Production of virus from infected cell cultures was monitored by
measuring the levels of p27gag antigen in
cell-free culture supernatants. Production of SIVmac239 generally did not exceed 1 ng of p27 per ml from these tissue macrophage cultures. Figure 1B and C show the results of
independent analyses with BAL cultures from two different rhesus
monkeys. Peak Gag antigen production resulting from infection by
SIVmac239/316E was 100 to 1,000 times higher than
what was observed with SIVmac239 (Fig. 1B and C), consistent
with our prior results (42, 43). Increasing the multiplicity
of infection (MOI) over a 100-fold range did not appear to affect the
peak height of production of either virus, but it did appear to affect
the kinetics of appearance of p27 antigen for both viruses (Fig. 1B
and C). Virus production peaked much earlier with
SIVmac239 than with SIVmac239/316E (Fig. 1B and
C). The titers of infectious virus produced early after infection
paralleled the p27 antigen measurements (Fig.
2).
Although replication of SIVmac239 is severely restricted in
alveolar macrophages, synthesis of viral DNA by reverse transcription was previously found to be normal or near normal for 24 h of
observation after exposure of cells to SIVmac239 (42,
43). We extended these earlier findings by extending the period
of observation for up to 8 days. As was observed previously, levels of
newly synthesized viral DNA at 1 day postinfection (dpi) were
comparable between SIVmac239 and SIVmac239/316E
(Fig. 3A to C). After 1 dpi, however, the
patterns diverged dramatically. Levels of viral DNA in macrophages
infected with SIVmac239/316E increased dramatically between 1 and 6 dpi concomitant with the spread of the infection through the culture (see also infectious center assays described below). Levels of viral DNA did not increase when dissemination of
infection was blocked by reverse transcriptase inhibitors (RTIs) (Fig.
3B). Levels of viral DNA in macrophages infected with
SIVmac239 decreased dramatically after 1 day (Fig. 3A). The
pattern of decreasing SIVmac239 DNA levels occurred similarly
with or without the addition of RTIs at 24 h postinfection (p.i.).
These differences in viral DNA kinetics were observed in four of four
independent experiments (Fig. 3D). These results suggested instability
of SIVmac239 DNA in infected cells or a loss of infected
cells from the cultures by cell death. They also indicated a failure of
SIVmac239 to spread through the cultures.
We next examined the numbers of infected cells over time in these
macrophage cultures. The proportion of infected cells varied with the
macrophage preparation (Table 1). Whereas
25 and 1.6% of cells were infected with SIVmac239/316E
at 1 dpi with two independent macrophage preparations, 3.1 and 0.9% of
the macrophages were infected with SIVmac239 at the same
time in the same two macrophage preparations (Table 1). By 3 dpi,
most of the cells in the culture were infected by
SIVmac239/316E. However, the numbers of cells infected
with SIVmac239 did not increase over the same period; in
fact, they decreased (Table 2). These
results are consistent with a small proportion of cells susceptible to
SIVmac239 and a failure of SIVmac239 to spread to
the vast majority of cells in the cultures.
We also examined the susceptibility of cells to reexposure to
SIVmac239 subsequent to the initial round of infection by
SIVmac239. Aliquots of the macrophages for each of these
experiments were exposed to SIVmac239 on day 0 (2 days after
plating), and identical aliquots were left unexposed. Levels of viral
DNA were measured at days 1, 2, 3, 4, and 5 (Table 2, experiment A). On
day 5, cells (both previously exposed and unexposed) were again given SIVmac239, and levels of viral DNA were measured 1 day later. The results indicated that on day 5, SIVmac239 was able to
infect a susceptible population of cells in the cultures that had not been previously exposed to virus, but few or no susceptible cells remained in the cultures that had been previously exposed to
SIVmac239. An independent test gave similar results (Table 2,
experiment B). When SIVmac239/316E was used as the second
infecting virus strain, it was able to establish infection in an
uncompromised fashion (data not shown). These results are consistent
with the above findings indicating that only a small minority of cells are susceptible to SIVmac239 in these alveolar macrophage cultures.
We analyzed levels of viral RNA by RNA PCR with the two sets of primers
shown in Fig. 4A. RNAs for gag
and tat/rev were analyzed. In
SIVmac239/316E-infected cells, viral RNA levels
increased from days 1 to 2 and remained high 2 and 3 dpi during
the spreading infection (Fig. 4B). In SIVmac239-infected cells,
levels of viral RNA increased until 2 dpi and then decreased
(Fig. 4B), consistent with the viral DNA analysis. Viral RNA
levels at 2 days after infection with SIVmac239/316E were
approximately five times higher than those observed with
SIVmac239 (Fig. 3B), consistent with slightly increased
levels of SIVmac239/316E DNA at 1 dpi.
Flow cytometric analysis of BAL specimens revealed two populations of
cells as shown in Fig. 5, alveolar
macrophages (R2) and a minor population of lymphocytes (R1). The
phenotype of these cells was confirmed by demonstrating that cells
within R1, the lymphocyte gate, were predominantly
CD3+ CD4+ or CD3+
CD4
, consistent with helper or cytotoxic lymphocyte
populations, respectively. The larger cells were shown to express
the mannose receptor, consistent with a population of macrophages
(33, 50). However, more than 99% of these macrophages did
not express CD4 detectably on their surface, as determined by flow
cytometry. Using multiple replicate analyses of BAL samples from three
rhesus monkeys, the frequency of CD4+ macrophages was
0.24% ± 0.2%. The SIV coreceptor CCR5 was shown to be expressed on a
percentage of the CD4-negative alveolar macrophages (Fig.
6). BAL samples were divided into
three groups: uninfected, infected with SIVmac239, and
infected with SIVmac239/316E. BAL samples infected with
SIVmac239/316E demonstrated a profound loss of
CCR5+ cells as early as 3 dpi. This observation
suggests that CCR5+ CD4 cells are the
principal early targets of SIVmac239/316E in these alveolar macrophage cultures.
The antibodies for these flow cytometric determinations have been used
repeatedly by our laboratories and the laboratories of others to
identify CD4 and CCR5 on the surface of rhesus monkey cells with
good sensitivity (2, 34, 35, 48, 49, 53, 55, 66). For
the present experiments, we used appropriate CD4/CCR5-positive and
-negative controls to establish gating and sensitivity. The level of
CD4 on BAL that we report here is markedly reduced compared to rhesus
monkey peripheral blood monocytes/macrophages. In fact, we have
routinely used the same OKT4 monoclonal antibody and flow cytometry
methods to routinely identify CD4 on the surface of blood monocytes
from rhesus monkeys (56). Thus, the macrophages from BAL
fluid are indeed significantly different from
monocytes/macrophages from peripheral blood in the level of
surface CD4 expression.
Finally, we examined the ability of monoclonal antibody to CD4 to block
replication of SIVmac239/316E. High concentration of the
19thy5D7 monoclonal antibody significantly slowed the kinetics of virus
appearance, but the CD4 blockage was not highly effective at inhibiting
viral replication (Fig. 7). These results
are consistent with previous testing in which the same monoclonal
antibody inhibited but did not completely block the early appearance of
newly synthesized SIVmac239/316E DNA after exposure of
virus to alveolar macrophage cultures (42).
The vast majority of lung macrophages from BAL samples appear to be
refractory to infection by SIVmac239. The establishment of
early infection by SIVmac239 in these alveolar macrophage
cultures appears to be due to a small population of susceptible cells
beyond which SIVmac239 cannot spread significantly. Although
we have not shown directly what these susceptible cells are, it is
likely that they are the small fraction of CD4+ macrophages
in these cultures. Consistent with this, entry of SIVmac239
into the early susceptible population and viral DNA synthesis
were effectively blocked by antibody to CD4 (42). Some of
these susceptible cells could very well express levels of CD4
below our ability to detect them by flow cytometry. Thus, the block to
replication of SIVmac239 in alveolar macrophages appears to
relate to the absence of sufficient CD4 on the surface of the vast
majority of these cells.
It is not entirely clear to what extent the decay in
SIVmac239 DNA levels is actually due to DNA instability
versus the die-off of productively infected cells. The decay in numbers
of infectious cells over time (Table 1), the inability to get expected
levels of SIVmac239 DNA at day 4 or 5 after an initial
exposure to SIVmac239 (Table 2), and the die-off of
CCR5-positive cells infected with SIVmac239/316E all
argue that die-off of the small numbers of SIVmac239-infected
cells is likely to be a major factor in the loss of SIVmac239
DNA over time. However, in addition to major limitations in the amount
of CD4 needed to allow SIV239 entry into the vast majority of alveolar
macrophages, intracellular blocks may also exist.
About 30 to 50% of monkeys that die with AIDS from SIVmac239
infection have marked, characteristic tissue lesions in brain and/or
lung (12). The infected cell type that vastly predominates in the pathologic lesions in these tissues is the macrophage. The
appearance of SIV encephalitis and giant cell pneumonia in monkeys is
uniformly associated with the evolution of virus variants with high
replicative capacity for tissue macrophages not only at the New England
Regional Primate Research Center with strain SIVmac239
(12, 30, 43) but also at other research centers (3). Strains with specific sequence changes that allow high replicative capacity in macrophages have been characterized as able to
infect cells in a fashion that is less dependent, or independent, of
the presence of CD4 (15-17, 41, 58).
Our studies indicate that the ability of SIVmac239/316E
and similar strains to replicate efficiently in cultured tissue
macrophages appears to relate to the low or absent levels of CD4 on
these cells and the decreased dependence of these strains on CD4. Thus, SIVmac239/316E may be able to replicate in tissue
macrophages lacking detectable CD4 because of an ability to infect
independent of this receptor. Evidence has also been presented that
SIVmac239/316E has a higher affinity for CD4 than
SIVmac239 (58; R. E. Means and
R. C. Desrosiers, unpublished data). Furthermore, strains of SIV
that have been described as "CD4 independent" usually, but not
always, infect much better with CD4 present on the surface of the cell
than in its absence (15-17, 41, 58). Thus, the increased
affinity of SIVmac239/316E for CD4 may allow infection of
tissue macrophages with an extremely low density of CD4 (below our
ability to detect by flow cytometry) in a CD4-dependent fashion. It is
important to note that these properties, and the sequence changes that
are required to bring them about, are not an unusual feature of one SIV
isolate or one monkey but a quite uniform feature of virtually all
monkeys that die with SIV encephalitis or giant cell pneumonia (3,
12, 15, 30, 43, 54).
Our results clearly predict the evolution of HIV-1 strains with the
properties of relative CD4 independence in the brains and lungs of at
least some people with tissue-specific HIV-1 disease manifestations.
The dearth of CD4 on the surface of human alveolar macrophages, similar
to rhesus monkey alveolar macrophages, has been reported (37,
64). Resident macrophages in brains of both monkeys and humans
are also largely low for CD4 expression (63), and the same
sorts of SIV envelope sequence changes that allow high replicative
capacity in lung macrophages occur in the brain (3, 30). It
is not clear if analyses have been done appropriately to determine
whether such changes or properties occur with some regularity at a
terminal stage of brain or lung disease in humans (6, 22, 26, 37,
47, 60).
We thank Prabhat Sehgal for help with lung lavages, Ken Williams
for helpful advice, and H. Yoshikura for critical comments.
This work was supported by grants from the Ministry of Health and
Welfare and the Science and Technology Agency of Japan, the Health
Sciences Foundation and Organization for Pharmaceutical Safety and
Research in Japan, and the Public Health Service (AI 25328 and RR00168).
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Abstract
Text
) or a monoclonal antibody against CD4 (19thy5D7; kindly provided
by S. Schlossman, Dana-Farber Cancer Institute) serially diluted 1:10
(0.1 mg/ml; 
), 1:25 (
), 1:50 (
), 1:100 (
), or 1:200 (
).
Cells were then infected with SIVmac239/316E (10 ng of
p27 antigen) for 2 h. The cells were washed with Hanks balanced
salt solution and then maintained in medium with the same concentration
of antibody as was used for the pretreatment.
p27gag antigen level in the culture supernatant
collected at 3-day intervals was determined with a commercial
p27gag antigen assay kit (Coulter).