Biology Reference
In-Depth Information
number per cell. Often, investigators analyzing cells for chemokine receptors
obtain histograms that are not clear and de®nitive. The histograms that repre-
sent cells stained with the isotype and subtype control Ig and histograms that
represent cells stained with the antibodies speci®c for chemokine receptors are
not clearly distinct from each other. There may be shoulders or smears or shifts
that make the interpretations of these data di½cult and tentative. In other
cases, a failure to demonstrate any di¨erence between the histograms obtained
with control and speci®c Ig is dubious. The sensitivity of ¯ow cytometry for
the surface expression of molecules has been measured to be approximately
2000 molecules per cell (Loken and Herzenberg, 1975), but signal transduction
through receptors has been shown to be more in the order of several hundred
molecules per cell ( Harding and Unanue, 1990). Thus, the failure to detect ex-
pression using a suboptimal technology does not mean that the targeted mole-
cule, such as chemokine receptors, is not present or that it is not active.
Again, EAS has the potential to clarify the data concerning chemokine re-
ceptor expression. The added resolving power of EAS can be used to de®ni-
tively assess the expression of chemokine receptors on speci®c cells. Clear-cut,
distinct peaks can be obtained with EAS and these data, which are easier to
interpret and thereby have the potential to give us a more accurate description
of the expression of these important molecules. It is likely that some of the dif-
®culties that have been encountered in detecting chemokine receptors on cells
have been related to antibodies that do not possess a½nities su½cient to sup-
port the protocol. EAS may render these low-a½nity antibodies more useful
and may thereby widen the reagents that can be utilized in the analysis of cell
surface expression of chemokine receptors.
Cytokines and their receptors have also been shown to be important in HIV-
1 pathogenesis. Several cytokines, tumor necrosis factor ( TNF )-a ( Israel et al.,
1989), IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF ),
and macrophage colony-stimulating factor (M-CSF ) ( Koyanagi et al., 1988),
are known to enhance viral production. Other cytokines induced by infection,
such as IL-10 (Akridge et al., 1994; Borghi et al., 1995), IL-16 (Zhou et al.,
1997), IL-13, and interferon (IFN )-g (Bailer et al., 1999), have been shown
to inhibit viral replication. The expression of many of these cytokines that in-
¯uence viral production are also induced by viral proteins. Moreover, many
cytokines have been implicated in the immune response to the virus (Appay et
al., 2000). The production of cytokines by antigen-speci®c cells has also been
used to track these cells (Pitcher et al., 1999). Thus, the detection of cytokine-
producing cells and cytokine receptor-expressing cells is valuable in the inves-
tigation of HIV-1 disease pathogenesis.
Like chemokines, cytokines and their receptors are active at low copy num-
bers per cell. Consequently, they are often di½cult to assess accurately and
sensitively on a cell-by-cell basis. For these reasons, it is likely that analyses of
cytokines and cytokine receptors by ¯ow cytometry would bene®t from the
greater sensitivity and resolving power that has been demonstrated by EAS.
