as previously reported.
43 44 Thus, to unravel
the role of NO in the natural resistance to HSV infections, it may be
crucial to determine the stage of viral disease in which this molecule
exerts its major effect.
The authors thank Juan Flo for valuable help in molecular biology.