Componentes forrajeros de la dieta experimental a) La avena ( Avena sativa L )

The time course of the response in the CD45RA enriched population, did not suggest a pure ‘primary’ T cell response to HRV. However in order to confirm that HRV could not stimulate either a typical primary response in vitro, nor a superantigen type response, the response to HRV was tested using UCBMC, which represent a unique population of naive T cells. As shown in figure 4.15, even after 10 days in culture these naive cells were unable to respond to either HRV or IFZ A.

4.6 Discussion

The experiments in this chapter demonstrate that stimulation of tonsil derived T cells with HRV in vitro results in the activation of CD4 Thl type T cells, within both CD45RA and CD45RO T cell subsets.

The finding that HRV induces predominantly CD4 T cells is consistent with the dependency of the response on the presence of MHC class n+ cells. There is a possibility that the conditions of this in vitro assay may have favoured the survival of CD4 T cells over CDS T cells. Indeed evidence was found to suggest that CDS T cells may also make a small contribution to the HRV response. However, previous studies in HRV immunised humans (Levandowski et al. 1986) and mice (Hastings et al. 1991) also showed the preferential induction of the CD4 T cell subset. A protective role for CD4 T cells has been found against IFZ A and RSV infection, both in vivo and in vitro

(Liang et al. 1994; Lukacher et al. 1986; Graham et al. 1994, 1991; Alwan et al. 1994). Presumably, the activation of CD4 T cells in vivo, may be required to provide help for B cells in order to produce the neutralising antibodies required to confer protection on re­ exposure.

The high levels of IL-2 and IFNy produced in response to HRV stimulation would suggest the activation of a Thl type response. Again it is possible that the conditions of the in vitro assay may have favoured the preferential expansion of the Thl T cell subset since the stimulator population used were found to be comprised of activated B cells which are modest producers of IL-12 and DC which are extremely potent producers of IL-12. However, the induction of Thl cytokines is also consistent with a previous report on HRV that show a similar production of IL-2 and IFNy on stimulation of PBMC from infected individuals (Hsia et al. 1990). In IFZ A and RSV infections the induction of a Thl type response has been shown to have a protective role (Graham et al. 1994; Tang et al. 1994). In the case of HRV, one study showed that the prophylactic use of IFNa and IFNp but not IFNy was effective at protecting against reinfection (Higgins et al. 1988). However, it is possible that the overall effect of Thl cytokines can confer protection, and indeed this is substantiated by the protective effect of IFN a (Higgins et

al. 1988), which in addition to its own anti-viral activity, can further promote the production of other anti-viral cytokines by promoting Thl responses and inhibiting Th2 responses (Finkelman et al. 1991; Brinkman et al. 1992, 1993; Recht et al. 1991; Pene et al. 1988; W enneiet al. 1996).

Most studies indicate that the Th2 cytokines IL-5 and IL-6, are important for production of IgA (Kopf et al. 1995). In contrast, IFNy has been found to inhibit the proliferation and priming of naive T cells to differentiate into Th2 T cells (Sedar et al. 1992; Gajewski et al. 1988, 1989; Scott et al. 1991; Fernando-Botran et al. 1988; Betz et al.

1990), and inhibit the production of IgA by IL-5 (Eckman et al. 1992). It is therefore possible that this inhibitory effect may contribute to the slow production of IgA following HRV infection (Cate et al. 1966; Barclay et al. 1989). This would suggest that the Thl cytokines found in this study would not be conducive to the production of IgA that is found in nasal secretions and the serum of protected individuals (Barclay et al.

1989). However, there is also evidence to suggest that human Thl cells, may be able to support the production of IgA, IgG, IgM and IgD but not IgE, at high B cell to T cell ratios and display a cytolytic function at higher T cell numbers (Del Prete et al. 1991). Thus according to this model, the induction of a Thl response enables the immune system to provide protection against the pathogen on two levels, first via anti-viral cytokines during the acute phase of the response when infiltration of T cells is maximum and later when the T cell numbers have declined after the symptomatic phase, the production of neutralising antibodies can confer protection against reinfection.

Although IFNy can upregulate the expression of the major group receptor ICAM-1 on a variety of cells such as endothelial and epithelial cells (Rothlein et al. 1988; Bloemen et al. 1993; Subauste et al. 1993; Dustin et al. 1986), a recent study has shown that IFNy itself, does not facilitate viral infection. Thus IFNy and IFN a were found to reduce the susceptibility of HRV 14 and HRV2 to infect a bronchial epithelium cell line but TNFa (another Thl cytokine) was able to enhance infectivity of HRV 14 (Subauste et al. 1995). Since both TN Fa and IFNy can upregulate ICAM-1 expression, this suggests that the spread of infection is not determined only by the ability of a cytokine to upregulate the cellular receptor. It has been suggested that the relative levels of these cytokines could

determine the susceptibility to infection in any particular individual (Subauste et al. 1995).

HRV has been found to be the major viral precipitant of asthma (Johnston et al. 1995, 1996b; Nicholson et al. 1993). However, in contrast to the Thl profile induced by HRV in this study, the major cytokine profile induced in asthmatic individuals is a Th2 subtype (Kline et al. 1994; Walker et al. 1992, 1994; Robinson et al. 1996). This discrepancy may be explained by the ability of other URT viruses such as RSV and IFZ A (which are also précipitants of asthma) to induce both Thl and Th2 profiles under different conditions. Thus it is possible that HRV may also induce a Th2 profile under certain conditions or may switch from a the Thl profile seen above to a Th2 profile during the course of the response. In asthmatic individuals, it is likely that the presence of high concentrations of IL-4 and IL-10 may prevent the natural development of Thl cells in response to HRV and induce an immediate expansion of Th2 cells. Many studies have shown that IL-4 can overcome the effects of Thl promoting cytokines such as IL- 12, to promote the development of Th2 cells (Seder et al. 1993; Hsieh et al. 1993; Schmitt et al. 1994b; Wu et al. 1994).

There is also evidence to suggest that in asthmatic individuals, HRV may contribute to the pathology of an asthmatic response by its ability to produce inflammatory cytokines and mediators such as IL-1, IL-8, GM-CSF, histamine, prostaglandins and bradykinin (Welliver et al. 1981; Bardin et al. 1992; Johnston et al. 1995a; Gwaltney et al. 1995). The ability of these cytokines and mediators to promote the infiltration and activation of eosinophils and the mediators to promote airway hyperreactivity and obstruction, may trigger and potentiate the inflammatory response induced in asthpaatic individuals. Indeed many of these features are augmented after a viral infection in asthmatic individuals (Teran et al. 1994; Calhoun et al 1991, 1994; Fraenkel et al. 1995; Bardin et al. 1995, Lemanske et al. 1989). Thus many factors, which include Th2 cytokines may contribute to the inflammatory response in asthma following a viral infection. However in non asthmatic individuals, the absence of a Th2 response, combined with the anti­ viral effects of the Thl cytokines, may limit the pathology and promote viral clearance.

One of the most surprising findings of this study was the presence of an HRV specific response in both CD45RA and CD45RO enriched T cells. When optimal conditions were used, the CD45RO and CD45RA enriched populations were quantitatively and qualitatively comparable. However at suboptimal conditions, the CD45RA enriched population displayed particularly high background proliferation when compared to other responder populations, and which reduced the HRV specific response. On removal of MHC class n cells and addition of optimal concentrations of exogenous APC back to the CD45RA enriched T cells, proliferative responses with high stimulation indices could be observed in the large number of tonsils tested. Indeed in some cases, a higher HRV specific response was observed in the^^EMSRA enriched population compared to the CD45RO enriched population. There a few studies that have also found higher responses in the CD45RA T cell subseTto non-recall antigens (Fern et al. 1991; Plebanski et al. 1992), which is consistent with these cells representing a naive population. However, as discussed in chapter 3, the high prevalence of HRV and the ability to activate cross reactive T cells in this study, would suggest previous exposure to the virus. Furthermore, there was no difference between the CD45RA and CD45RO enriched T cells in either the frequency of responders or the time course of the response, when the two populations were compared under optimal conditions. This is in contrast to previous studies where the CD45RA T cells respond at suboptimal levels compared to the CD45RO T cells to recall antigens (Merkenschlager et al. 1988; Pilling et al.

1996).

As a result of the difference in kinetics in the loss of CD45RA and gain of CD45RO, it is possible that a percentage of T cells can express both markers (Wallace et al. 1990; Picker et al. 1993). Therefore the response observed in the CD45RA enriched population in response to HRV and IFZ A may be due to the presence of a small population of T cells expressing both CD45RA and CD45RO. However, this is unlikely since the frequency of the responders in the CD45RA enriched population were within the same range as that observed for the CD45RO enriched population.

Taken together the results from this chapter suggest that the response to HRV or IFZ A in the tonsil derived CD45RA enriched T cells examined, was not representative of a pure naive population. Indeed the results from a number of previous studies suggest that

CD45RA expressing T cells may not always represent a naive population. Thus many marker of activation such as CD25 (Kanegane et al. 1991), CD44 (Lightstone et al.

1991) and CD29 (Pilarski 1991) were found to be co-expressed on CD45RA T cells. In addition, there have been several reports suggesting that CD45RO T cells may revert to a CD45RA phenotype in vivo (Michie et al. 1992; Mclean et al. 1995) and in vitro re­ expression of CD45RA was noted after prolonged culture of either CD45RA T cell lines or in PB derived CD45RA T cells activated to express CD45RO (Rothestein et al. 1990; Warren et al. 1991; Wallace et al. 1990). Furthermore, in contrast to studies that have shown that activation of CD45RA T cells requires both TcR and costimulatory signalling (Horgan et al. 1990; Azuma et al. 1993), one study found that CD45RA T cells could be activated by IL-2 alone to express CD45RO as well as other markers of activation (Roth et al. 1994). The finding that CD45RA expression was higher in both T cell clones and freshly isolated PB derived T cells clones in the G2/S/M phases of

activation compared to those in the Gq/Gi phases (La Salle et al. 1991), further supports the idea that CD45RA T cells may not always be completely unactivated cells.

It is therefore possible that the CD45RA T cells responding after 3 days in culture, represent a primed population of cells that have reverted from a CD45RO to a CD45RA phenotype. Cross reactive epitopes may be responsible for activating the secondary type response that is seen after 3 days in both CD45RA and CD45RO T cell subsets, whilst novel epitopes to the serotypes used may induce a primary response in the CD45RA T cell subset which may require the longer periods of 7-8 days to reach maximal levels.

A number of studies have shown that a functional distinction between CD45RA and CD45RO T cells can only be made under suboptimal costimulatory conditions (Horgan et al. 1990; Azuma et al. 1993). However, when optimal costimulatory condition were provided, CD45RA T cells were found to respond to non specific stimulation at equivalent or greater levels than CD45RO T cells (Horgan et al. 1990; Merkenschlager et al. 1991; Fischer et al. 1992; Unutmaz et al. 1994; Kalinski et al. 1995). Furthermore, in the presence of anti-CD28, CD45RA T cells were able to respond to specific stimulation with recall antigens (Pilling et al 1996). Thus it is possible that the conditions of this in vitro assay, may have provided optimal conditions under which the CD45RA enriched T cells responded to HRV at an equivalent or higher SI compared to

the CD45RO enriched T cells. This is substantiated by the finding in chapter 3, that the B cell enriched population used as the basic APC population in these studies were of equivalent or superior stimulatory capacity to the DC enriched population (figure 3.22).

Most of the studies defining the CD45RA/CD45RO naive/memory paradigm in humans have been demonstrated using T cells derived from PB. Thus the discrepancies found in this study may be accounted for by differences in the migratory patterns in T cells derived from PB compared to tonsils. Therefore in order to ensure that these results were not unique to tonsil, the HRV response in CD45RA T cells derived from peripheral blood was also examined in chapter 5.

Figure 4.1 Phenotypic comparison of CD4 and CDS depleted HD cells 3% 20%

I

3% u 17% LogjQ Fluorescence IkwkMaHMtaa

Figure 4.1; The phenotypic profiles of CD4 depleted HD cells (column 1) and CDS depleted HD cells (column 2) were compared. The phenotypic markers used included: a negative control (a-b); CD3 (panels c-d); CD4 (panels e-f); and CDS (panels g-h). The percentage of positive cells obtained with each marker is included in each panel n=5.

Figure 4.2 The HRV specific T cell response is dependent on the presence of CD4 T cells a. 60000 •E E

r

X 5 0 000 -- 4 0 000 -- 30000 20000 10000 - - HD

I

CD4 R esp o n d er population CDS ■ Medium BHRV15 □ HRV1A 35000 30000 25000 " ^ 20000 15000 10000 CD4 R espon d er population 70000 60000 50000 40000 3 0000 -- 20000 - - 10000 - C D4 CDS R esp o n d er population

Figure 4.2 T h e p roliferative resp on ses o f unseparated H D c e lls and C D 4 or C D 8 d ep leted H D

c e lls w ere com pared. T h e results from three separate ton sils a, b, and c are sh ow n a b o v e n=5.

Cells: C D 4 d ep leted H D c e lls (la b elled as C D S ), C D S d ep leted H D c e lls (la b e lle d as C D 4 ) and

unseparated H D c e lls (lab elled as H D ) at 4x10^ c e lls / w ell

1.2 1 0.8 E O) 5 0.6 CM 1 0 .4 0.2 0

Figure 4.3 The cytokine profile of the HRV response

b 4 — Medium HRV15 HRV1A Antigen 6 5 — 4 E TO 5 3 + - 2- à ■ Medium □ HRV15 □ HRV1A Medium HRV15 HRV1A Antigen 0.7 0.6 0.5 0.2 0.1 1 3 7 2.5 O) 0.5 3 1 7 •M edium ■HRV15 ■HRV1A days days

Figure 4.3: Supernatants w ere c o lle c te d from cultures o f E + H D c e lls after 1, 3, and 7 days in

culture and stored at -20^C until su bsequ en tly thaw ed and assayed for the p resen ce o f IL-2 and

IFNy. T h e results sh ow the m ean and standard error o f the m ean o f 7 to n sils after 1 day in

culture for IL -2 (a) and 7 days in culture for IF N y (b); the tim e cou rse for the r e le a se o f the IL -2

(c) and IF N y (d) for on e representative in dividu al.

Responders: E-i- H D c e lls at 4x10^ c e lls /w e ll

Stim ulators: E- L D c e lls at 10^ c e lls /w e ll.

Figure 4.4 Phenotypic analysis of CD45RO. CD14. CD19 depleted HD cells

I

8% a 64% 38% e 25% g 32% 1 38% 10% 85% 10% 14% 15% 22% 81% 0 Logjo Fluorescence

Figure 4.4: The phenotypic profiles of HD cells (column 1) and CD45RO, CD 14, CD 19 depleted HD cells (column 2) were compared. The phenotypic markers used included: a negative control (a-b); CD3 (c-d); HLA-DR (e-f); CD19 (g-h); CD14 (i-j); CD45RO (k-1); and CD45RA (m-n). The percentage of positive cells obtained with each marker is included in each panel n=3.

Figure 4.5 The HRV response with CD45RO. CD14. CD19. depleted HD cells 1 5000 1 2500 10000 7 5 0 0 2 5 0 0 ■ Medium 0 H R V 1 5 □ HRV1A CD3 CD45RA R esponder population

Figure 4.5: T h e p roliferative resp on ses o f C D 1 4 , C D 1 9 d ep leted and C D 4 5 R A , C D 14, C D 1 9

d ep leted H D c e lls w ere com pared n=4.

Cells: C D 14, C D 19 d ep leted H D c e lls (lab elled as C D 3) and C D 4 5 R O , C D 14, C D 19, d ep leted

H D c e lls (lab elled as C D 4 5 R A ) at 4x10^ c e lls / w ell.

Figure 4.6 Phenotypic analysis of CD45RO. CD14. CD19. M HC class II depleted HD cells 2% 2% 1% 4% U 8% 90% 0 Log,Q Fuorescence

Figure 4.6: The phenotypic profile of CD45RO, CD14, CD19, MHC class II depleted HD cells is shown above. The phenotypic markers used included: a negative control (a); CDS (b); HLA- DR (c); CD19 (d); CD14 (e); CD45RO ( f); and CD45RA (g). The percentage of positive cells obtained with each marker is included in each paneli n=8.

Figure 4.7 The APC requirement of

CD45RO. CD14. CD19. MHC class II depleted HD cells

1 6 0 0 0 0 1 4 0 0 0 0 120000 o 100000 8 0 0 0 0 6 0 0 0 0 4 0 0 0 0 20000 0 .1 3 3 0.4 APC concentration xIO® cells/w ell

■ Medium ^ H R V 1 5 □ HRV1A

Figure 4.7: Irradiated E- L D c e lls w ere titrated over a range o f con cen tration s from 0.133x10^'

to 10^ c e lls /w e ll in the p resen ce o f a fix ed num ber o f C D 45R O , C D 14, C D 19, M H C cla ss II

d ep leted H D c e lls n=12.

Responders: C D 45R O , C D 1 4 , C D 19, M H C cla ss II d ep leted H D c e lls at 4x10^ c e lls /w e ll

Figure 4.8 Comparison of the HRV response in CD45RO. CD14. CD19 depleted and CD45RA. CD14. CD19 depleted HD cells

60000 50000 --

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